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

Abstract

The present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) (including peripheral blood lymphocytes and bone marrow-infiltrating lymphocytes) from the blood and / or bone marrow of patients with hematological malignancies (e.g., liquid tumors, including lymphomas and leukemias), and the use of the TILs so expanded in treating diseases (e.g., cancer and hematological malignancies).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 504,337 filed on May 10, 2017; U.S. Provisional Application No. 62 / 530,681 filed on July 10, 2017; U.S. Provisional Application No. 62 / 550,398 filed on August 25, 2017; U.S. Provisional Application No. 62 / 590,034 filed on November 22, 2017; U.S. Provisional Application No. 62 / 621,462 filed on January 24, 2018; U.S. Provisional Application No. 62 / 621,798 filed on January 25, 2018; and U.S. Provisional Application No. 62 / 647,367 filed on March 23, 2018, the entire contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] Disclosed herein are methods for expanding tumor-infiltrating lymphocytes (TILs) from the blood and / or bone marrow of patients with hematological malignancies (e.g., liquid tumors, including lymphomas and leukemias), and compositions comprising the TIL populations thus obtained. In addition, disclosed herein are therapeutic uses of TILs expanded from the blood or bone marrow of patients with hematological malignancies (e.g., liquid tumors), including uses in the treatment of such hematological malignancies. Background Art

[0004] The use of adoptive autologous transfer of tumor infiltrating lymphocytes (TIL) to treat large, refractory cancers represents an effective method for treating patients with poor prognosis. Gattinoni et al., Nat. Rev. Immunol., 2006, 6, 383-393. TIL is mainly T cells, and IL-2-based TIL expansion followed by "rapid expansion process" (REP) has become the preferred method for TIL expansion because of its speed and efficiency. Dudley et al., Science, 2002, 298, 850-54; Dudley et al., J. Clin. Oncol., 2005, 23, 2346-57; Dudley et al., J. Clin. Oncol., 2008, 26, 5233-39; Riddell et al., Science 1992, 257, 238-41; Dudley et al., J Immunother., 2003, 26, 332-42. Many approaches have been explored to improve the response of melanoma to TIL therapy and to extend TIL therapy to other tumor types, but the success achieved is limited and the field remains challenging. 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. Early methods of expanding TILs from B-cell lymphomas produced poor results, with only 2 of 12 attempts to culture TILs providing potential antitumor activity. Schwartzentruber et al., Blood 1993, 82, 1204-1211. There is an urgent need to provide more effective treatments for many hematological malignancies, including acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL).

[0005] The present invention provides the unexpected discovery that a TIL expansion process can result in an effective population of TILs obtained from hematological malignancies (e.g., liquid tumors, including lymphomas or leukemias). Summary of the invention

[0006] In one embodiment, the present invention provides a method for treating cancer using a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

[0007] (a) optionally, pre-treating the patient with a regimen comprising at least one kinase inhibitor;

[0008] (b) obtaining a tumor from a patient by resection, biopsy, needle aspiration or apheresis, wherein the tumor comprises a first TIL population;

[0009] (c) optionally, fragmenting or dissociating the tumor, obtaining tumor fragments, and contacting the tumor fragments with a first cell culture medium;

[0010] (d) initially expanding the first TIL population in a first cell culture medium to obtain a second TIL population; wherein the number of the second TIL population is at least 5 times higher than the number of the first TIL population; wherein the first cell culture medium comprises IL-2; and wherein the initial expansion is performed for less than 21 days;

[0011] (e) amplifying the second TIL population for a second time in a second cell culture medium to obtain a third TIL population; wherein, 7 days after the start of the second expansion, the number of the third TIL population is at least 50 times higher than the number of the second TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC); and wherein the second expansion is performed for less than 14 days;

[0012] (f) harvesting a third TIL population; and

[0013] (g) administering a therapeutically effective amount of the third TIL population to the cancer patient;

[0014] wherein the tumor is a liquid tumor; and wherein the cancer is a hematological malignancy.

[0015] In one embodiment, the present invention provides a method for treating cancer using a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

[0016] (a) optionally, pre-treating the patient with a regimen comprising at least one kinase inhibitor;

[0017] (b) obtaining a tumor from a patient by resection, biopsy, needle aspiration or apheresis, wherein the tumor comprises a first TIL population;

[0018] (c) optionally, fragmenting or dissociating the tumor, obtaining tumor fragments, and contacting the tumor fragments with a first cell culture medium;

[0019] (d) initially expanding the first TIL population in a first cell culture medium to obtain a second TIL population; wherein the number of the second TIL population is at least 5 times higher than the number of the first TIL population; wherein the first cell culture medium comprises IL-2; and wherein the initial expansion is performed for less than 21 days;

[0020] (e) amplifying the second TIL population for a second time in a second cell culture medium to obtain a third TIL population; wherein, 7 days after the start of the second expansion, the number of the third TIL population is at least 50 times higher than the number of the second TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC); and wherein the second expansion is performed for less than 14 days;

[0021] (f) harvesting a third TIL population; and

[0022] (g) administering a therapeutically effective amount of the third TIL population to the cancer patient;

[0023] wherein the tumor is a liquid tumor; and wherein the cancer 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's lymphoma, Waldenstrom's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndrome, myelofibrosis, chronic myeloid leukemia, follicle-center lymphoma, indolent NHL, human immunodeficiency virus (HIV)-associated B-cell lymphoma, and Epstein-Barr virus (EBV)-associated B-cell lymphoma.

[0024] In one embodiment of the invention, the method further comprises adding an ITK inhibitor. In one embodiment, during at least one of steps (d) and (e), the ITK inhibitor is added to the cell culture medium. In one embodiment of the invention, the ITK inhibitor is a covalent ITK inhibitor that covalently and irreversibly binds to ITK. In one embodiment of the invention, the ITK inhibitor is an allosteric ITK inhibitor that binds to ITK. In another embodiment, the ITK inhibitor is selected from: aminothiazole ITK inhibitors, benzimidazole ITK inhibitors, aminopyrimidine ITK inhibitors, 3-aminopyridine-2-one ITK inhibitors, indolyl indazole ITK inhibitors, pyrazolyl indole 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 ((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 sources, including Tocris Bioscience, Inc. (Minneapolis, Minnesota, USA), Selleckchem, Inc. (Houston, Texas, 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 one 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.

[0025] In one embodiment of the present invention, a method for expanding peripheral blood lymphocytes (PBL) from peripheral blood comprises:

[0026] a. obtaining a sample of peripheral blood mononuclear cells (PBMC) from peripheral blood; wherein the sample is optionally cryopreserved;

[0027] b. isolating PBL from the sample by screening and removing CD19+ B cells;

[0028] c. Optionally, co-culturing the PBL with the CD19+B cells;

[0029] d. stimulating the PBL in the first cell culture medium with IL-2 and anti-CD3 / anti-CD28 antibodies for a period of about 2 days to about 6 days in a gas permeable container;

[0030] e. culturing the PBL in step (d) with IL-2 and anti-CD3 / anti-CD28 antibodies for a period of about 2 days to about 6 days;

[0031] f. separating the PBL bound to the antibody from the culture of step (e);

[0032] g. removing the antibodies from the PBL isolated in step (e); and

[0033] h. Harvesting PBLs.

[0034] In one embodiment of the invention, the method further comprises adding IL-2 after step (d) and replacing the first culture medium with a second cell culture medium. In another embodiment, the method further comprises adding IL-2 after step (e) and replacing the second culture medium with a third culture medium. In one embodiment, the first cell culture medium, the second cell culture medium or the third culture medium is selected from: CM-2, CM-4 and AIM-V. In another embodiment, the first cell culture medium and the second cell culture medium are the same. In another embodiment, the first cell culture medium and the second cell culture medium are different. In one embodiment of the invention, one or more of the first cell culture medium, the second cell culture medium and the third cell culture medium are the same. In another embodiment, the first cell culture medium, the second cell culture medium and the third cell culture medium are all different.

[0035] In one embodiment, the optional co-culture of said PBLs and said CD19+ B cells is performed for a period of 1 hour to 3 days.

[0036] In one embodiment of the present invention, the ratio of T cells to B cells in step (c) is 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: 0.1:1, 1:1 and 10:1 (B cells: T cells).

[0037] In one embodiment of the present invention, at the beginning of step (d), the starting cell number of PBL is at least about 1×10 5 to about 10×10 5 In another embodiment, at the beginning of step (d), the starting number of PBLs is at least about 2.5×10 5 Up to 10×10 5 In another embodiment, at the beginning of step (d), the starting number of PBLs is at least 5×10 5 PBLs.

[0038] In one embodiment of the invention, the IL-2 concentration used in each of steps (c) and (d) is about 1000 IU / mL to about 6000 IU / mL. In another embodiment, the IL-2 concentration used in each of steps (c) and (d) is about 3000 IU / mL.

[0039] In one embodiment of the present invention, the anti-CD3 / anti-CD28 antibody is coated on magnetic beads. In one embodiment, the method comprises co-culturing anti-CD3 / anti-CD28 antibody (coupled) magnetic beads with PBLs at a ratio of about 1:1 magnetic beads:PBLs in each of steps (c) and (d).

[0040] In one embodiment of the invention, the method comprises adding an ITK inhibitor. In one embodiment, the ITK inhibitor is added during at least one of steps (c), (d) and (e). In one embodiment of the invention, the ITK inhibitor is selected from the group consisting of aminothiazole ITK inhibitors, benzimidazole ITK inhibitors, aminopyrimidine ITK inhibitors, 3-aminopyridine-2-one ITK inhibitors, indolyl indazole ITK inhibitors, pyrazolyl indole 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.

[0041] In one embodiment of the present invention, any of the aforementioned methods for preparing PBLs is performed in a closed sterile system.

[0042] In one embodiment of the present invention, a method for expanding peripheral blood lymphocytes (PBL) from peripheral blood comprises:

[0043] a. obtaining a sample of PBMC from peripheral blood; wherein the sample is optionally cryopreserved;

[0044] b. isolating PBL from the sample by screening and removing CD19+ B cells;

[0045] c. co-culturing the PBL and the CD19+B cells for 4 days;

[0046] d. Approximately 2.5×10 5 About 5×10 5 The cells were added to CM-2 cell culture medium in a gas permeable container, and the PBLs were stimulated with IL-2 at a concentration of 3000 IU / mL and anti-CD3 / anti-CD28 antibodies immobilized on magnetic beads for about 4 days;

[0047] e. Replace CM-2 with AIM-V cell culture medium and approximately 3000 IU / mL of IL-2;

[0048] f. Culturing the PBL of step (e) with IL-2 and anti-CD3 / anti-CD28 antibodies immobilized on magnetic beads for an additional approximately 3 days;

[0049] g. separating the antibody-bound PBL from the culture of step (f);

[0050] h. removing the antibodies from the PBL isolated in step (g); and

[0051] i. Harvesting PBLs.

[0052] In one embodiment of the present invention, a method for treating a hematological malignancy comprises:

[0053] a. Obtain PBMC samples from peripheral blood of patients with hematological malignancies;

[0054] b. isolating PBL from the sample by screening and removing CD19+ B cells;

[0055] c. Optionally, co-culturing the PBL with the CD19+B cells;

[0056] d. stimulating the PBLs in the first cell culture medium with IL-2 and anti-CD3 / anti-CD28 antibodies for a period of at least about 4 days in a gas permeable container;

[0057] e. Culturing the PBL in step (d) with IL-2 and anti-CD3 / anti-CD28 antibodies for 3 days;

[0058] f. separating the PBL bound to the antibody from the culture of step (e);

[0059] g. removing the antibodies from the PBL isolated in step (e);

[0060] h. Harvesting PBL; and

[0061] i. administering to a patient a therapeutically effective amount of PBL to treat the hematological malignancy.

[0062] In one embodiment of the invention, the method further comprises obtaining a PBMC sample from a patient pretreated with an ITK inhibitor. In one embodiment of the invention, the ITK inhibitor is selected from the group consisting of aminothiazole ITK inhibitors, benzimidazole ITK inhibitors, aminopyrimidine ITK inhibitors, 3-aminopyridine-2-one ITK inhibitors, indolyl indazole ITK inhibitors, pyrazolyl indole inhibitors, thienopyrazole inhibitors, and ITK inhibitors targeting cysteine ​​442 in the ATP pocket. In one embodiment of the 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 pretreated with at least three rounds of ibrutinib regimens.

[0063] In one embodiment of the present invention, 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 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's lymphoma, Waldenstrom's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndrome, myelofibrosis, chronic myeloid leukemia, follicle 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 chronic lymphocytic leukemia (CLL). In one embodiment of the invention, about 0.1×10 9 About 15×10 9 PBLs were administered in an amount of 1 PBL.

[0064] In one embodiment of the present invention, the method for expanding marrow-infiltrating lymphocytes (MIL) from bone marrow comprises:

[0065] a. obtaining a sample of peripheral blood mononuclear cells (PBMC) from the bone marrow; wherein the sample is optionally cryopreserved;

[0066] b. Sorting CD3+, CD33+, CD20+ and CD14+ cell fractions (MIL fraction) and non-CD3+, non-CD33+, non-CD20+, non-CD14+ cell fractions (AML primitive cell fraction);

[0067] c. Optionally, disrupting the AML blast cell fraction;

[0068] d. adding the optionally disrupted AML primitive cell fraction to the MIL fraction at a cell number ratio of about 0.1:1 to about 10:1;

[0069] e. culturing one or both cell portions in a first cell culture medium comprising IL-2 in a gas permeable container;

[0070] f. Stimulate MIL with anti-CD3 / anti-CD28 antibodies to obtain expansion of MIL;

[0071] g. restimulate MIL with IL-2 and anti-CD3 / anti-CD28 antibodies for another period of about 2 days to about 6 days;

[0072] h. Cultivating the MIL with additional IL-2 for an additional period of about 1 to about 3 days; and

[0073] i. Harvesting the MIL.

[0074] In one embodiment of the invention, the method further comprises adding IL-2 after step (e) and replacing the culture medium with a second cell culture medium. In one embodiment, the first cell culture medium and the second cell culture medium are selected from: CM-2, CM-4 and AIM-V. In another embodiment, the first cell culture medium and the second cell culture medium are the same. In another embodiment, the first cell culture medium and the second cell culture medium are different.

[0075] In one embodiment, at the beginning of step (e), the gas permeable container contains at least about 2×10 4 About 5×10 5 In another embodiment, at the beginning of step (e), the gas permeable container contains at least about 2.8×10 4 to 3.4×10 5 In another embodiment, at the beginning of step (e), the gas permeable container contains at least 5×10 5 A MIL.

[0076] In one embodiment of the invention, in step (e), IL-2 is present at a concentration of 1000 IU / mL to 6000 IL / mL. In another embodiment, IL-2 is present at a concentration of about 6000 IU / mL. In another embodiment, in step (g), IL-2 is present at a concentration of about 3000 IU / mL. In another embodiment, in step (h), IL-2 is present at a concentration of about 3000 IU / mL.

[0077] In one embodiment of the invention, the culturing in step (e) is performed for about 3 days. In one embodiment, the stimulation in step (f) is performed for about 4 days. In one embodiment, the stimulation in step (g) is performed for about 7 days.

[0078] In one embodiment of the invention, the optionally broken cell part is broken using a method selected from the group consisting of sonication, homogenization, vortexing, vibration and lysis. In one embodiment of the invention, a suitable lysis method is used to lyse the non-CD3+, non-CD33+, non-CD20+, non-CD14+ cell part (AML original part), and the lysis method includes: high temperature lysis, chemical lysis (e.g., organic alcohol), enzyme lysis and other cell lysis methods known in the art.

[0079] In one embodiment of the present invention, in each of steps (f) and (g), the anti-CD3 / anti-CD28 antibodies are coated on magnetic beads, and the ratio of MIL:magnetic beads is about 1:1.

[0080] In one embodiment of the invention, the method is performed in a closed sterile system.

[0081] In one embodiment of the present invention, the method for expanding bone marrow infiltrating lymphocytes (MIL) from bone marrow comprises:

[0082] a. obtaining a sample of peripheral blood mononuclear cells (PBMC) from the bone marrow; wherein the sample is optionally cryopreserved;

[0083] b. Sorting CD3+, CD33+, CD20+ and CD14+ cell fractions (MIL cell fraction) and non-CD3+, non-CD33+, non-CD20+, non-CD14+ cell fractions (AML primitive cell fraction);

[0084] c. disrupting the AML primitive cell fraction, and adding the disrupted AML primitive cell fraction to the MIL cell fraction at a cell number ratio of about 1:1;

[0085] d. culturing the cell portion for about 3 days in a gas permeable container with a first cell culture medium comprising IL-2 at a concentration of about 6000 IU / mL;

[0086] e. adding the anti-CD3 / anti-CD28 antibodies immobilized on magnetic beads to the cell culture at a ratio of about 1:1 (MIL: magnetic beads), and culturing the MIL and antibodies for about 1 day;

[0087] f. replacing the first cell culture medium with a second cell culture medium comprising an additional IL-2 at a concentration of about 3000 IU / mL;

[0088] g, incubate the antibodies and MIL for another approximately 3 days;

[0089] h. re-stimulating the MIL with IL-2 and anti-CD3 / anti-CD28 antibodies immobilized on magnetic beads for at least about 4 days;

[0090] i. replacing the second cell culture medium with a third cell culture medium comprising an additional IL-2 at a concentration of about 3000 IU / mL for an additional period of at least about 3 days;

[0091] j. Harvesting the MIL.

[0092] In one embodiment of the present invention, a method for treating a hematological malignancy comprises:

[0093] a. obtaining a sample of peripheral blood mononuclear cells (PBMC) from the bone marrow; wherein the sample is optionally cryopreserved;

[0094] b. Sorting CD3+, CD33+, CD20+ and CD14+ cell fractions (MIL fraction) and non-CD3+, non-CD33+, non-CD20+, non-CD14+ cell fractions (AML primitive cell fraction);

[0095] c. Optionally, disrupting the AML blast fraction;

[0096] d. adding the optionally disrupted AML primitive cell fraction to the MIL fraction at a cell number ratio of about 0.1:1 to about 10:1;

[0097] e. culturing one or both cell portions in a first cell culture medium comprising IL-2 in a gas permeable container;

[0098] f. stimulating the sample with anti-CD3 / anti-CD28 antibodies;

[0099] g. restimulate MIL with IL-2 and anti-CD3 / anti-CD28 antibodies for at least another 4 days;

[0100] h. Cultivating the MIL with additional IL-2 for at least about 3 additional days;

[0101] i. harvesting the MIL; and

[0102] j. administering a therapeutically effective amount of the MIL to a patient to treat a hematological malignancy.

[0103] In one embodiment of the present invention, 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 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's lymphoma, Waldenstrom's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndrome, myelofibrosis, chronic myeloid leukemia, follicle 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 invention, about 4×10 8 About 2.5×10 9 The amount of MIL is applied. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] The foregoing Summary, as well as the following Detailed Description, will be better understood when read in conjunction with the appended drawings.

[0105] Figure 1 Pathological information of lymphoma tumors is shown.

[0106] Figure 2 A comparison of different subsets of lymphoma TILs and melanoma TILs is shown, showing that the effector memory (EM) subset in lymphoma TILs is significantly higher than the EM subset in melanoma TILs.

[0107] Figure 3 A comparison of different subsets of lymphoma TILs and melanoma TILs is shown, showing CD28 in lymphoma TILs + CD4 + The subpopulations were significantly higher than those in melanoma TILs.

[0108] Figure 4 CD4 of non-Hodgkin lymphoma TIL and melanoma TIL are shown + Comparison of T cell subsets, showing 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.

[0109] Figure 5 CD8 TILs of non-Hodgkin lymphoma and melanoma TILs are shown+ Comparison of T cell subsets, showing 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.

[0110] Figure 6 CD4 of non-Hodgkin lymphoma TIL and melanoma TIL are shown + Comparison of T cell subsets showing 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.

[0111] Figure 7 CD8 TILs of non-Hodgkin lymphoma and melanoma TILs are shown + Comparison of T cell subsets showing 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.

[0112] Figure 8 A comparison of the cell types of 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.

[0113] Fig. 9 Shown are the results of bioluminescent redirected lysis assay (BRLA).

[0114] Fig.10 Shown are the results of interferon-γ (IFN-γ) enzyme-linked immunosorbent assay (ELISA) of lymphoma TILs and melanoma TILs.

[0115] Fig.11 Shown are the results of an ELISpot assay of lymphoma TILs.

[0116] Fig.12 Shown are the results of ELIspot assay of melanoma TILs.

[0117] Fig.13 Results of NANOSTRING NCOUNTER analysis are shown, showing that lymphoma TILs express higher levels of RORC IL17A (TH17 phenotype) and GATA3 (Th2 phenotype) compared to melanoma TILs. The individual genes are highlighted in the red boxes in the heatmap.

[0118] Fig.14 The TIL expansion and processing process is shown. Step 1 involves adding 4 tumor fragments to 10 G-Rex 10 flasks. In step 2, approximately 40×10 6 More than 100 TILs are obtained. In step 3, (the TILs obtained in step 2) are divided into 36 G-Rex100 flasks for REP. In step 4, the TILs are harvested by centrifugation. After a total processing time of about 43 days, a fresh TIL product is obtained in step 5, at which time the TILs can be infused into the patient.

[0119] Fig.15 A treatment regimen of the present disclosure using TILs obtained from lymphoma is shown. Surgery (and tumor resection) is performed initially, and lymphocyte depleting chemotherapy refers to "non-myeloablative lymphocyte depletion with chemotherapy" as described elsewhere herein.

[0120] Figure 16 shows the results of flow cytometry analysis using the standard phenotypic panel DF2 as described below in Example 4. Lymphoma and melanoma TILs were stained using the standard phenotypic panel DF2 as described in Example 4. The data shown represent different subsets of total CD4 and CD8 T cells in the TILs. Fig.16A The proportions of CD4 and CD8 cells in naive T cell subsets are shown; Fig. 16B is the ratio of CD4 and CD8 cells in the central memory T cell subset (CM); Fig. 16C is the ratio of CD4 and CD8 cells in the effector memory T cell subset (EM), Fig.16D is the ratio of CD4 and CD8 cells in terminally differentiated effector memory T cell subsets. P values ​​were calculated using a two-tailed Mann-Whitney test (unpaired). The average ratio of cell subsets is represented by horizontal bars.

[0121] Figure 17 shows the results of flow cytometry analysis using the standard phenotypic panel DF1 as described in Example 4 below. Lymphoma and melanoma TILs were stained using the standard phenotypic panel DF1 as described in Example 4. The data shown represent the different CD27+ ( Fig.17A ) and CD28+( Fig. 17B ) subsets, indicating that the proportion of CD4 T cells expressing the costimulatory molecule CD28 was higher in lymphoma TILs. P values ​​were calculated using a two-tailed Mann-Whitney test (unpaired).

[0122] Figure 18 shows the results of an interferon-γ (IFN-γ) assay performed according to Example 4 below. Fig.18AResults using ELISpot are shown. ELISpot data are expressed as: IFN-γ producing cells / 10 6 TILs. Fig.18B Results using ELISA are shown. ELISA data are expressed as 5 × 10 5 IFN-γ levels in TIL culture supernatants of 1 TIL / well (log values). P values ​​were calculated using a two-tailed Mann-Whitney test (unpaired).

[0123] Figure 19 shows the lytic potential of TILs. Fig.19A Co-culture (TIL effector cells and GFP+P815 target cells) for 4 hours ( Fig.19A ) and 24 hours ( Fig.19B ) 50 (relative to 10 6 TILs were normalized).

[0124] FIG. 20 shows the cytolytic activity of different TILs against allogeneic and autologous tumor types. Fig. 20A The cytolytic activity of melanoma TILs against allogeneic 526 target cells is shown. Fig. 20B The cytolytic activity of lymphoma TILs against autologous tumor cells as determined by 7-AAD uptake is shown. Fig. 20A and Fig. 20B The data in are shown as the percentage of dead cells in co-cultures with an effector cell:target cell (E:T) ratio of 50:1. Fig. 20C represents the percentage of target cell killing induced by melanoma TIL. Fig.20D Represents the percentage of target cell killing induced by lymphoma TILs at different E:T ratios.

[0125] Fig.21 is a heat map showing the gene expression profiles of lymphoma TILs and melanoma TILs. The expression profiles were determined using the 579plex nCounter GX Human Immunology V2 CSO Panel from NanoString. This heat map shows the fold change in expression of a specific set of genes in lymphoma TILs compared to melanoma TILs, demonstrating higher expression of IL-17A and RORC in TILs from lymphomas. The cancers shown in this figure include follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), and mantle cell lymphoma (MCL).

[0126] Fig. 22 is a schematic diagram of the 2A process showing arrangements for preparing TILs, harvesting, and shipping.

[0127] Fig.23 is a flow chart showing the 2A process for preparing TIL.

[0128] FIG. 24 is a flow chart showing three different methods for expanding peripheral blood lymphocytes (PBL).

[0129] Figures 25A-25C Three different methods for expanding bone marrow infiltrating lymphocytes (MIL) from the bone marrow are shown.

[0130] Fig.26 Figure 2 shows the fold expansion of PBL isolated from fresh peripheral blood mononuclear cells (PBMC) and cryopreserved PBMC. Cryopreserved PBMC were from CLL patients who had not received (PreRx PBL) or had received (PostRx PBL) ibrutinib regimen. Figure 26-3 In each of the graphs in Figure 4, each dot is a patient. The PBL of the red dot patient was expanded using PBL method 1. The PBL of the green dot patient was expanded using PBL method 2; the PBL of the black dot patient was expanded using PBL method 3.

[0131] Fig. 27 A graph showing IFN-γ producing cells of PBL isolated from fresh PBMCs and cryopreserved PBMCs. Among cryopreserved PBMCs, PreRx PBLs and PostRx PBLs are also shown.

[0132] Fig.28 Shown are the proportions of CD4+ and CD8+ T cell subsets in PreRx PBL and PostRx PBL (using melanoma TILs as comparators).

[0133] Figures 29A-29D and Figures 30A-30D Comparison of CD4 ( FIG. 29 ) and CD8 ( FIG. 30 ) memory subsets of PreRx PBL and PostRx PBL is shown (using melanoma TILs as comparators). Fig.29A and 30A Data for naive (CCR7+ / CD45RA+) are shown; Fig.29B and 30B Data are shown for central memory T cells (CM) (CCR7+ / CD45RA-); Fig.29C and 30C Data are shown for effector memory T cells (EM) (CCR7- / CD45RA-); Fig.29D and 30D Data are shown for terminally differentiated effector memory cells (TEMRA) (CCR7- / CD45RA+).

[0134] Fig.31A and31B CD4( Fig.31A ) and CD8( Fig.31B ) Comparison of the CD27 subset of the p-terminal TILs (using melanoma TILs as comparator).

[0135] Fig.32A and 32B CD4( Fig.32A ) and CD8( Fig.32B ) Comparison of the CD28 subset of the subpopulation (using melanoma TIL as a comparator).

[0136] Fig.33A and 33B CD4( Fig.33A ) and CD8( Fig.33B )Comparison of LAG3+ subpopulations within the group.

[0137] Fig.34A and 34B CD4( Fig.34A ) and CD8( Fig.34B )Comparison of PD1+ subpopulation within the group.

[0138] Fig.35A and 35B The results showed that the PreRx PBL ( Fig.35A ) and PostRxPBL( Fig.35B ) of the cytolytic activity. Cytotoxicity was measured in LU 50 The number of PBLs required to kill 50% of target cells was measured.

[0139] Fig.36A and 36B MIL isolated from the bone marrow (MIL) or peripheral blood (PBL) of AML patients ( Fig.36A ) and PBL( Fig.36B ). MIL 1.1 was expanded using MIL Method 1, MIL 1.2 was expanded using MIL Method 2, and MIL 1.3 was expanded using MIL Method 3. MIL2 and MIL3 were expanded using MIL Method 3. All PBLs were expanded using PBL Method 3. The starting cell number for MIL1.3 was 138,000 cells, the starting cell number for MIL2 was 62,000 cells, and the starting cell number for MIL3 was 28,000 cells. The starting cell number for PBL2 was 338,000 cells, and the starting cell number for PBL3 was 336,000 cells.

[0140] Fig.37A and 37B Shows MIL( Fig.37A ) and PBL( Fig.37B ) of IFN-γ producing cells.

[0141] Figures 38A-38F showed that MILs isolated from AML patients ( Figures 38A-38C ) and PBL( Figures 38D-38F ) in Figure 3. Fig.38A and 38D TCRαβ+ subsets are shown. Fig.38B and 38E CD4+ subsets are shown. Fig.38C and 38F CD8 subsets are shown. PBL are shown at day 0 and day 14.

[0142] Figures 39A-39D Diagram showing CD4 memory subsets of MILs isolated from AML patients. Fig.39A Data for naive (CCR7+ / CD45RA+) are shown; Fig.39B Data are shown for central memory T cells (CM) (CCR7+ / CD45RA-); Fig.39C Data are shown for effector memory T cells (EM) (CCR7- / CD45RA-); Fig.39D Data are shown for terminally differentiated effector memory cells (TEMRA) (CCR7- / CD45RA+).

[0143] Figures 40A-40D Diagram showing CD4 memory subsets of PBL isolated from AML patients. Fig.40A Data for naive (CCR7+ / CD45RA+) are shown; Fig.40B Data are shown for central memory T cells (CM) (CCR7+ / CD45RA-); Fig.40C Data are shown for effector memory T cells (EM) (CCR7- / CD45RA-); Fig.40D Data are shown for terminally differentiated effector memory cells (TEMRA) (CCR7- / CD45RA+).

[0144] Figures 41A-41D Diagram showing CD8 memory subsets of MILs isolated from AML patients. Fig.41A Data for naive (CCR7+ / CD45RA+) are shown; Fig.41B Data are shown for central memory T cells (CM) (CCR7+ / CD45RA-); Fig.41CData are shown for effector memory T cells (EM) (CCR7- / CD45RA-); Fig.41D Data are shown for terminally differentiated effector memory cells (TEMRA) (CCR7- / CD45RA+).

[0145] Figures 42A-42D Diagram showing the CD8 memory subset of PBL isolated from AML patients. Fig.42A Data for naive (CCR7+ / CD45RA+) are shown; Fig.42B Data are shown for central memory T cells (CM) (CCR7+ / CD45RA-); Fig.42C Data are shown for effector memory T cells (EM) (CCR7- / CD45RA-); Fig.42D Data are shown for terminally differentiated effector memory cells (TEMRA) (CCR7- / CD45RA+).

[0146] Fig.43A and 43B Display MIL( Fig.43A ) and PBL( Fig.43B ) of the CD27 subset of CD4 and CD8 cell populations.

[0147] Fig.44A and 44B Display MIL( Fig.44A ) and PBL( Fig.44B ) of the CD28 subset of the CD4 and CD8 cell populations.

[0148] Fig.45A and 45B Display MIL( Fig.45A ) and PBL( Fig.45B ) of the PD1+ subsets of the CD4 and CD8 cell populations.

[0149] Fig.46A and 46B Display MIL( Fig.46A ) and PBL( Fig.46B ) of the LAG3+ subsets of CD4 and CD8 cell populations.

[0150] Fig.47 A timeline is shown for an exemplary embodiment 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, the addition of IL-2 can be performed in the bracketed area.

[0151] Fig.48A timeline is shown for an exemplary embodiment of MIL method 3. In this figure, the addition of IL-2 can occur at any point in time during the period shown; in an exemplary embodiment, the addition of IL-2 can be performed in the bracketed area.

[0152] Brief Description of Sequence Listing

[0153] SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

[0154] SEQ ID NO: 2 is the amino acid sequence of the light chain of muromonab.

[0155] SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

[0156] SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

[0157] SEQ ID NO: 5 is the amino acid sequence of recombinant human IL-4 protein.

[0158] SEQ ID NO: 6 is the amino acid sequence of recombinant human IL-7 protein.

[0159] SEQ ID NO: 7 is the amino acid sequence of recombinant human IL-15 protein.

[0160] SEQ ID NO: 8 is the amino acid sequence of recombinant human IL-21 protein. DETAILED DESCRIPTION

[0161] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. All patents and publications mentioned herein are incorporated herein by reference in their entirety.

[0162] definition

[0163] As used herein, the terms "co-administration", "co-administering", "administered in combination with", "administering in combination with", "simultaneous", and "concurrent" include administering two or more active pharmaceutical ingredients to a subject such that the active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. Co-administration includes administering different compositions simultaneously, administering different compositions at different times, or administering a composition in which two or more active pharmaceutical ingredients are present. Preferred are simultaneous administration of different compositions and administration of a composition in which two agents are present.

[0164] The term "in vivo" refers to events that occur within the body of a mammalian subject.

[0165] The term "ex vivo" refers to an event that occurs outside the body of a mammalian subject in an artificial environment.

[0166] The term "in vitro" refers to an event that occurs in a test system. In vitro assays include cell-based assays that may employ live or dead cells, and may also include cell-free assays that do not employ intact cells.

[0167] The term "rapid expansion" refers to an increase in the number of antigen-specific TILs by at least about 3 times (or 4 times, 5 times, 6 times, 7 times, 8 times or 9 times) within a week, more preferably by at least about 10 times (or 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times or 90 times) within a week, or most preferably by at least about 100 times within a week. Many rapid expansion protocols are described herein.

[0168] The terms "fragmenting," "fragment," and "fragmented" as used herein to describe methods of disrupting tumors include mechanical disruption methods such as crushing, slicing, dividing, and pulverizing tumor tissue, as well as any other method that disrupts the physical structure of tumor tissue.

[0169] The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. Optionally, peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells, which are antigen presenting cells. The term "PBL" refers to peripheral blood lymphocytes, which are T cells expanded from peripheral blood. The terms PBL and TIL are used interchangeably herein.

[0170] The term "anti-CD3 antibody" refers to an antibody or its variant (e.g., monoclonal antibody), including: human, humanized, chimeric or murine antibodies against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3 (also known as muromonab) and UCHT-1. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0171] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody or a biosimilar or variant thereof, including human, humanized, chimeric or murine antibodies directed against the CD3 receptor in the T cell antigen receptor of mature T cells, and also includes commercially available forms such as OKT-3 (30 ng / mL, pure MACS GMP CD3, Miltenyi Biotech, Inc., San Diego, California, USA) and muromonab or a variant thereof, conservative amino acid substitution, glycoform or biosimilar. The amino acid sequences of the heavy and light chains of muromonab are given in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). The hybridoma capable of producing OKT-3 is deposited in the American Type Culture Collection (ATCC) and is assigned the ATCC accession number CRL 8001. The OKT-3 producing hybridoma was also deposited in the European Collection of Authenticated Cell Cultures (ECACC) and assigned catalogue number 86022706.

[0172] Table 1: Amino acid sequence of muromonab

[0173]

[0174] The term "IL-2" (also referred to herein as "IL2") refers to a T cell growth factor known as interleukin-2, including all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars and variants thereof. IL-2 is described in, for example, Nelson, J Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of a recombinant human IL-2 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 3). For example, the term IL-2 includes human recombinant forms of IL-2, for example, aldesleukin (PROLEUKIN, commercially available from multiple suppliers, 22 million IU per vial for single use), and recombinant forms of IL-2 commercially available from CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, New Jersey, USA (Catalog No. CYT-209-b) and other commercial equivalents from other suppliers. 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 given in Table 2 (SEQ ID NO: 4). The term IL-2 also includes IL-2 in a PEGylated form as described herein, including PEGylated IL2 prodrug NKTR-214, commercially available from Nektar Therapeutics, South San Francisco, California, USA. NKTR-214 and PEGylated IL-2 suitable for the present invention are described in U.S. Patent Application Publication No. US2014 / 0328791A1 and International Patent Application Publication No. WO2012 / 065086A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 suitable for the present invention are described in U.S. Patents 4,766,106, 5,206,344, 5,089,261 and 4,902,502, the disclosures of which are incorporated herein by reference. IL-2 preparations suitable for the present invention are described in U.S. Patent No. 6,706,289, the disclosures of which are incorporated herein by reference.

[0175] Table 2: Amino acid sequences of interleukins

[0176]

[0177] The term "IL-4" (also referred to herein as "IL4") refers to a cytokine called interleukin 4, which is produced by Th2 T cells and eosinophils, basophils and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) to Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. After being activated by IL-4, Th2 T cells then produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, and induces B cell class conversion to IgE and IgG1 expression. Recombinant human IL-4 suitable for the present invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, New Jersey, USA (Catalog No. CYT-211) and ThermoFisher Scientific, Inc., Waltham, Massachusetts, USA (human IL-15 recombinant protein, catalog No. Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 5).

[0178] The term "IL-7" (also referred to herein as "IL7") refers to a glycosylated tissue-derived cytokine called interleukin 7, which can be obtained from stromal cells and epithelial cells as well as dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate the growth of T cells. IL-7 binds to the IL-7 receptor, which is a heterodimer composed of IL-7 receptor α and common gamma chain receptor, which plays an important role in T cell development and peripheral survival in the thymus in a series of signals. Recombinant human IL-4 suitable for the present invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, New Jersey, USA (Catalog No. CYT-254) and ThermoFisher Scientific, Inc., Waltham, Massachusetts, USA (human IL-7 recombinant protein, catalog No. Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 6).

[0179] The term "IL-15" (also referred to herein as "IL15") refers to a T cell growth factor known as interleukin-15, and includes all forms of IL-15, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. 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-chain, 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 a number of suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, New Jersey, USA (Catalog No. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, Massachusetts, USA (Human IL-15 Recombinant Protein, Catalog No. 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 7).

[0180] The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic cytokine protein called interleukin-21, and includes all forms of IL-21, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated herein by reference. IL-21 is primarily produced by natural killer T cells and activated human CD4 + T cell production. Recombinant human IL-21 is a single-chain non-glycosylated polypeptide chain containing 132 amino acids with a molecular weight of 15.4 kDa. Recombinant human IL-21 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, New Jersey, USA (Catalog No. CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, Massachusetts, USA (Human IL-21 Recombinant Protein, Catalog No. 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for the present invention is given in Table 2 (SEQ ID NO: 8).

[0181] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of these pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Unless any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the present invention is contemplated. Other active pharmaceutical ingredients (e.g., other drugs) may also be incorporated into the described compositions and methods.

[0182] The term "antibody" and its plural form "antibodies" refer to intact immunoglobulins and any antigen-binding fragments thereof ("antigen-binding portions") or single chains thereof. "Antibody" also 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 consists of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as V L ) and the light chain constant region. The light chain constant region consists of a domain C L Composition. Antibody V H and V L The V regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs) or hypervariable regions (HVRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V L It consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigenic epitopes. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0183] The term "antigen" refers to a substance that induces an immune response. In some embodiments, an antigen is a molecule that can be combined with an antibody or TCR if presented by a major histocompatibility complex (MHC) molecule. As used herein, the term "antigen" also includes T cell epitopes. Antigens can also be recognized by the immune system. In some embodiments, antigens can induce a humoral immune response or a cellular immune response that causes B lymphocytes and / or T lymphocyte activation. In some cases, this may require that the antigen include or connect Th cell epitopes. Antigens can also have one or more epitopes (e.g., B- and T- epitopes). In some embodiments, an antigen preferably reacts with its corresponding antibody or TCR (usually in a highly specific and selective manner), and not with a variety of other antibodies or TCR reactions that can be induced by other antigens.

[0184] The terms "monoclonal antibody", "mAb", "monoclonal antibody composition" or their plural forms refer to antibody molecules of a prepared single molecule composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies specific for certain receptors can be prepared using knowledge and techniques in the art: a test subject is injected with a suitable antigen, and then a hybridoma expressing the antibody (having the desired sequence or functional characteristics) is isolated. The DNA encoding the monoclonal antibody is easily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that are able to specifically bind to genes encoding the heavy and light chains of the monoclonal antibodies). Hybridoma cells are preferred sources of such DNA. Once isolated, the DNA can be placed in an expression vector, which is then transfected into a host cell (e.g., an E. coli cell, a simian COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not produce immunoglobulins) to obtain synthesized monoclonal antibodies in a recombinant host cell. The recombinant production of antibodies will be described in more detail below.

[0185] As used herein, the terms "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "fragment") refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of a full-length antibody can perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, which is a fragment consisting of V L 、V H , C L and CH1 domains; (ii) a F(ab′)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment, which consists of a V Hand CH1 domains; (iv) Fv fragment, which consists of the V L and V H (v) domain antibody (dAb) fragments (Ward et al., Nature, 1989, 341, 544-546), which can be composed of V H or V L (vi) separate complementary determining regions (CDRs). In addition, although the two domains (V L and V H ) are encoded by different genes, but they can be connected by a synthetic linker by recombinant methods, which enables them to be prepared as a single protein chain; wherein V L and V H The regions are paired to form a monovalent molecule, which is called a single-chain Fv (scFv); see, for example, Bird et al., Science, 1988, 242, 423-426; and Huston et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 5879-5883. It is also intended that such scFv antibodies be included in the term "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 utility of these fragments is screened in the same manner as intact antibodies.

[0186] As used herein, the term "human antibody" is intended to include antibodies having variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present invention may contain amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). As used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) are grafted onto human framework sequences.

[0187] The term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity having a variable region in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody is produced by a hybridoma comprising a B cell obtained from a transgenic non-human animal (e.g., a transgenic mouse) fused to an immortalized cell, the genome of the B cell comprising a human heavy chain transgene and a human light chain transgene.

[0188] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, produced or isolated by recombinant methods, for example: (a) antibodies isolated from animals (such as mice) that are transgenic or transchromosomal animals of human immunoglobulin genes or hybridomas prepared therefrom (described further below); (b) antibodies isolated from host cells (such as transfectomas) that are transformed to express human antibodies; (c) antibodies isolated from recombinant, combinatorial human antibody libraries; and (d) antibodies prepared, expressed, produced or isolated by any other means that involve splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework regions and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when transgenic animals for human Ig sequences are used, in vivo somatic cell mutagenesis) and thus, the recombinant antibody V H and V L The amino acid sequence of the region (although derived from and similar to human germline V H and V L sequences) may not naturally exist in the human antibody germline repertoire in vivo.

[0189] As used herein, "isotype" refers to the antibody class (eg, IgM or IgG1) encoded by the heavy chain constant region genes.

[0190] The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0191] The term "human antibody derivative" refers to any modified form of a human antibody, including a conjugate of an antibody with another active pharmaceutical ingredient or antibody. The term "conjugate", "antibody-drug conjugate", "ADC" or "immunoconjugate" refers to an antibody or fragment thereof conjugated to another therapeutic moiety, which can be conjugated to the antibodies described herein using methods available in the art.

[0192] The terms "humanized antibody", "humanized antibodies" and "humanization" refer to antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) are grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the hypervariable regions of the receptor are replaced by residues from 15 hypervariable regions of a non-human species (e.g., mouse, rat, rabbit or non-human primate) (donor antibody) with the desired specificity, affinity and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may contain residues that are not found in the receptor antibody or the donor antibody. These modifications are made to further improve antibody performance. Typically, a humanized antibody will comprise substantially all of at least one (usually two) variable domains in which all or substantially all hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all FR regions are those of a human immunoglobulin sequence. Optionally, a humanized antibody also comprises at least a portion of an 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 et al., Curr. Op. Struct. Biol., 1992, 2, 593-596. The antibodies described herein may also be modified to use any Fc variant known to have improved (e.g., reduced) effects on effector function and / or FcR binding.For example, Fc variants may include: International Patent Application Publication Nos. WO1988 / 07089A1, WO1996 / 14339A1, WO1998 / 05787A1, WO1998 / 23289A1, WO1999 / 51642A1, WO99 / 58572A1, WO2000 / 09560A2, WO2000 / 32767A1, WO2000 / 42072A2, WO O2002 / 44215A2, WO2002 / 060919A2, WO2003 / 074569A2, WO2004 / 016750A2, WO2004 / 029207A2 , WO2004 / 035752A2, WO2004 / 063351A2, WO2004 / 074455A2, WO2004 / 099249A2, WO2005 / 040217 A2, WO2005 / 070963A1, WO2005 / 077981A2, WO2005 / 092925A2, WO2005 / 123780A2, WO2006 / 019447A1, WO2006 / 047350A2 and WO2006 / 085967A2; and U.S. Pat. Nos. 5,648,260, 5,739,277, 5 any one of the amino acid substitutions disclosed in, for example, 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.

[0193] The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, for example, 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.

[0194] "Diabodies" are small antibody fragments with two antigen-binding sites. The fragments contain the light chain variable domain (V L ) is connected to the heavy chain variable domain (V H )(V H -V L or V L -V H). By using a linker that is too short to allow the two domains on the same chain to pair, the domains are paired with the complementary domains of another chain to create two antigen-binding sites. For example, European Patent No. EP404,097, International Patent Publication No. WO93 / 11161 and Bolliger et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 6444-6448 more fully describe diabodies.

[0195] The term "glycosylation" refers to a modified antibody derivative. An aglycoslated antibody is not glycosylated. For example, glycosylation can be altered to increase the affinity of the antibody for the antigen. For example, such carbohydrate modification can be achieved by changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made, which lead to the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at the site. As described in U.S. Pat. Nos. 5,714,350 and 6,350,861, glycosylation can increase the affinity of the antibody for the antigen. Additionally or alternatively, antibodies with altered glycosylation types can be prepared, for example, hypofucosylated antibodies with reduced amounts of fucosyl residues, or antibodies with increased bisecting GlcNac structures. It has been shown that such altered glycosylation patterns increase the ability of the antibody. For example, such carbohydrate modification can be achieved by expressing the antibody in a host cell (with an altered glycosylation mechanism). Cells with altered glycosylation machinery have been described in the art and can be used as host cells that express the recombinant antibodies of the present invention to produce antibodies with altered glycosylation. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (α(1,6) fucosyltransferase), so that the antibodies expressed by the Ms704, Ms705, and Ms709 cell lines lack fucose on carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were generated by targeted disruption of the FUT8 gene of CHO / DG44 cells using two replacement vectors (see, e.g., U.S. Patent Publication No. 2004 / 0110704 or Yamane-Ohnuki et al., Biotechnol. Bioeng., 2004, 87, 614-622). As another example, European Patent No. EP1,176,195 describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase, such that antibodies expressed by such a cell line exhibit hypofucosylation (by reducing or eliminating the enzyme associated with α1,6 bonds); the patent also describes a cell line having low or no enzymatic activity for adding fucose to N-acetylglucosamine bound to the Fc region of an antibody, for example, the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). International Patent Publication No. WO03 / 035835 describes a variant CHO cell line Lec13 cells, which has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in the host cell (see also Shields et al., J. Biol. Chem., 2002, 277, 26733-26740).International Patent Publication No. WO99 / 54342 describes a cell line engineered to express a glycoprotein modifying glycosyltransferase (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell line exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibody (see Umana et al., Nat. Biotech., 1999, 17, 176-180). Alternatively, fucosidase can be used to cleave the fucose residues of the antibody. For example, as described in Tarentino et al., BioChem., 1975, 14, 5516-5523, fucosidase (α-L-fucosidase) removes the fucose residues of the antibody.

[0196] "PEGylation" refers to the reaction of a modified antibody or fragment thereof with polyethylene glycol (PEG) (e.g., an active ester or aldehyde derivative of PEG) under conditions where more than one PEG group is attached to the antibody or antibody fragment. Pegylation can, for example, increase the biological (e.g., serum) half-life of the antibody. Preferably, PEGylation is performed by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a 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-C1 10 ) alkoxy-polyethylene glycol or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. The antibody to be pegylated can be a non-glycosylated antibody. Methods of pegylation are known in the art and can be applied to the antibodies described in the present invention, such as described in European Patent Nos. EP0154316 and EP 0401384 and U.S. Patent No. 5,824,778, the disclosures of which are incorporated herein by reference.

[0197] The term "fusion protein" or "fusion polypeptide" refers to a protein that combines the properties of two or more individual proteins. Such proteins have at least two heterologous polypeptides covalently linked directly or through an amino acid linker. The polypeptides that form the fusion protein are usually C-terminal to N-terminal, although they can also be C-terminal to C-terminal, N-terminal to N-terminal or N-terminal to C-terminal. The polypeptides of the fusion protein can be in any order and can include more than one constituent polypeptide or both constituent polypeptides. The term includes conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, interspecies homologs and immunogenic fragments of antigens that constitute the fusion protein. The fusion protein of the present disclosure may also contain additional copies of the component antigen or its immunogenic fragment. The fusion protein may contain more than one binding domain that is linked together and further linked to an Fc domain (e.g., an IgG Fc domain). The fusion protein may be further linked together to mimic a monoclonal antibody and provide more than six binding domains. The fusion protein may be produced by recombinant methods known in the art. The preparation of fusion proteins is known in the art and is described in, for example, International Patent Application Publication Nos. WO1995 / 027735A1, WO2005 / 103077A1, WO2008 / 025516A1, WO2009 / 007120A1, WO2010 / 003766A1, WO2010 / 010051A1, WO2010 / 078966A1, U.S. Patent Application Publication Nos. US2015 / 0125419A1 and US2016 / 0272695A1, and U.S. Patent No. 8,921,519, the disclosures of each of which are incorporated herein by reference.

[0198] The term "heterologous" when used in reference to a nucleic acid or protein portion means that the nucleic acid or protein comprises two or more subsequences that do not exist in the same relationship to each other in nature. For example, nucleic acids are often recombinantly produced, with two or more sequences from unrelated genes that are arranged to produce 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 means that the protein comprises two or more subsequences that do not exist in the same relationship to each other in nature (e.g., a fusion protein).

[0199] The term "conservative amino acid substitution" refers to an amino acid sequence modification that does not disrupt the binding of an antibody or fusion protein to an antigen. Conservative amino acid substitutions include replacing an amino acid in a class with an amino acid of the same class; wherein a class is defined by common physicochemical amino acid side chain properties and a high frequency of substitution in homologous proteins found in nature (as determined by, for example, a standard Dayhoff frequency exchange matrix or a BLOSUM matrix). Amino acid side chains are divided into six major classes, 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, replacing Asp with another Class III residue (e.g., Asn, Gln, or Glu) is a conservative substitution. Therefore, a non-essential amino acid residue expected in an antibody is preferably replaced by another amino acid residue from the same class. Methods for identifying conservative amino acid substitutions that do not abolish antigen binding are well known in the art (see, e.g., Brummell et al., Biochemistry 1993, 32, 1180-1187; Kobayashi et al., Protein Eng. 1999, 12, 879-884; and Burks et al., Proc. Natl. Acad. Sci., USA, 1997, 94, 412-417).

[0200] In the case of two or more nucleic acids or polypeptides, the terms "sequence identity", "percent identity" and "percent sequence identity" (or their synonyms, e.g., "99% identical") refer to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotides or amino acid residues, without considering any conservative amino acid substitutions as part of the sequence identity, when compared and aligned for maximum correspondence (if necessary, introducing gaps). Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art for obtaining alignments 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 U.S. government. Comparisons between two sequences can be performed using the BLASTN or BLASTP algorithms. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign available from DNASTAR are other publicly available software programs that can be used to align sequences. One skilled in the art can determine appropriate parameters for maximum alignment by specific alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0201] As used herein, the term "variant" includes, but is not limited to, antibodies or fusion proteins that contain an amino acid sequence that is different from the amino acid sequence of a reference antibody by one or more substitutions, deletions, and / or additions at certain positions within or near the amino acid sequence of the reference antibody. A variant may contain one or more conservative substitutions in its amino acid sequence compared to the amino acid sequence of the reference antibody. Conservative substitutions may involve, for example, substitutions of similarly charged or uncharged amino acids. The variant retains the ability to specifically bind to the antigen of the reference antibody. The term "variant" also includes pegylated antibodies or proteins.

[0202] Nucleic acid sequences implicitly include conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly indicated sequences. In particular, degenerate codon substitutions can be achieved by generating such sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues. Batzer et al., Nucleic Acid Res., 1991, 19, 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 can be used interchangeably with cDNA, mRNA, oligonucleotides and polynucleotides.

[0203] The term "biosimilar" refers to a biological product, including a monoclonal antibody or protein, which is highly similar to a reference biological product licensed in the United States, despite minor differences in clinically inactive ingredients, and there are no clinically meaningful differences between the biological product and the reference product in terms of product safety, purity and efficacy. In addition, a biosimilar or "biosimilar" drug is a biological pharmaceutical that is similar to another biological pharmaceutical that has been authorized for use by the European Medicines Agency. The term "biosimilar" is also used synonymously by other national and regional regulatory agencies. Biological products or biopharmaceuticals are drugs made or derived from biological sources (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 drug regulatory agency for aldesleukin is "biosimilar to" aldesleukin or a "biosimilar" of aldesleukin. In Europe, a biosimilar or "biosimilar" drug is a biological pharmaceutical that is similar to another biological pharmaceutical that has been authorized for use by the European Medicines Agency (EMA). As amended, the relevant legal basis for similar biological applications in Europe is Article 6 of Regulation (EC) No. 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC, and therefore in Europe, biosimilars may be authorised or allowed to be authorised or be the subject of an application for authorisation in accordance with Article 6 of Regulation (EC) No. 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. An authorised original biological medicinal product may be referred to in Europe as a "reference medicinal product". Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP Guidelines for Biosimilar Products. In addition, product specific guidance (including guidance related to monoclonal antibody biosimilars) is provided by the EMA on a product-by-product basis and published on its website. The biosimilars described herein may be similar to the reference medicinal product in terms of quality characteristics, biological activity, mechanism of action, safety and / or efficacy. In addition, the biosimilar may be used or intended for use in the treatment of the same condition as the reference medicinal product. Therefore, the biosimilars described herein may be considered to have similar or highly similar quality characteristics to the reference medicinal product. Alternatively, or in addition, it can be considered that the biosimilars as described herein have similar or highly similar biological activity to the reference medicinal product. Alternatively, or in addition, it can be considered that the biosimilars as described herein have similar or highly similar safety to the reference medicinal product. Alternatively, or in addition, it can be considered that the biosimilars as described herein have similar or highly similar efficacy to the reference medicinal product. As described herein, European biosimilars are compared with reference medicinal products that have been authorized by the EMA. However, in some cases, biosimilars can be compared with biological medicinal products that have been authorized outside the European Economic Area (non-European Economic Area authorized "comparator") in certain studies.These studies include, for example, certain clinical and in vivo nonclinical studies. As used herein, the term "biosimilar" also refers to a biopharmaceutical product that has been or can be compared with a non-EEA authorized comparator. Certain biosimilars are proteins, such as antibodies, antibody fragments (e.g., antigen binding portions) and fusion proteins. Protein biosimilars may have an amino acid sequence with minor modifications (including, for example, deletions, additions and / or substitutions of amino acids) in the amino acid structure that do not significantly affect the function of the polypeptide. A biosimilar may include an amino acid sequence that has more than 97% sequence identity with the amino acid sequence of its reference drug product, such as 97%, 98%, 99% or 100%. A biosimilar may include one or more post-translational modifications that are different from the post-translational modifications of the reference drug product, such as, but not limited to, glycosylation, oxidation, deamidation and / or truncation, provided that the difference does not result in changes in the safety and / or efficacy of the drug. A biosimilar may have a glycosylation pattern that is the same or different from that of the reference drug product. In particular, although not exclusively, a biosimilar may have a different glycosylation pattern if the difference relates to or is intended to address a safety concern associated with the reference medicinal product. In addition, a biosimilar may deviate from the reference medicinal product in, for example, its strength, pharmaceutical form, formulation, excipients and / or presentation, provided that the safety and efficacy of the medicinal product are not compromised. A biosimilar may include, for example, differences in the pharmacokinetic (PK) and / or pharmacodynamic (PD) profile compared to the reference medicinal product, but is still considered sufficiently similar to the reference medicinal product to be authorized or deemed suitable for authorization. In some cases, biosimilars exhibit different binding properties compared to the reference medicinal product; wherein regulatory agencies such as the EMA consider that the different binding properties are not an obstacle to the authorization of biosimilar products. The term "biosimilar" is also used synonymously by other national and regional regulatory agencies.

[0204] The term "hematological malignancies" refers to mammalian cancers and tumors of hematopoietic and lymphoid tissues, including but not limited to the blood, bone marrow, lymph nodes, and tissues of the lymphatic system. Hematological malignancies may result in the formation of "liquid tumors". Hematological malignancies include but are not limited to acute lymphocytic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma and non-Hodgkin's lymphoma. The term "B-cell hematological malignancies" refers to hematological malignancies that affect B cells.

[0205] The term "liquid tumor" refers to an abnormal cell mass that is essentially fluid. Liquid tumor cancers include, but are not limited to, leukemias, myelomas, and lymphomas, as well as other hematological malignancies. TILs obtained from liquid tumors (including liquid tumors residing in the bone marrow) may also be referred to herein as bone marrow infiltrating lymphocytes (MILs). TILs obtained from liquid tumors (including liquid tumors circulating in peripheral blood) may also be referred to herein as PBLs. The terms MIL, TIL, and PBL are used interchangeably herein, and they differ only based on the tissue type from which the cells are derived.

[0206] The term "biopsy" refers to any medical procedure used to obtain cancer cells, including a bone marrow biopsy.

[0207] The term "acute myeloid leukemia" or "AML" refers to a cancer of the myeloid blood cell lineage, which is also referred to in the art as acute myeloid leukemia and acute non-lymphocytic leukemia. Although AML is a liquid tumor, certain manifestations of AML (including extramedullary manifestations, such as chloroma) exhibit characteristics of solid tumors, but AML is classified as a liquid tumor herein.

[0208] As used herein, the term "microenvironment" can refer to the entire solid or blood tumor microenvironment, or to a single cell subpopulation within the microenvironment. As used herein, the tumor microenvironment refers to a complex mixture of "cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that promote tumor transformation, support tumor growth and invasion, protect tumors from host immunity, promote treatment resistance, and provide niches for the propagation of dominant metastatic lesions", as described in Swartz et al., Cancer Res., 2012, 72, 2473. Although tumors express antigens that should be recognized by T cells, the immune system rarely clears tumors due to the immunosuppression of the microenvironment.

[0209] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds as described herein that is sufficient to achieve the intended application, including but not limited to disease treatment. The therapeutically effective amount may vary depending on the intended application (in vitro or in vivo) or the subject and disease condition being treated (e.g., the subject's weight, age, and sex), the severity of the disease condition, or the mode of administration. The term also applies to a dose that will induce a specific response in the target cell (e.g., a decrease in platelet adhesion and / or cell migration). The specific dose will depend on the specific compound selected, the administration regimen to be followed, whether the compound is administered in combination with other compounds, the time of administration, the administration tissue, and the physical delivery system that carries the compound.

[0210] The term "therapeutic effect" as used herein includes therapeutic benefits and / or prophylactic benefits. Prophylactic effects include: delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, stopping or reversing the progression of a disease or condition, or any combination thereof.

[0211] The terms "treatment", "treating", "treat" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or an adverse reaction caused by the disease. As used herein, "treatment" includes any treatment of a disease in mammals, particularly humans, including: (a) preventing the occurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (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 meant to include the delivery of an agent to provide a pharmacological effect, even in the absence of a disease or condition. For example, "treatment" includes the delivery of a composition that can elicit an immune response or confer immunity in the absence of a disease, such as in the case of a vaccine.

[0212] The term "QD", "qd" or "qd" means once a day, once a day or once daily. The term "BID", "bid" or "bid" means twice a day, twice a day or twice daily. The term "TID", "tid" or "tid" means three times a day, three times a day or three times daily. The term "QID", "qid" or "qid" means four times a day, four times a day or four times daily.

[0213] As used herein, "tumor infiltrating lymphocytes" or "TILs" refer to a population of cells that were originally obtained as leukocytes and have left the bloodstream of a subject and migrated to a tumor. TILs include, but are not limited to, CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 +T cells, natural killer cells, dendritic cells and M1 macrophages. TIL includes primary TIL and secondary TIL. "Primary TIL" is those obtained from patient tissue samples as described herein (sometimes referred to as "freshly harvested"), and "secondary TIL" is any TIL cell population that has been expanded or propagated as discussed herein, including but not limited to a large number of TIL (bulk TIL) and amplified TIL ("REP TIL") and "reREP TIL" as discussed herein.

[0214] 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 expressing one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally or alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors after reintroduction into the patient. TILs can also be characterized by potency—for example, if, for example, the release of interferon (IFN) is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL, TILs can be considered to be effective.

[0215] "Cryopreserved TILs" (or cryopreserved MILs or PBLs) herein refer to primary, bulk or expanded TILs (REP TILs) processed and stored in the range of about -150°C to -60°C. General methods for cryopreservation are also described elsewhere herein (including in the Examples). For clarity, "cryopreserved TILs" can be distinguished from frozen tissue samples that can be used as a source of primary TILs.

[0216] "Thawed cryopreserved TILs" (or thawed MILs or PBLs) herein refers to a population of TILs that were previously cryopreserved and then processed to return to room temperature or higher temperature (including but not limited to cell culture temperature or a temperature at which the TILs can be administered to a patient).

[0217] The term "cell population" (including TIL population) herein refers to a number of cells having common characteristics. Usually, the number of the population is approximately 1×10 6 Up to 1×10 10 Different TIL populations contain different numbers. For example, initial growth of primary TIL in the presence of IL-2 produces approximately 1×10 8 Typically, REP expansion is performed to provide 1.5 × 10 TILs for infusion. 9 to 1.5×10 10 Groups of cells.

[0218] Typically, TILs are initially obtained from patient tumor samples ("primary TILs"), then expanded into larger populations for further manipulation as described herein, optionally cryopreserved, restimulated as outlined herein, and optionally assessed for phenotypic and metabolic parameters as indicators of TIL health.

[0219] Often, the harvested cell suspension is referred to as a "primary cell population" or a "freshly harvested" cell population.

[0220] Typically, as described herein, TILs are initially prepared by obtaining a primary population of TILs ("primary cell population" or "first cell population") from a tumor removed from a patient as described herein. After this, cells are cultured with IL-2 for initial mass expansion to form a second cell population (sometimes referred to herein as "mass TIL population" or "second population").

[0221] The term "cytotoxic lymphocytes" includes: cytotoxic T (CTL) cells (including CD8 + Cytotoxic T lymphocytes and CD4 + Helper T lymphocytes), natural killer T (NKT) cells and natural killer (NK) cells. For example, cytotoxic lymphocytes may include: peripheral blood-derived αβTCR-positive or γδTCR-positive T cells activated by tumor-associated antigens and / or transduced by tumor-specific chimeric antigen receptors or T cell receptors, and tumor infiltrating lymphocytes (TILs).

[0222] The term "central memory T cells" refers to cells that are CD45RO+ and constitutively express CCR7 (CCR7 高 ) and CD62L (CD62 高 ) of human T cells. 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 BMII. After TCR triggering, central memory T cells mainly secrete IL-2 and CD40L as effector molecules. Central memory T cells are mainly present in the CD4 compartment of the blood and are proportionally enriched in lymph nodes and tonsils in the human body.

[0223] The term "effector memory T cells" refers to a subpopulation of human or mammalian T cells that, like central memory T cells, are CD45R0+ but lack the constitutive expression of CCR7 (CCR7 低 ), and heterogeneous or low expression of CD62L (CD62L 低). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R) and IL-15R. Transcription factors of central memory T cells include BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines, including interferon-γ, IL-4 and IL-5, after antigen stimulation. Effector memory T cells are mainly present in the CD8 compartment of the blood and are proportionally enriched in the lungs, liver and intestines in the human body. 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 in the methods of the present invention. Closed systems include, for example, but are not limited to, closed G containers. Once tumor fragments are added to the closed system, the system will not be open to the external environment until the TILs are ready to be administered to the patient.

[0224] In some embodiments, the methods of the present disclosure also include a "pre-REP" stage, in which tumor tissue or cells from tumor tissue are grown in standard laboratory culture medium (including but not limited to RPMI) and treated with reagents (e.g., irradiated feeder cells and anti-CD3 antibodies) to achieve the desired effect, such as an increase in the number of TILs and / or an enrichment of cell populations containing desired cell surface markers or other structural, biochemical or functional characteristics. The pre-REP stage can use laboratory-grade reagents (assuming that the laboratory-grade reagents are diluted in subsequent REP stages), making it easier to adopt alternative strategies to increase the production of TILs. Therefore, in some embodiments, during the pre-REP stage, the disclosed TLR agonists and / or peptides or peptidomimetics can be included in the culture medium. In some embodiments, the pre-REP culture can include IL-2. In a preferred aspect, the present invention relates to novel methods for expanding TILs using one or more additional restimulation protocols (also referred to herein as "restimulation rapid expansion protocols" or "reREP"), which unexpectedly result in expanded memory T cell subsets (including memory effector T cell subsets) and / or enhance glycolytic respiratory markers of restimulated TILs (sometimes referred to herein as "reTILs") compared to freshly harvested TILs or thawed cryopreserved TILs. In other words, by using the reREP procedure on cryopreserved TILs, patients can receive highly metabolically active, healthy TILs, thereby obtaining better outcomes.

[0225] When referring to an "anti-tumor effective amount", "tumor suppressive effective amount" or "therapeutic amount", the exact amount of the composition of the present invention to be administered can be determined by the physician, taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject). Generally speaking, the pharmaceutical composition comprising the genetically modified cytotoxic lymphocytes described herein can be administered in an amount of 10 4 Up to 10 11cells / kg body weight (e.g. 10 5 Up to 10 6 , 10 5 Up to 10 10 , 10 5 Up to 10 11 , 10 6 Up to 10 10 , 10 6 Up to 10 11 , 10 7 Up to 10 11 , 10 7 Up to 10 10 , 10 8 Up to 10 11 , 10 8 Up to 10 10 , 10 9 Up to 10 11 or 10 9 Up to 10 10 The genetically modified cytotoxic lymphocyte composition can also be administered multiple times at these doses. The genetically modified cytotoxic lymphocytes can be administered using infusion techniques well known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. Med. 319: 1676, 1988). Those skilled in the medical field can easily determine the optimal dosage and treatment regimen for a particular patient by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0226] For the avoidance of doubt, it is intended that specific features (e.g., integers, features, values, uses, diseases, formulations, compounds or groups) described in conjunction with a particular aspect, embodiment or example of the invention be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith. Therefore, these features may be used in conjunction with any definition, claim or embodiment defined herein, as appropriate. All features disclosed in this specification (including any accompanying claims, abstracts and drawings) and / or all steps of any method or process so disclosed may be combined in any combination, except combinations in which at least some of the features and / or steps are mutually exclusive. The present invention is not limited to any details of any disclosed embodiment. The present invention extends to any new feature or new combination of features disclosed in this specification (including any accompanying claims, abstracts and drawings), or to any new step or new combination of any step of any method or process so disclosed.

[0227] The terms "about" and "approximately" mean within a statistically significant numerical range. Such a range can be within an order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, more preferably within 10%, and even more preferably within 5%. The permissible variations contained in the terms "about" or "approximately" depend on the specific system under study, and can be easily understood by those of ordinary skill in the art. In addition, as used herein, the terms "about" and "approximately" represent that size, dimensions, formulas, parameters, shapes and other properties and characteristics are not and do not have to be precise, but can be approximate and / or larger or smaller as needed, reflecting tolerances, conversion factors, rounding, measurement errors, etc., as well as other factors known to those skilled in the art. Typically, size, dimensions, formulas, parameters, shapes or other properties or characteristics are "about" or "approximate", whether or not explicitly stated as such. It should be noted that embodiments with very different sizes, shapes and sizes can adopt the described arrangements.

[0228] When used in the appended claims in their original and amended forms, the transitional terms "comprising," "consisting essentially of," and "consisting of" define the scope of the claims with respect to unrecited additional claim elements or steps, if any, that are excluded from the scope of the claims. The term "comprising" is intended to be inclusive or open-ended, and does not exclude any additional, unrecited elements, methods, steps, or materials. The term "consisting of" does not include any elements, steps, or materials other than those described in the claim, and in the case of materials, the term "consisting of" does not include ordinary impurities associated with the specified material. The term "consisting essentially of" limits the scope of the claim to the specified elements, steps, or materials and those that do not materially affect the basic and novel characteristics of the claimed invention. In alternative embodiments, all compositions, methods, and kits embodying the present invention described herein may be more specifically defined by any of the transitional terms "comprising," "consisting essentially of," and "consisting of."

[0229] Embodiments of methods for expanding therapeutic T cells comprising peripheral blood (PBL) and / or bone marrow (MIL)

[0230] Method for expanding peripheral blood lymphocytes (PBL) from peripheral blood

[0231] PBL Method 1. In one embodiment of the invention, PBLs are expanded using the methods described herein. In one embodiment of the invention, the method comprises obtaining a PBMC sample from whole blood. In one embodiment, the method comprises enriching T cells by isolating pure T cells from PBMCs using negative selection of the non-CD19+ fraction. On day 0, anti-CD3 / anti-CD28 antibodies are added at a 1:1 ratio (beads: cells). Pure T cells were cultured with 3000 IU / mL IL-2. On day 4, an additional 3000 IU / mL IL-2 was added to the culture. On day 7, anti-CD3 / anti-CD28 antibodies were added at a 1:1 ratio (beads: cells). The cultures were stimulated again and an additional 3000 IU / mL IL-2 was added to the cultures. On day 14, the PBLs were harvested, the magnetic beads were removed, and the PBLs were counted and phenotyped. In one embodiment, the method comprises enriching T cells by isolating pure T cells from PBMCs using negative selection of the non-CD19+ fraction based on magnetic beads.

[0232] In one embodiment of the present invention, PBL method 1 is performed as follows: On day 0, the cryopreserved PBMC sample is thawed and the PBMCs are counted. T cells are isolated using a Human Pan T cell isolation kit and LS columns (Miltenyi Biotec). The isolated T cells are counted and counted according to 5×10 5 T cells were inoculated at a ratio of 1:1 with (anti-CD3 / anti-CD28) and 3000 IU / mL IL-2 were co-cultured, with a total of 8 mL of CM2 medium in each well. On day 4, the medium in each well was replaced from CM2 to fresh AIM-V containing 3000 IU / mL IL-2. On day 7, the expanded cells were harvested, counted, and then plated at 15×10 6 The density of cells / flask was 1000 IU / mL IL-2 and 1:1 ratio (beads: cells). The cells were cultured in GRex 10M flasks with a total of 100 ml of AIM-V medium. On day 11, the medium was replaced with fresh CM-4 medium supplemented with 3000 IU / mL IL-2. On day 14, the cells were cultured using DynaMag Magnet (DynaMag TM -15) Remove And count the cells.

[0233] In one embodiment of the present invention, PBL method 1 is performed as follows: On day 0, the cryopreserved PBMC sample is thawed and the PBMCs are counted. T cells are isolated using a Human Pan T cell isolation kit and LS columns (Miltenyi Biotec). The isolated T cells are counted and counted according to 5×10 5 T cells were inoculated at a ratio of 1:1 with (anti-CD3 / anti-CD28) and 3000 IU / mL IL-2, with a total of 8 mL of CM2 medium per well. On day 4, the medium in each well was replaced from CM2 to fresh AIM-V containing 3000 IU / mL IL-2. On day 7, PBLs were harvested, counted, and then plated at 1×10 6 The cells were seeded at a density of 100 cells / well in a 1:1 ratio (beads: cells) containing 3000 IU / mL IL-2. A total of 8 mL of AIM-V medium was added to a new GRex-24-well plate. On day 11, the medium was replaced with fresh CM-4 medium supplemented with 3000 IU / mL IL-2. On day 14, DynaMag Magnet (DynaMag TM -15) Remove And count the cells.

[0234] PBL Method 2. In one embodiment of the invention, PBLs are expanded using PBL method 2, which includes obtaining a PBMC sample from whole blood. T cells are enriched from PBMCs by incubating the PBMCs at 37°C for at least three hours and then separating the non-adherent cells. Non-adherent cells are expanded similarly to PBL method 1, i.e., on day 0, anti-CD3 / anti-CD28 antibodies are added at a 1:1 ratio (beads: cells). Non-adherent cells were cultured with 3000 IU / mL IL-2. On day 4, an additional 3000 IU / mL IL-2 was added to the culture. On day 7, anti-CD3 / anti-CD28 antibodies were added at a 1:1 ratio (beads: cells). The cultures were re-stimulated and an additional 3000 IU / mL IL-2 was added to the cultures. On day 14, PBLs were harvested, the beads removed, and the PBLs were counted and phenotyped.

[0235] In one embodiment of the present invention, PBL method 2 is performed as follows: On day 0, the cryopreserved PMBC samples are thawed and 6×10 6 PBMC cells were seeded in 6-well plates in CM-2 medium at a density of 10 cells / well and incubated at 37°C for 3 hours. After 3 hours, non-adherent cells (i.e., PBLs) were removed and counted. Anti-CD3 / anti-CD28 antibodies were used in a 1:1 ratio (beads: cells). and 3000 IU / mL IL-2 to culture PBL, 1×10 6cells / well, with a total of 7 ml of CM-2 medium in each well of the GRex24-well plate. On day 4, the medium in each well was replaced with AIM-V medium and fresh 3000 IU / mL IL-2. On day 7, the expanded cells were harvested, counted, and then plated at 15 × 10 6 The density of cells / flask was 1:1 ratio (T cells: magnetic beads). The cells were cultured in GRex 10M flasks supplemented with 100 mL AIM-V medium and 3000 IU / mL IL-2. On day 11, the medium was changed to CM-4 medium supplemented with fresh IL-2 (3000 IU / mL). On day 14, DynaMag TM Magnet(DynaMag TM -15) Remove the DynaBeads and count the cells.

[0236] In one embodiment of the present invention, PBL method 2 is performed as follows: On day 0, the cryopreserved PMBC samples are thawed and 6×10 6 PBMC cells were seeded in 6-well plates in CM-2 medium at a density of 10 cells / well and incubated at 37°C for 3 hours. After 3 hours, non-adherent cells (i.e., PBLs) were removed and counted. Anti-CD3 / anti-CD28 and 3000 IU / mL IL-2 to culture PBL, 1×10 6 cells / well, with a total of 7 ml of CM-2 medium in each well of the GRex 24-well plate. On day 4, the medium in each well was replaced with AIM-V medium and fresh 3000 IU / mL IL-2. On day 7, the expanded cells were harvested, counted, and then plated at 1 × 10 6 The cells were plated at a density of 10 cells / well in a 1:1 ratio (T cells: beads) containing 3000 IU / mL IL-2. A total of 8 mL of AIM-V medium was inoculated in a new GRex-24-well plate. On day 11, the medium was replaced with CM-4 medium supplemented with fresh IL-2 (3000 IU / mL). On day 14, DynaMag TM Magnet(DynaMag TM -15) Remove the DynaBeads and count the cells.

[0237] PBL method 3.In one embodiment of the invention, PBLs are expanded using PBL method 3, which includes obtaining a PBMC sample from peripheral blood. B cells are isolated using CD19+ selection, and T cells are selected using negative selection of the non-CD19+ portion of the PBMC sample. On day 0, T cells and B cells are incubated with anti-CD3 / anti-CD28 antibodies at a 1:1 ratio (beads: cells). On day 4, an additional 3000 IU / mL IL-2 was added to the culture. On day 7, anti-CD3 / anti-CD28 antibodies were added at a 1:1 ratio (beads: cells). The cultures were restimulated and an additional 3000 IU / mL IL-2 was added to the cultures. On day 14, PBLs were harvested, the beads removed, and the PBLs were counted and phenotyped.

[0238] In one embodiment of the invention, PBL method 3 is performed as follows: On day 0, cryopreserved PBMCs derived from peripheral blood are thawed and counted. CD19+ B cells are sorted using CD19 Multisort Kit, Human (Miltenyi Biotec). In the non-CD19+ cell fraction, T cells are purified using Human Pan T Cell Isolation Kit and LS chromatography columns (Miltenyi Biotec). T cells (PBL) and B cells are co-cultured at different ratios in a Grex 24-well plate containing approximately 8 mL of CM2 medium in the presence of approximately 3000 IU / mL IL-2. The ratios of B cells:T cells are 0.1:1, 1:1, and 10:1. Anti-CD3 / anti-CD28 antibodies are used in a 1:1 ratio (magnetic beads: cells). Stimulation of T cell / B cell co-cultures. On day 4, the medium was changed from CM2 to AIM-V medium, and an additional 3000 IU / mL IL-2 was added to the culture. On day 7, cells were harvested, counted, and then plated at approximately 1.5 × 10 5 About 4×10 5 Cells were replated in new Grex 24-well plates containing AIM-V medium at a density of 10 cells / well and incubated with anti-CD3 / anti-CD28 antibodies at a 1:1 ratio (beads: cells). Stimulation was performed with an additional 3000 IU / mL IL-2. On day 14, DynaMag TM Magnet(DynaMag TM -15) Remove the DynaBeads and count the cells.

[0239] In one embodiment, PBMCs are isolated from a whole blood sample. In one embodiment, a PBMC sample is used as a starting material for amplifying PBLs. In one embodiment, the sample is cryopreserved prior to the amplification process. In another embodiment, a fresh sample is used as a starting material for amplifying PBLs. In one embodiment of the 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 LS columns. In one embodiment of the invention, T cells are isolated from PBMCs using antibody screening methods known in the art (e.g., CD19 negative screening).

[0240] In one embodiment of the invention, the process is carried out for 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 for about 7 days. In another embodiment, the process is carried out for about 14 days.

[0241] 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 can be used in the present invention. In one embodiment of the present invention, the commercially available product used is In one embodiment, In another embodiment, the antibody is cultured with PBMC at a ratio of 1:1 (magnetic beads: cells). In another embodiment, the antibody is 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 (magnetic beads: cells). In one embodiment of the invention, the antibody culture step and / or the step of restimulating the cells with the antibody is performed for a period of about 2 to about 6 days, about 3 to about 5 days, or about 4 days. In one embodiment of the invention, the antibody culture step is performed for a period of about 2 days, 3 days, 4 days, 5 days or 6 days.

[0242] In one embodiment, the PBMC sample is cultured with IL-2. In one embodiment of the invention, the cell culture medium for expanding PBL from PBMC comprises IL-2, and the concentration of IL-2 is selected from: 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. / 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 2400 IU / mL, about 2500 IU / mL, about 2600 IU / mL, about 2700 IU / mL, about 2800 IU / mL, about 2900 IU / mL, about 3000 IU / mL, about 3100 IU / mL mL, about 3200 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 5,0 In some embodiments, the present invention relates to about 10,000 IU / mL, about 10,000 IU / mL, about 10,000 IU / mL, about 10,000 IU / mL, about 10,000 IU / mL, about 10,000 IU / mL, about 10,000 IU / mL, about 10,000 IU / mL, about 10,000 IU / mL, about 10,000 IU / mL, and about 10,000 IU / mL.

[0243] In one embodiment of the invention, the starting cell number of PBMCs used in the expansion 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 6 In one embodiment of the invention, the starting cell number of PBMC is about 138,000, 140,000, 145,000 or more. In another embodiment, the starting cell number of PBMC is about 28,000. In another embodiment, the starting cell number of PBMC 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.

[0244] In one embodiment of the invention, cells are cultured in GRex 24-well plates. In one embodiment of the invention, comparable well plates are used. In one embodiment, the starting material for expansion is approximately 5×10 5 T cells / well. In one embodiment of the present invention, there are 1×10 6 In one embodiment of the invention, the number of cells per well is sufficient to seed the well and expand T cells.

[0245] In one embodiment of the invention, the fold expansion of PBL 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 invention, the fold expansion is about 25%. In another embodiment of the invention, the fold expansion is about 50%. In another embodiment, the fold expansion is about 75%.

[0246] In one embodiment of the invention, additional IL-2 may be added to the culture on one or more days throughout the process. In one embodiment of the invention, additional IL-2 is added on day 4. In one embodiment of the invention, additional IL-2 is added on day 7. In one embodiment of the invention, additional IL-2 is added on day 11. In another embodiment, additional IL-2 is added on day 4, day 7 and / or day 11. In one embodiment of the invention, the cell culture medium may be replaced on one or more days throughout the cell culture process. In one embodiment, the cell culture medium is replaced on day 4, day 7 and / or day 11 of the process. In one embodiment of the invention, the PBLs are cultured with additional IL-2 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. In one embodiment of the invention, the PBLs are cultured for a period of 3 days after each addition of IL-2.

[0247] In one embodiment, the cell culture medium is replaced at least once during the method. In one embodiment, the cell culture medium is replaced while adding additional IL-2. In another embodiment, the cell culture medium is replaced on at least one 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 invention, the cell culture medium used throughout the method may be the same or different. In one embodiment of the invention, the cell culture medium is CM-2, CM-4, or AIM-V.

[0248] In one embodiment of the invention, T cells can be restimulated with anti-CD3 / anti-CD28 antibodies on one or more days throughout the 14-day expansion process. In one embodiment, T cells are restimulated on day 7. In one embodiment, GRex 10M flasks are used for the restimulation step. In one embodiment of the invention, comparable flasks are used.

[0249] In one embodiment of the invention, as further described in the Examples below, DynaMag TM Magnet removal The cells are counted and analyzed using phenotypic and functional assays. In one embodiment of the invention, antibodies are separated from PBLs or MILs using methods known in the art. In any of the foregoing embodiments, magnetic bead-based screening of TILs, PBLs or MILs is used.

[0250] In one embodiment of the invention, the PBMC sample is incubated at a desired temperature for a period of time to effectively identify non-adherent cells. In one embodiment of the invention, the incubation time is about 3 hours. In one embodiment of the invention, the temperature is about 37° C. The non-adherent cells are then expanded using the above process.

[0251] In one embodiment of the invention, PBMCs are obtained from patients who have been treated with ibrutinib or another ITK or kinase inhibitor (such as such ITK and kinase inhibitors described elsewhere herein). In one embodiment of the invention, the ITK inhibitor is a covalent ITK inhibitor that covalently and irreversibly binds to ITK. In one embodiment of the invention, the ITK inhibitor is an allosteric ITK inhibitor that binds to ITK. In one embodiment of the invention, before obtaining a PBMC sample for any of the aforementioned methods (including PBL method 1, PBL method 2, or PBL method 3), PBMCs are obtained from patients who have been treated with ibrutinib or other ITK inhibitors (including ITK inhibitors described elsewhere herein). In one embodiment of the invention, ITK inhibitor treatment has been performed at least 1 time, at least 2 times, or at least 3 times or more. In one embodiment of the invention, PBLs expanded from patients pretreated with ibrutinib or other ITK inhibitors contain fewer LAG3+, PD-1+ cells than patients not pretreated with ibrutinib or other ITK inhibitors. In one embodiment of the invention, PBLs expanded from patients pretreated with ibrutinib or other ITK inhibitors comprise increased levels of IFNγ production compared to patients not pretreated with ibrutinib or other ITK inhibitors. In one embodiment of the invention, PBLs expanded from patients pretreated with ibrutinib or other ITK inhibitors comprise increased lytic activity at a lower effector cell:target cell ratio compared to patients not pretreated with ibrutinib or other ITK inhibitors. In one embodiment of the invention, patients pretreated with ibrutinib or other ITK inhibitors have a higher fold expansion compared to untreated patients.

[0252] In one embodiment of the invention, the method includes the step of adding an ITK inhibitor to the cell culture. In one embodiment, the ITK inhibitor is added on one or more days 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 during the method of changing the days of the cell culture medium. 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 when IL-2 is added. In one embodiment, the ITK inhibitor is added on day 0, day 4, day 7 and optionally on day 11 of the method. In one embodiment of the invention, the ITK inhibitor is added on day 0 and day 7 of the method. In one embodiment of the invention, the ITK inhibitor is an ITK inhibitor known in the art. In one embodiment of the invention, the ITK inhibitor is an ITK inhibitor described elsewhere herein.

[0253] In one embodiment of the invention, the concentration of the ITK inhibitor used in the method is about 0.1 nM to about 5 μM. In one embodiment, the concentration of the ITK inhibitor used in the method is 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.

[0254] In one embodiment of the present invention, when the PBMCs are from patients who have not been previously treated with an ITK inhibitor (eg, ibrutinib), the method comprises the step of adding an ITK inhibitor.

[0255] In some embodiments, the PBMC sample is from a subject or patient who has been pretreated with a regimen comprising a kinase inhibitor or ITK inhibitor, optionally. In some embodiments, the tumor sample is from a subject or patient who has been pretreated with a regimen comprising a kinase inhibitor or ITK inhibitor. In some embodiments, the PBMC sample is from a subject or patient who has been pretreated with a regimen comprising a kinase inhibitor or ITK inhibitor for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or 1 year or more of treatment. In another embodiment, the PBMC is from a patient who is currently undergoing a regimen of an ITK inhibitor (e.g., ibrutinib).

[0256] In some embodiments, the PBMC sample is from a subject or patient who has been pretreated with a regimen comprising a kinase inhibitor or ITK inhibitor and who is refractory to treatment with the kinase inhibitor or ITK inhibitor (eg, ibrutinib).

[0257] In some embodiments, the PBMC sample is from a subject or patient who has been pretreated with a regimen comprising a kinase inhibitor or ITK inhibitor, but the subject or patient is no longer receiving treatment with the kinase inhibitor or ITK inhibitor. In some embodiments, the PBMC sample is from a subject or patient who has been pretreated with a regimen comprising a kinase inhibitor or ITK inhibitor, but the subject or patient is no longer receiving treatment with the kinase inhibitor or ITK inhibitor and has not been treated for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year or more. In another embodiment, the PBMC is from a patient who has been previously exposed to an ITK inhibitor but has not been treated for at least 3 months, at least 6 months, at least 9 months, or at least 1 year.

[0258] In one embodiment of the invention, at day 0, CD19+ cells are selected and sorted accordingly. In one embodiment of the invention, magnetic beads bound to antibodies are used for screening. In one embodiment of the invention, pure T cells are isolated from PBMCs at day 0. In one embodiment of the invention, at 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 invention, at day 4, IL-2 is added to the culture. In one embodiment of the invention, at day 7, the culture is restimulated with anti-CD3 / anti-CD28 antibodies and additional IL-2. In one embodiment of the invention, at day 14, PBLs are harvested.

[0259] In one embodiment of the invention, for patients not pretreated with ibrutinib or other ITK inhibitors, 10-15 mL of buffy coat will produce approximately 5×10 9 PBMCs, which in turn will generate approximately 5.5 × 10 7 of starting cell material and about 11 × 10 9 In one embodiment of the present invention, about 54×10 6 PBMCs will generate approximately 6 × 10 5 starting materials and about 1.2×10 8 MIL (approximately 205-fold amplification).

[0260] In one embodiment of the invention, for patients pretreated with ibrutinib or other ITK inhibitors, the expansion process will generate approximately 20×10 9 In one embodiment of the present invention, 40.3×10 6 PBMCs will generate approximately 4.7 × 10 5 starting cell material and about 1.6×10 8 PBL (approximately 338-fold expansion).

[0261] In one embodiment of the present invention, the clinical dose of PBL useful for chronic lymphocytic leukemia (CLL) patients of the present invention is about 0.1×10 9 About 15×10 9 PBL, about 0.1×10 9 About 15×10 9 PBL, about 0.12×10 9 About 12×10 9 PBL, about 0.15×10 9 About 11×10 9 PBL, about 0.2×10 9 to about 10×10 9 PBL, about 0.3×10 9 About 9×10 9 PBL, about 0.4×10 9 About 8×10 9 PBL, about 0.5×10 9 About 7×10 9 PBL, about 0.6×10 9 About 6×10 9 PBL, about 0.7×10 9 About 5×10 9 PBL, about 0.8×109 About 4×10 9 PBL, about 0.9×10 9 About 3×10 9 PBL or about 1×10 9 About 2×10 9 PBLs.

[0262] In any of the foregoing embodiments, the PBMCs may be from a whole blood sample, apheresis, buffy coat, or from any other method known in the art for obtaining PBMCs.

[0263] Method for expanding bone marrow infiltrating lymphocytes (MIL) from bone marrow-derived PBMCs

[0264] MIL Method 1 In one embodiment of the invention, a method for expanding MILs from bone marrow derived PBMCs is described. In one embodiment of the invention, the method is performed for 14 days. In one embodiment, the method comprises obtaining bone marrow PBMCs and cryopreserving the PBMCs. On day 0, anti-CD3 / anti-CD28 antibodies are used in a 1:1 ratio (beads: cells). PBMCs were cultured with 3000 IU / mL IL-2. On day 4, an additional 3000 IU / mL IL-2 was added to the cultures. On day 7, anti-CD3 / anti-CD28 antibodies were added at a 1:1 ratio (beads: cells). The cultures are re-stimulated and an additional 3000 IU / mL IL-2 is added to the cultures. On day 14, the MILs are harvested, the magnetic beads are removed, and the MILs are optionally counted and phenotyped.

[0265] In one embodiment of the present invention, MIL method 1 is performed as follows: On day 0, a cryopreserved PBMC sample from bone marrow is thawed and the PBMCs are counted. 5 Incubate PBMCs with anti-CD3 / anti-CD28 antibodies at a 1:1 ratio Co-culture was performed in GRex24-well plates with approximately 8 mL of CM-2 cell culture medium (including RPMI-1640, human AB serum, l-glutamine, 2-mercaptoethanol, gentamicin sulfate, AIM-V medium) per well. On day 4, the cell culture medium was replaced with AIM-V supplemented with an additional 3000 IU / mL IL-2. On day 7, the expanded MILs were counted. 1×10 6 The cells / well were transferred to a new GRex 24-well plate and incubated with anti-CD3 / anti-CD28 antibodies at a 1:1 ratio in the presence of 3000 IU / mL IL-2 in approximately 8 mL AIM-V medium / well. The cells were cultured. On day 11, the cell culture medium was changed from AIM-V to CM-4 (including AIM-V medium, 2 mM Glutamax and 3000 IU / mL IL2). On day 14, DynaMag Magnet (DynaMag TM 15) Remove Count the MILs.

[0266] MIL Method 2. In one embodiment of the invention, the method is performed for 7 days. In one embodiment, the method comprises obtaining PBMCs from bone marrow and cryopreserving PBMCs. On day 0, anti-CD3 / anti-CD28 antibodies are added at a ratio of 3:1 (beads: cells). PBMCs were cultured with 3000 IU / mL IL-2. On day 7, MILs were harvested, the magnetic beads were removed, and the MILs were optionally counted and phenotyped.

[0267] In one embodiment of the present invention, MIL method 2 is performed as follows: On day 0, the cryopreserved PBMC sample is thawed and the PBMCs are counted. In the presence of 3000 IU / mL of IL-2, 5×10 5 Incubate PBMCs with anti-CD3 / anti-CD28 antibodies at a 1:1 ratio Co-culture was performed in GRex 24-well plates with approximately 8 mL of CM-2 cell culture medium (including RPMI-1640, human AB serum, l-glutamine, 2-mercaptoethanol, gentamicin sulfate, AIM-V medium) per well. On day 7, DynaMag Magnet (DynaMag TM 15) Remove Count the MILs.

[0268] MIL Method 3. In one embodiment of the invention, the method comprises obtaining PBMCs from bone marrow. On day 0, PBMCs are screened for CD3+ / CD33+ / CD20+ / CD14+, and the PBMCs are sorted, the non-CD3+ / CD33+ / CD20+ / CD14+ cell fraction is sonicated, and a portion of the sonicated cell fraction is added back to the screened cell fraction. 3000 IU / mL IL-2 is added to the cell culture. On day 3, anti-CD3 / anti-CD28 antibodies are used at a 1:1 ratio (magnetic beads: cells). PBMCs were cultured with 3000 IU / mL IL-2. On day 4, an additional 3000 IU / mL IL-2 was added to the cultures. On day 7, anti-CD3 / anti-CD28 antibodies were added at a 1:1 ratio (beads: cells). The cultures are stimulated again and an additional 3000 IU / mL IL-2 is added to the cultures. On day 11, 3000 IU / mL IL-2 is added to the cultures. On day 14, the MILs are harvested, the magnetic beads are removed, and the MILs are optionally counted and phenotyped.

[0269] In one embodiment of the present invention, MIL method 3 is performed as follows: On day 0, the cryopreserved PBMC sample is thawed and the PBMCs are counted. The cells are stained with CD3, CD33, CD20 and CD14 antibodies and sorted using S3eCell Sorter (Bio-Rad). The cells are divided into two parts - the immune cell part (or MIL part) (CD3+CD33+CD20+CD14+) and the AML primitive cell part (non-CD3+CD33+CD20+CD14+). A certain number of cells from the AML primitive cell part (approximately equal to the number of cells in the immune cell part (or MIL part) to be inoculated on a Grex 24-well plate) are suspended in 100 μL of culture medium and sonicated. In this example, approximately 2.8×10 4 to about 3.38×10 5 cells, suspended in 100 μL CM2 medium, and then sonicated for 30 seconds. 100 μL of the sonicated AML blast fraction was added to the immune cell fraction in a Grex 24-well plate. In the presence of 6000 IU / mL IL-2, immune cells were cultured at a rate of approximately 2.8 × 10 4 to about 3.38×10 5 The amount of cells / well was present in about 8 mL of CM-2 cell culture medium / well, and the immune cells were cultured with this portion of AML primitive cells for about 3 days. On the 3rd day, a 1:1 ratio of anti-CD3 / anti-CD28 antibodies was added. Add to each well and culture for about 1 day. On day 4, replace the cell culture medium with AIM-V supplemented with an additional 3000 IU / mL IL-2. On day 7, count the expanded MILs. 5 Up to 4×10 5 The cells / well were transferred to a new GRex 24-well plate and incubated with anti-CD3 / anti-CD28 antibodies in a 1:1 ratio in the presence of 3000 IU / mL IL-2 in approximately 8 mL AIM-V medium / well. On day 11, the cell culture medium was changed from AIM-V to CM-4 (supplemented with 3000 IU / mL IL-2). On day 14, DynaMag Magnet (DynaMag TM 15) Remove Optionally count the MIL.

[0270] In one embodiment of the present invention, PBMCs are obtained from bone marrow. In one embodiment, PBMCs are obtained from bone marrow by single collection, aspiration, puncture biopsy or other similar methods known in the art. In one embodiment, PBMCs are fresh. In another embodiment, PBMCs are cryopreserved.

[0271] In one embodiment of the invention, the method is performed for 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 method is performed for about 7 days. In another embodiment, the method is performed for about 14 days.

[0272] 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 can be used in the present invention. In one embodiment of the present invention, the commercially available product used is In one embodiment, In another embodiment, the antibody is 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 (magnetic beads: cells). In any of the foregoing embodiments, screening of a magnetic bead-based immune cell fraction (or MIL fraction) (CD3+CD33+CD20+CD14+) or AML primitive cell fraction (non-CD3+CD33+CD20+CD14+) is used. In one embodiment of the invention, the antibody culture step and / or the step of restimulating the cells with antibodies is performed for a period of about 2 to about 6 days, about 3 to about 5 days, or about 4 days. In one embodiment of the invention, the antibody culture step is performed for a period of about 2 days, 3 days, 4 days, 5 days, or 6 days.

[0273] In one embodiment of the invention, the ratio of the number of cells from the AML primitive cell portion to the number of cells from the immune cell portion (or MIL portion) is about 0.1: 1 to about 10: 1. In another embodiment, the ratio is about 0.1: 1 to about 5: 1, about 0.1: 1 to about 2: 1 or about 1: 1. In one embodiment of the invention, the AML primitive cell portion is optionally broken to destroy cell aggregation. In one embodiment, ultrasound, homogenization, cell lysis, vortexing or vibration are used to break up the AML primitive cell portion. In another embodiment, ultrasound is used to break up the AML primitive cell portion. In one embodiment of the invention, a suitable lysis method is used to lyse the non-CD3+, non-CD33+, non-CD20+, non-CD14+ cell portion (AML primitive cell portion), including high temperature lysis, chemical lysis (e.g., organic alcohol), enzymatic lysis, and other cell lysis methods known in the art.

[0274] In one embodiment of the present invention, about 0.2×10 5 About 2×10 5 Cells from the AML blast fraction are suspended at a concentration of about 0.5×10 cells / 100 μL and added to the cell culture along with the immune cell fraction. In another embodiment, the concentration is about 0.5×10 5 About 2×10 5 cells / 100μL, about 0.7×10 5 About 2×10 5 cells / 100μL, about 1×10 5 About 2×10 5 cells / 100 μL or approximately 1.5 × 10 5 About 2×10 5 cells / 100 μL.

[0275] In one embodiment, the PBMC sample is cultured with IL-2. In one embodiment of the present invention, the cell culture medium for expanding MIL contains IL-2, and the concentration of IL-2 is selected from: about 100 IU / mL, about 200 IU / mL, about 300 IU / mL, about 400 μL 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 2500 IU / mL, about 2600 IU / mL, about 2700 IU / mL, about 2800 IU / mL, about 2900 IU / mL, about 3000 IU / mL, about 3100 IU / mL, about 3200 IU / mL, about 3,300 IU / mL, about 3,400 IU / mL, about 3,50 0 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, In some embodiments, the present invention relates to an aqueous solution of at least about 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.

[0276] In one embodiment of the invention, additional IL-2 may be added to the culture on one or more days throughout the method. In one embodiment of the invention, additional IL-2 is added on day 4. In one embodiment of the invention, additional IL-2 is added on day 7. In one embodiment of the invention, additional IL-2 is added on day 11. In another embodiment, additional IL-2 is added on day 4, day 7 and / or day 11. In one embodiment of the invention, the MILs are cultured with additional IL-2 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. In one embodiment of the invention, the MILs are cultured for a period of 3 days after each addition of IL-2.

[0277] In one embodiment, during the method, the cell culture medium is replaced at least once. In one embodiment, the cell culture medium is replaced while adding additional IL-2. In another embodiment, the cell culture medium is replaced on at least one of the 1st day, 2nd day, 3rd day, 4th day, 5th day, 6th day, 7th day, 8th day, 9th day, 10th day, 11th day, 12th day, 13th day or 14th day. In one embodiment of the invention, the cell culture medium used throughout the method may be the same or different. In one embodiment of the invention, the cell culture medium is CM-2, CM-4 or AIM-V. In one embodiment of the invention, the cell culture medium replacement step on day 11 is optional. In one embodiment of the invention, the starting cell number of PBMCs used in the expansion 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 invention, the starting cell number of PBMC is about 138,000, 140,000, 145,000 or more. In another embodiment, the starting cell number of PBMC is about 28,000. In another embodiment, the starting cell number of PBMC 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.

[0278] In one embodiment of the invention, the fold expansion of the 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 invention, the fold expansion is about 25%. In another embodiment of the invention, the fold expansion is about 50%. In another embodiment, the fold expansion is about 75%.

[0279] In one embodiment of the invention, MILs are expanded from 10-50 ml of bone marrow aspirate. In one embodiment of the 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.

[0280] In one embodiment of the present invention, the number of PBMCs generated from about 10-50 ml of bone marrow aspirate is about 5×10 7 to about 10×10 7 In another embodiment, the number of PBMCs produced is about 7×10 7 PBMCs.

[0281] In one embodiment of the present invention, about 5×10 7 to about 10×10 7 PBMCs generate approximately 0.5 × 10 6 About 1.5×10 6 In one embodiment of the present invention, about 1×10 6 Amplification starting cell material.

[0282] In one embodiment of the invention, the total number of MILs harvested at the end of the expansion period is about 0.01×10 9 About 1×10 9 , about 0.05×10 9 About 0.9×10 9 , about 0.1×10 9 About 0.85×10 9 , about 0.15×10 9 About 0.7×10 9 , about 0.2×10 9 to about 0.65×10 9 , about 0.25×10 9 About 0.6×10 9 , about 0.3×10 9 About 0.55×10 9 , about 0.35×10 9 About 0.5×10 9 or about 0.4×10 9 About 0.45×10 9 .

[0283] In one embodiment of the invention, 12×106 PBMCs generate approximately 1.4 × 10 5 of starting cell material, which at the end of the expansion process yields approximately 1.1 × 10 7 A MIL.

[0284] In one embodiment of the present invention, MILs expanded from bone marrow PBMCs using the above-mentioned MIL method 3 contain a high proportion of CD8+ cells and a lower number of LAG3+ and PD1+ cells compared to MILs expanded using MIL method 1 or MIL method 2. In one embodiment of the present invention, PBLs expanded from blood PBMCs using the above-mentioned MIL method 3 contain a high proportion of CD8+ cells and an increased level of IFNγ production compared to PBLs expanded using MIL method 1 or MIL method 2.

[0285] In one embodiment of the present invention, the clinical dose of MIL useful for acute myeloid leukemia (AML) patients is about 4×10 8 About 2.5×10 9 In another embodiment, the number of MILs provided in the pharmaceutical composition of the present invention is 9.5×10 8 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 amount of MIL provided in the pharmaceutical composition of the present invention is 2.2×10 9 .

[0286] In any of the foregoing embodiments, the PBMCs may be from a whole blood sample, bone marrow, apheresis, buffy coat, or any other method known in the art for obtaining PBMCs.

[0287] Method for expanding TIL using the "2A process"

[0288] In one embodiment of the invention, the invention provides a device and method for expanding T cells from bone marrow and / or peripheral blood. In one embodiment of the invention, T cells have enhanced tumor specificity from the bone marrow microenvironment in a polyclonal but highly tumor-specific manner. In one embodiment, the bone marrow microenvironment is used to maintain and expand T cells. In one embodiment of the invention, during the expansion process of 7 days or 14 days, TIL expands by about 25 to 100 times. In one embodiment, the multiple expansion of TIL is about 30-90 times. In one embodiment, the multiple expansion is about 35-85 times. In one embodiment, the multiple expansion is about 40-80 times. In one embodiment, the multiple expansion is about 45-75 times. In another embodiment, the multiple expansion is about 40-70 times. In another embodiment, the multiple expansion is about 45-65 times. In another embodiment, the fold expansion is about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, or about 100-fold expansion.

[0289] In one embodiment of the invention, the T cell preparation process does not require any intervention to select tumor specificity. In one embodiment of the invention, the T cell preparation process does not require the presence of tumors in the bone marrow and / or peripheral blood when the T cells are expanded. In one embodiment, T cells are expanded in the presence of almost complete bone marrow.

[0290] In one embodiment, the present invention provides a method for extracting T cells from bone marrow and / or peripheral blood as described in the examples, described in WO2010 / 062742 (particularly Example 21), the contents of which are 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, the contents of which are incorporated herein by reference.

[0291] In one embodiment, methods known to those skilled in the art for obtaining bone marrow and / or peripheral blood can be used in the present invention. In one embodiment of the present invention, 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 syringe containing heparin and stored overnight at room temperature. In one embodiment of the present invention, after storage, the contents of the syringe are combined together in a sterile container and quality tested. Mononuclear cells (MNCs) in the bone marrow are enriched using lymphocyte separation media (LSM) and centrifuged using COBE Spectra. Cells in the gradient are collected until red blood cells and washed with HBSS. MNCs are cryopreserved using a hydroxyethyl starch antifreeze supplemented with 2% HSA and 5% DMSO, and some MNCs are retained for quality control. QC vials are thawed and the CD3+ and CD38+ / 138+ cell content of the MNC product is determined. It is important to note that the collection of bone marrow does not constitute a limitation of the present invention.

[0292] In one embodiment of the invention, the bone marrow is aspirated and fractionated in a density gradient of a "lymphocyte separation fluid" to collect cells that are almost at the red blood cell sedimentation layer. In one embodiment, this fractionation method substantially removes red blood cells and neutrophils, providing an almost complete bone marrow. In one embodiment, the resulting fractionated material is T cells and tumor cells. In one embodiment of the invention, the method can be performed without a T cell-specific separation step and without a tumor cell separation step, for example, without labeling T cells with antibodies or other cell type-specific detectable markers, and without using fluorescence activated cell sorting (FACS) for sorting.

[0293] In one embodiment of the present invention, the obtained bone marrow is subjected to Ficoll, or the peripheral blood is subjected to 1×10 6 Cells / mL were suspended in 200 μL / well of AIM-V medium under serum-free conditions.

[0294] In one embodiment of the invention, bone marrow is collected from a subject in incomplete remission. In one embodiment of the invention, bone marrow is collected from a subject in complete remission.

[0295] In one embodiment of the invention, bone marrow may be obtained and frozen. In one embodiment, bone marrow may be obtained and immediately used to extract T cells.

[0296] In other embodiments, and according to any of the above, the present invention provides a method of expanding TIL, the method comprising contacting a TIL population comprising at least one TIL obtained from a liquid tumor. All discussions herein about expanding TIL are applicable to expanding TIL obtained from bone marrow, peripheral blood and / or hematological malignancies (including liquid tumors).

[0297] In one embodiment, the present invention provides a method for preparing a tumor infiltrating lymphocyte (TIL) population from a tumor, the method comprising the following steps:

[0298] (a) contacting the fragmented tumor with a first cell culture medium;

[0299] (b) performing initial expansion (pre-REP) on the first TIL population in a first cell culture medium to obtain a second TIL population; wherein the number of the second TIL population is at least 5 times higher than the number of the first TIL population; wherein the first cell culture medium comprises IL-2;

[0300] (c) amplifying the second TIL population for a second time in a second cell culture medium to obtain a third TIL population; wherein, 7 days after the start of the second amplification, the number of the third TIL population is at least 50 times higher than the number of the second TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), and irradiated allogeneic peripheral blood mononuclear cells (PBMC); and wherein the second amplification is performed for less than 14 days;

[0301] (d) harvesting a third TIL population; and

[0302] wherein the tumor is a liquid tumor; and wherein the cancer is a hematological malignancy.

[0303] In one embodiment, the present invention provides a method for expanding a TIL population, the method comprising a first pre-rapid expansion (pre-REP) process and then a second expansion process (which may be a rapid expansion process - REP); wherein the cell culture medium used for expansion comprises IL-2, and the concentration of IL-2 is selected from: 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 and 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. L, 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.

[0304] In one embodiment, the present invention provides a method for expanding a TIL population, the method comprising a pre-rapid expansion (pre-REP) process and a rapid expansion process (REP); wherein the cell culture medium for expansion comprises IL-2, and the concentration of IL-2 is selected from: 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. 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 1100 IU / mL, about 1200 IU / mL, about 1300 IU / mL, about 1400 IU / mL, about 1500 IU / mL, about 1600 IU / mL, about 1700 IU / mL, about 1800 IU / mL, about 1900 IU / mL, about 2,000 IU / mL, about 2,100 IU / mL, about 2,200 IU / mL, about 2300 IU / mL, about 2400 IU / mL, about 2500 IU / mL, about 2600 IU / mL, about 2700 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, ab out 3400 IU / mL, about 3500 IU / mL, about 3600 IU / mL , about 3700 IU / mL, about 3800 IU / mL, about 3900 IU / mL, about 4000 IU / mL, about 4100 IU / mL, about 4200 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 5,3 In some embodiments, the present invention relates to about 10,000 IU / mL of the present invention and about 10,000 IU / mL of the present invention. In some embodiments, the present invention relates to about 10,000 IU / mL of the present invention and about 10,000 IU / mL of the present invention.

[0305] In one embodiment, the present invention provides a method for expanding a TIL population, the method comprising a pre-rapid expansion (pre-REP) process. In one embodiment, the present invention provides a pre-REP process for expanding a TIL population, the pre-REP process comprising the step of contacting a TIL population obtained from a liquid tumor with a cell culture medium; wherein the cell culture medium further comprises IL-2 at an initial concentration of 1000 IU / mL to 6000 IU / mL.

[0306] In one embodiment, the present invention provides a pre-REP process for expanding a TIL population, comprising the step of contacting a TIL population obtained from a liquid tumor with a cell culture medium; wherein the cell culture medium further comprises IL-2 at an initial concentration of about 6000 IU / mL.

[0307] In one embodiment, REP can be performed in a permeable container using TILs obtained from a liquid tumor according to the present disclosure by any suitable method. For example, TILs can be rapidly expanded using non-specific T cell receptor stimulation in the presence of interleukin 2 (IL-2) or interleukin-15 (IL-15). 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, New Jersey or Miltenyi Biotech, Auburn, California). Optionally in the presence of T cell growth factors such as 300 IU / mL IL-2 or IL-15, TIL can be rapidly expanded by further stimulating TIL in vitro with more than one cancer antigen (including its antigenic portion, such as epitope), which can be optionally expressed by a vector, such as human leukocyte antigen A2 (HLA-A2) binding peptide, such as 0.3 μM MART-1: 26-35 (27L) or gpl 00: 209-217 (210M). Other suitable antigens may include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2 and VEGFR2, or antigenic portions thereof. TIL can also be rapidly expanded by re-stimulating with the same cancer antigen pulsed with antigen presenting cells expressing HLA-A2. Alternatively, TIL can be further re-stimulated with, for example, irradiated autologous lymphocytes or with irradiated HLA-A2 + allogeneic lymphocytes and IL-2.

[0308] In one embodiment, a method for expanding TILs may include using about 5000 mL to about 25000 mL of cell culture medium, about 5000 mL to about 10000 mL of cell culture medium, or about 5800 mL to about 8700 mL of cell culture medium. In one embodiment, the method for expanding TIL may include using about 1000mL to about 2000mL of cell culture medium, about 2000mL to about 3000mL of cell culture medium, about 3000mL to about 4000mL of cell culture medium, about 4000mL to about 5000mL of cell culture medium, about 5000mL to about 6000mL of cell culture medium, about 6000mL to about 7000mL of cell culture medium, about 7000mL to about 8000mL of cell culture medium, about 8000mL to about 9000mL of cell culture medium, about 9000mL to about 10000mL of cell culture medium, about 10000mL to about 15000mL of cell culture medium, about 15000mL to about 20000mL of cell culture medium, or about 20000mL to about 25000mL of cell culture medium. In one embodiment, the quantity of TIL is expanded using no more than one type of cell culture medium. Any suitable cell culture medium can be used, such as AIM-V cell culture medium (L-glutamine, 50 μM streptomycin sulfate and 10 μM gentamicin sulfate) cell culture medium (Invitrogen, Carlsbad CA). In this regard, the method of the present invention advantageously reduces the amount of culture medium required for the number of amplified TILs and the number of culture medium types. In one embodiment, the number of amplified TILs can include feeding cells at a frequency no more than once every three days or once every four days. Amplifying the number of cells with a permeable container simplifies the steps required for the number of amplified cells by reducing the feeding frequency required for the amplified cells.

[0309] In one embodiment, a second expansion is performed using a gas permeable container. Such an embodiment allows the cell population to be expanded from approximately 5×10 5 cells / cm 2 Expanded to 10×10 6 Up to 30×10 6 cells / cm 2. In one embodiment, this amplification is not fed. In one embodiment, as long as the culture medium is at a height of about 10 cm in the gas permeable flask, this amplification is not fed. In one embodiment, no feeding is performed but more than one cytokine is added. In one embodiment, the cytokine can be added as a bolus without mixing the cytokine with the culture medium. Such containers, devices and methods are known in the art and have been used to amplify TILs, and are included in U.S. Patent Application Publication No. US2014 / 0377739A1, International Patent Application Publication No. WO2014 / 210036A1, U.S. Patent Application Publication No. US2013 / 0115617A1, International Publication No. WO2013 / 188427A1, U.S. Patent Application Publication No. US2011 / 0136228A1, U.S. Patent No. 8,809,050, International Patent Application Publication No. WO2011 / 072 The invention relates to a method for treating a cervical cancer in a patient with a cervical cancer. The invention relates to a method for treating a cervical cancer in a patient with a cervical cancer. The invention relates to a method for treating a cervical cancer in a patient with a cervical cancer. The invention relates to a method for treating a cervical cancer in a patient with a cervical cancer. The invention relates to a method for treating a cervical cancer in a patient with a cervical cancer.

[0310] In one embodiment, the gas permeable container is a G-Rex 10 flask (Wilson Wolf Manufacturing Corporation, New Brighton, Minnesota, USA). In one embodiment, the gas permeable container has a 10 cm 2 In one embodiment, the gas permeable container has a cell culture medium capacity of 40 mL. In one embodiment, the gas permeable container provides 100 million to 300 million TILs after 2 medium changes.

[0311] In one embodiment, the gas permeable container is a G-Rex 100 flask (Wilson Wolf Manufacturing Corporation, New Brighton, Minnesota, USA). In one embodiment, the gas permeable container has a 100 cm 2In one embodiment, the gas permeable container has a cell culture medium capacity of 450 mL. In one embodiment, the gas permeable container provides 1 billion to 3 billion TILs after 2 medium changes.

[0312] In one embodiment, the gas permeable container is a G-Rex 100M flask (Wilson Wolf Manufacturing Corporation, New Brighton, Minnesota, USA). In one embodiment, the gas permeable container has a 100 cm 2 In one embodiment, the gas permeable container has a 1000 mL cell culture medium capacity. In one embodiment, the gas permeable container provides 1 billion to 3 billion TILs without changing the culture medium.

[0313] In one embodiment, the gas permeable container is a G-Rex 100L flask (Wilson Wolf Manufacturing Corporation, New Brighton, Minnesota, USA). In one embodiment, the gas permeable container has a 100 cm 2 In one embodiment, the gas permeable container has a 2000 mL cell culture medium capacity. In one embodiment, the gas permeable container provides 1 billion to 3 billion TILs without changing the culture medium.

[0314] In one embodiment, the gas permeable container is a G-Rex 24-well plate (Wilson Wolf Manufacturing Corporation, New Brighton, Minnesota, USA). In one embodiment, the gas permeable container comprises a plate having wells, wherein each well has a 2 cm 2 In one embodiment, the gas permeable container comprises a plate having wells, wherein each well has a cell culture medium capacity of 8 mL. In one embodiment, the gas permeable container provides 20 million to 60 million cells per well after 2 medium changes.

[0315] In one embodiment, the gas permeable container is a G-Rex 6-well plate (Wilson Wolf Manufacturing Corporation, New Brighton, Minnesota, USA). In one embodiment, the gas permeable container comprises a plate having wells, wherein each well has a 10 cm 2 In one embodiment, the gas permeable container comprises a plate having wells, wherein each well has a 40 mL cell culture medium capacity. In one embodiment, the gas permeable container provides 100 million to 300 million cells per well after 2 medium changes.

[0316] In one embodiment, the cell culture medium in the first gas permeable container and / or the second gas permeable container is not filtered. Using unfiltered cell culture medium can simplify the steps required to expand the number of cells. In one embodiment, the cell culture medium in the first gas permeable container and / or the second gas permeable container does not have beta-mercaptoethanol (BME).

[0317] In one embodiment, the duration of the method includes: obtaining a tumor tissue sample from a mammal; culturing the tumor tissue sample in a first gas permeable container containing a cell culture medium; obtaining TILs from the tumor tissue sample; and expanding the number of TILs in a second gas permeable container containing a cell culture medium for about 14 days to about 42 days, for example, about 28 days.

[0318] In one embodiment, the cell culture medium comprises IL-2. In a preferred embodiment, the cell culture medium comprises about 3000 IU / mL of IL-2. In one embodiment, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL or about 8000 IU / mL of IL-2. In one embodiment, the cell culture medium comprises 1000 to 2000 IU / mL, 2000 to 3000 IU / mL, 3000 to 4000 IU / mL, 4000 to 5000 IU / mL, 5000 to 6000 IU / mL, 6000 to 7000 IU / mL, 7000 to 8000 IU / mL, or 8000 IU / mL of IL-2.

[0319] In one embodiment, the cell culture medium comprises OKT-3 antibody. In a preferred embodiment, the cell culture medium comprises about 30 ng / mL of OKT-3 antibody. In one embodiment, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL and about 1 μg / mL of OKT-3 antibody. In one embodiment, the cell culture medium comprises 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL, or 50 ng / mL to 100 ng / mL of OKT-3 antibody.

[0320] In one embodiment, TIL is expanded in a gas permeable container. Using methods known in the art, compositions and devices, including those described in U.S. Patent Application Publication No. US2005 / 0106717A1 (its disclosure is incorporated herein by reference), a gas permeable container is used to expand TIL with PBMC. In one embodiment, TIL is expanded in a gas permeable bag. In one embodiment, TIL is expanded using a cell expansion system, and the cell expansion system expands TIL in a gas permeable bag, such as Xuri Cell Expansion System W25 (GE Healthcare). In one embodiment, TIL is expanded using a cell expansion system, and the cell expansion system expands TIL in a gas permeable bag, such as WAVE bioreactor system, also referred to as Xuri Cell Expansion System W5 (GE Healthcare). In one embodiment, the cell expansion system comprises a gas permeable cell bag having a volume selected from the group consisting of about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, 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 system comprises a gas permeable cell bag having a volume selected from the group consisting of 50 to 150 mL, 150 to 250 mL, 250 to 350 mL, 350 to 450 mL, 450 to 550 mL, 550 to 650 mL, 650 to 750 mL, 750 to 850 mL, 850 to 950 mL, and 950 to 1050 mL. In one embodiment, the cell expansion system comprises a gas permeable cell bag having a volume selected from the group consisting of 1 L to 2 L, 2 L to 3 L, 3 L to 4 L, 4 L to 5 L, 5 L to 6 L, 6 L to 7 L, 7 L to 8 L, 8 L to 9 L, 9 L to 10 L, 10 L to 11 L, 11 L to 12 L, 12 L to 13 L, 13 L to 14 L, 14 L to 15 L, 15 L to 16 L, 16 L to 17 L, 17 L to 18 L, 18 L to 19 L, and 19 L to 20 L. In one embodiment, the cell expansion system comprises a gas permeable cell bag having a volume 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 system uses a rocketing 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 rocking time used by the cell expansion system is 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 rocking rate used by the cell expansion system is about 2 rocks / minute, about 5 rocks / minute, about 10 rocks / minute, about 20 rocks / minute, about 30 rocks / minute, and about 40 rocks / minute. In one embodiment, the rocking rate used by the cell expansion system is 2 rocks / minute to 5 rocks / minute, 5 rocks / minute to 10 rocks / minute, 10 rocks / minute to 20 rocks / minute, 20 rocks / minute to 30 rocks / minute, 30 rocks / minute to 40 rocks / minute. In one embodiment, the rocking angle used by the cell expansion system is 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 rocking angle used by the cell expansion system is 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°, 11° to 12°.

[0321] In one embodiment, the method of amplifying TILs obtained from liquid tumors also includes the step of selecting TILs with excellent tumor reactivity. Any selection method known in the art can be used. For example, the method described in U.S. Patent Application Publication No. 2016 / 0010058A1 (the disclosure of which is incorporated herein by reference) can be used to select TILs with excellent tumor reactivity.

[0322] In one embodiment, the present invention provides a method for expanding a TIL population from a liquid tumor, the method comprising 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 portion thereof may be placed in an enzyme medium and mechanically dissociated for about 1 minute. The mixture may then be incubated at 37° C., 5% CO2 for 30 minutes and then mechanically disrupted again for about 1 minute. After incubation at 37° C., 5% CO2 for 30 minutes, the tumor or a portion thereof may be mechanically disrupted for a third time for about 1 minute. If large pieces of tissue are present after the third mechanical disruption, 1 or 2 additional mechanical dissociations may be applied to the sample, with or without an additional 30-minute incubation at 37° C., 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 may be performed to remove these cells. TIL cultures were initiated in 24-well plates (Costar 24-well cell culture clusters, flat bottom; Corning Incorporated, Corning, NY) by seeding each well with 1 × 10 200 μL of IL-2 in 2 mL of complete culture medium (CM) containing IL-2 (6000 IU / mL; Chiron Corp., Emeryville, CA). 6 Tumor digestion cells or about 1-8mm in size 3 CM contains Roswell Park Memorial Institute (RPMI) 1640 buffer with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. 2 Cultures were started in gas-permeable flasks with a silicon bottom (G-Rex 10; Wilson Wolf Manufacturing, New Brighton). Each flask was loaded with 10–40 × 10 6 Live tumor digested cells or 5 to 30 tumor fragments. G-Rex 10 and 24-well plates can be cultured in a humidified incubator at 37°C and 5% CO2. Five days after the start of culture, half of the culture medium can be removed and replaced with fresh CM and IL-2. After the fifth day, half of the culture medium can be replaced every 2 to 3 days. As described elsewhere herein, TILs obtained from the liquid tumors of the present disclosure can be used to perform a second expansion protocol (REP) of TILs using T-175 flasks and permeable bags or permeable G-Rex flasks. For REP in T-175 flasks, 1×10 6Each TIL is suspended in 150 mL of culture medium in each flask. TILs 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). The T-175 flask can be incubated at 37°C, 5% CO2. Half of the culture medium can be replaced on day 5 with 50 / 50 culture medium containing 3000 IU / mL IL-2. On day 7, cells from 2 T-175 flasks can be combined in a 3L bag, and 300 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2 can be added to the 300 mL TIL suspension. The number of cells in each bag can be counted every day or every two days, and fresh culture medium can be added to keep the cell count at 0.5 to 2×10 6 cells / mL. 2 REP can be cultured in 400 mL of 50 / 50 medium in a 500 mL gas-permeable silicon-bottomed flask (e.g., G-Rex 100, WilsonWolf Manufacturing, as described elsewhere herein). 6 or 10×10 6 TIL, culture medium supplemented with 3000IU / mL IL-2 and 30ng / mL anti-CD3 antibody (OKT-3). G-Rex100 flasks can be incubated at 37°C, 5% CO2. On the 5th day, 250mL supernatant can be taken out and placed in a centrifuge bottle and centrifuged at 1500rpm (491g) for 10 minutes. The TIL precipitation obtained can be resuspended with 150mL of fresh 50 / 50 culture medium containing 3000IU / mL IL-2 and added back to the G-Rex 100 flask. When TIL is continuously amplified with G-Rex 100 flasks, on the 7th day, the TIL in each G-Rex 100 is suspended in the 300mL culture medium present in each flask, and the cell suspension is divided into three 100mL aliquots, which can be used to inoculate 3 G-Rex 100 flasks. Approximately 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2 can then be added to each flask. The G-Rex 100 flasks can then be incubated at 37°C, 5% CO2, and after 4 days, 150 mL of AIM-V containing 3000 IU / mL IL-2 can be added to each G-Rex 100 flask. Thereafter, the cells can be harvested on day 14 of culture to complete the REP.

[0323] In one embodiment, the method of amplifying or treating cancer includes the step of obtaining TILs from a patient tumor sample. Patient tumor samples can be obtained using methods known in the art. For example, tumor fragments (about 1 to about 8 mm in size) can be obtained from enzymatic tumor digestion fluid and sharp dissection. 3 ) to culture TIL. Such tumor digestion fluid can be produced by incubation in enzyme culture medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2mM glutamate, 10mcg / mL gentamicin, 30U / mL DNase, and 1.0mg / mL collagenase) and then mechanical dissociation (e.g., using a tissue dissociator). Tumor digestion fluid can be produced by placing the tumor in an enzyme culture medium and mechanically dissociating the tumor for about 1 minute, then incubating for 30 minutes at 37°C, 5% CO2, and then performing repeated cycles of mechanical dissociation and incubation under the above conditions until only small tissue fragments are present. At the end of the process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation can be performed using FICOLL branched hydrophilic polysaccharides to remove these cells. Alternative methods known in the art, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1, can be used, and the disclosure thereof is incorporated herein by reference. Any of the foregoing methods can be used in any embodiment of the method for amplifying TIL described herein or the method for treating cancer.

[0324] In one embodiment, the second / REP expansion process of TIL can be performed using a T-175 flask and a gas-permeable bag as described above (Tran et al., J. Immunother. 2008, 31, 742-51; Dudley et al., J. Immunother. 2003, 26, 332-42) or a gas-permeable culture vessel (G-Rex flask, available from Wilson Wolf Manufacturing Corporation, New Brighton, Minnesota, USA). For TIL expansion in a T-175 flask, 1×10 6TILs are added to each T-175 flask. TILs can be cultured in a 1:1 mixture of CM and AFM-V medium supplemented with 3000 IU (international units) / mL of IL-2 and 30 ng / mL of anti-CD3 antibodies (e.g., OKT-3). The T-175 flasks can be incubated at 37 ° C, 5% CO2. Half of the culture medium can be replaced on day 5 with a 50 / 50 culture medium containing 3000 IU / mL IL-2. On day 7, cells from two T-175 flasks are combined in a 3L bag, and 300 mL of AIM V containing 5% human AB serum and 3000 IU / mL IL-2 is added to 300 mL of TIL suspension. The number of cells in each bag is counted every day or every two days, and fresh culture medium is added to keep the cell count at 0.5 × 10 6 Up to 2.0×10 6 cells / mL.

[0325] In one embodiment, for a 100 cm 2 For the second / REP TIL expansion in a 500 mL gas-permeable silicon-bottomed flask (G-Rex100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA), 5 × 10 6 or 10×10 6 TIL, the culture medium is supplemented with 5% human AB serum, 3000IU / mL IL-2 and 30ng / mL anti-CD3 (OKT-3). G-Rex 100 flasks can be incubated at 37°C, 5% CO2. On the 5th day, 250mL of supernatant can be taken out and placed in a centrifuge bottle and centrifuged at 1500rpm (revolutions per minute; 491×g) for 10 minutes. The TIL precipitate can then be resuspended with 150mL of fresh culture medium containing 5% human AB serum, 3000IU / mL IL-2, and added back to the original G-Rex 100 flask. When TIL is continuously amplified in G-Rex 100 flasks, on the 7th day, the TIL in each G-Rex 100 flask is suspended in the 300mL culture medium present in each flask, and the cell suspension can be divided into three 100mL aliquots, which can be used to inoculate three G-Rex 100 flasks. Then, 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2 can be added to each flask. The G-Rex100 flask can be incubated at 37°C, 5% CO2, and after 4 days, 150 mL of AIM-V containing 3000 IU / mL IL-2 can be added to each G-Rex 100 flask. The cells can be harvested on day 14 of culture.

[0326] In one embodiment, TILs can be prepared as follows. Culture in complete medium (CM) for 2 mm 3 Tumor fragments, the CM contained AIM-V medium (Invitrogen Life Technologies, Carlsbad, California), the AIM-V medium supplemented with 2mM glutamine (Mediatech, Inc., Manassas, Virginia), 100U / mL penicillin (Invitrogen Life Technologies), 100μg / mL streptomycin (Invitrogen Life Technologies), 5% heat-inactivated human AB serum (Valley Biomedical, Inc., Winchester, Virginia) and 600IU / mL rhIL-2 (Chiron, Emeryville, California). For enzymatic digestion of liquid tumors, tumor specimens were cut into small pieces and added to RPMI-1640 medium, washed, centrifuged at 800rpm for 5 minutes at 15-22°C, and resuspended in enzymatic digestion buffer (RPMI-1640 medium containing 0.2mg / mL collagenase and 30U / mL DNase), and then rotated overnight at room temperature. TILs generated from the fragments were grown in CM for 3 to 4 weeks and either freshly expanded or cryopreserved in heat-inactivated HAB serum supplemented with 10% dimethyl sulfoxide (DMSO) and stored at −180°C until the time of study. Tumor-associated lymphocytes (TALs) obtained from ascites collections were cultured at 3 × 10 6 Cells / well were seeded in CM in 24-well plates. TIL growth was checked every other day using a low-power inverted microscope.

[0327] Exemplary Embodiment of TIL Preparation Process ("2A Process")

[0328] Fig. 22An exemplary TIL preparation / expansion process, referred to as process 2A, is schematically shown in FIG. In certain aspects, the present method produces TILs that are capable of increasing replication cycles after administration to a subject / patient, and thus the TILs produced by the present method may provide additional therapeutic benefits relative to older TILs (i.e., TILs that have undergone more rounds of replication before administration to a subject / patient). Characteristics of younger TILs have been described in the literature, e.g., Donia et al., Scandinavian Journal of Immunology, 75:157-167 (2012); Dudley et al., Clin Cancer Res, 16:6122-6131 (2010); Huang et al., J Immunother, 28(3):258-267 (2005); Besser et al., Clin Cancer Res, 19(17):OF1-OF9 (2013); Besser et al., J Immunother, 32:415-423 (2009); Bunds et al., J Immunother, 32:415-423 (2009); Robbins et al., J Immunol, 2004; 173:7125-7130; Shen et al., J Immunol, 2004; 174:7125-7130; Shen et al., J Immunol, 2004; 175:7125-7130; Shen et al., J Immunol, 2004; 176:7125-7130; Shen et al., J Immunol, 2004; 177:7125-7130; Shen et al., J Immunol, 2004; 178:7125-7130; Shen et al., J Immunol, 2004; 179:7125-7130; Shen et al., J Immunother, 2005; 180:7125-7130; Shen et al., J Immunother, 2005; 181:7125-7130; Shen et al., J Immunother, 2005 Immunother, 30: 123-129 (2007); Zhou et al., J Immunother, 28: 53-62 (2005) and Tran et al., J Immunother, 31: 742-751 (2008), the entire contents of which are incorporated herein by reference in their entirety.

[0329] As discussed herein, the present invention may include steps related to restimulation of cryopreserved TILs to increase their metabolic activity and thus relative health prior to transplantation into a patient, as well as methods for testing said metabolic health. As generally outlined herein, TILs are typically taken from a patient sample and manipulated to expand their number prior to transplantation into a patient. In some embodiments, TILs may optionally be genetically manipulated as described below.

[0330] In some embodiments, TILs can be cryopreserved. Once thawed, they can also be re-stimulated to increase their metabolic effects before infusion into the patient.

[0331] In some embodiments, as discussed in detail below and in the Examples and Figures, the first amplification (including a process called pre-REP) is shortened to 7 to 14 days, and the second amplification (including a process called REP) is shortened to 7 to 14 days, compared to conventional amplification methods.

[0332] Fig.23 An exemplary 2A process is shown. Fig.23 As shown and described in further detail below, in some embodiments, the first amplification (step B) is shortened to 11 days and the second amplification (step D) is shortened to 11 days. In some embodiments, as discussed in detail herein, the combination of the first amplification and the second amplification (step B and step D) is shortened to 22 days. It is understood that Fig.23 The processes shown and described below are exemplary, and the methods described herein include changes and additions to the steps and any combination thereof. An exemplary embodiment of the method is described in PCT Application No. PCT / US2018 / 012633, the contents of which are incorporated herein by reference in their entirety.

[0333] A. Step A: Obtaining Patient Tumor Samples

[0334] Typically, TILs are initially obtained from patient tumor samples ("primary TILs"), then expanded into larger populations as described herein for further manipulation, optionally cryopreserved as outlined herein, restimulated, and optionally assessed for phenotypic and metabolic parameters as indicators of TIL health.

[0335] Patient tumor samples can be obtained using methods known in the art, usually by surgical resection, puncture biopsy, single sampling or other means for obtaining samples containing tumors and TIL cell mixtures. Generally, tumor samples can be from any solid tumor, including primary tumors, invasive tumors or metastatic tumors. Tumor samples can also be liquid tumors, for example, tumors obtained from hematological malignancies. Solid tumors can be any cancer type, including but not limited to breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, kidney cancer, gastric cancer and skin cancer (including but not limited to squamous cell carcinoma, basal cell carcinoma and melanoma). In some embodiments, useful TIL is obtained from malignant melanoma tumors, because it is reported that these malignant melanoma tumors have a particularly high level of TIL. In some embodiments, the tumor is greater than about 1.5cm but less than about 4cm. In some embodiments, the tumor is less than 4cm.

[0336] Once the tumor sample is obtained, it is usually cut into 1 mm pieces using sharp dissection. 3 To about 8mm 3 Small pieces; among them, about 2mm 3 Up to 3mm 3It is particularly useful. TILs are cultured from these fragments using enzymatic tumor digestion fluid. Such tumor digestion fluid can be produced by incubation in enzyme culture medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2mm glutamate, 10mcg / mL gentamicin, 30U / mL DNase, and 1.0mg / mL collagenase) and then mechanical dissociation (e.g., using a tissue dissociator). Tumor digestion fluid can be produced by placing the tumor in an enzyme culture medium and mechanically dissociating the tumor for about 1 minute, then incubating for 30 minutes at 37°C, 5% CO2, and then performing repeated cycles of mechanical dissociation and incubation under the above conditions until only small tissue fragments are present. At the end of the process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation can be performed using FICOLL branched hydrophilic polysaccharides to remove these cells. Alternative methods known in the art, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1, can be used, and the disclosure thereof is incorporated herein by reference. Any of the foregoing methods can be used in any embodiment of the method of expanding TILs or the method of treating cancer described herein.

[0337] Typically, the harvested cell suspension is referred to as a "primary cell population" or a "freshly harvested" cell population.

[0338] In one embodiment, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from the patient.

[0339] In some embodiments, TILs are obtained from tumor fragments. Tumor fragments are obtained by sharp dissection. In some embodiments, tumor fragments are about 1 mm 3 Up to 10mm 3 In some embodiments, the tumor fragments are about 1 mm 3 Up to 8mm 3 In some embodiments, the tumor fragments are about 1 mm 3 In some embodiments, the tumor fragment is about 2 mm 3 In some embodiments, the tumor fragment is about 3 mm 3 In some embodiments, the tumor fragment is about 4 mm 3 In some embodiments, the tumor fragment is about 5 mm 3 In some embodiments, the tumor fragment is about 6 mm 3 In some embodiments, the tumor fragment is about 7 mm 3 In some embodiments, the tumor fragment is about 8 mm 3 In some embodiments, the tumor fragment is about 9 mm 3 In some embodiments, the tumor fragment is about 10 mm3 In some embodiments, the tumor fragments are about 8-27 mm 3 In some embodiments, the tumor fragments are about 10-25 mm 3 In some embodiments, the tumor fragments are about 15-25 mm 3 In some embodiments, the tumor fragments are about 8-20 mm 3 In some embodiments, the tumor fragment is about 15-20 mm 3 In some embodiments, the tumor fragments are about 8-15 mm 3 In some embodiments, the tumor fragments are about 8-10 mm 3 .

[0340] 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 size of the tumor fragments is about 8-27 mm. 3 And there are less than about 50 tumor fragments.

[0341] In some embodiments, TIL is obtained from tumor digestion fluid. In some embodiments, by incubation in enzyme culture medium (such as but not limited to RPMI 1640, 2mm GlutaMAX, 10mg / mL gentamicin, 30U / mL DNase and 1.0mg / mL collagenase), then mechanical dissociation (GentleMACS, MiltenyiBiotec, Auburn, California), tumor digestion fluid is produced. After the tumor is placed in the enzyme culture medium, the tumor can be mechanically dissociated for about 1 minute. Then the solution is incubated at 37°C, 5% CO2 for 30 minutes, and then mechanically broken again for about 1 minute. After incubating again at 37°C, 5% CO2 for 30 minutes, the tumor can be mechanically broken for the third time for about 1 minute. In some embodiments, after the third mechanical fragmentation, if there is a large piece of tissue, 1 or 2 additional mechanical dissociations are applied to the sample, with or without incubation at 37°C, 5% CO2 for another 30 minutes. In some embodiments, at the end of the final incubation, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using Ficoll can be performed to remove these cells.

[0342] B. Step B: First Amplification

[0343] After dissection or digestion of tumor fragments in step A, the resulting cells are cultured in serum containing IL-2 under conditions that favor the growth of TILs (relative to tumor and other cells). In some embodiments, tumor digests are incubated in 2 mL wells in culture medium containing inactivated human AB serum and 6000 IU / mL IL-2. This primary cell population is cultured for a period of time, typically 3 to 14 days, to generate a large population of TILs, typically about 1×10 8 In some embodiments, the primary cell population is cultured for 7 to 14 days to produce a large number of TIL populations, typically about 1×10 8 In some embodiments, the primary cell population is cultured for 10 to 14 days to produce a large number of TIL populations, typically about 1×10 8 In some embodiments, the primary cell population is cultured for about 11 days to produce a large number of TIL populations, typically about 1×10 8 In some embodiments, the primary cell population is cultured for about 11 days to produce a large number of TIL populations, typically less than or equal to about 200×10 6 A large number of TIL cells.

[0344] In a preferred embodiment, the expansion of TILs can be performed as follows: using an initial bulk TIL expansion step as described below and herein ( Fig.23 The method comprises the steps of: step B as shown in the above, which may include a process called pre-REP), followed by a second expansion as described in step D below and herein (step D, including a process called a rapid expansion protocol (REP) step), followed by optional cryopreservation, followed by a second step D as described below and herein (including a process called a restimulation REP step). Optionally, the TILs obtained from the process may be characterized by phenotypic characteristics and metabolic parameters as described herein.

[0345] In embodiments where TIL culture is initiated using 24-well plates, for example, using Costar 24-well cell culture plates, flat bottom (Corning Incorporated, Corning, NY), each well can be seeded with 1×10 10 TILs in 2 mL of complete medium (CM) containing IL-2 (6000 IU / mL; Chiron Corp., Emeryville, CA). 6 In some embodiments, the tumor fragment is about 1 mm 3 Up to 10mm 3 .

[0346] In some embodiments, the CM of step B consists of RPMI 1640 containing GlutaMAX, supplemented with 10% human AB serum, 25 mm HEPES, and 10 mg / mL gentamicin. 2 In one embodiment, the culture is started in a gas-permeable flask (e.g., G-RE×10; Wilson Wolf Manufacturing, New Brighton, Minnesota) ( Figure 1 ), each flask contained 10×10 6 Up to 40×10 6 Live tumor digested cells or 5 to 30 tumor fragments were incubated in a humidified incubator at 37°C and 5% CO2 for 10-24 well plates. After 5 days of culture, half of the medium was removed and replaced with fresh CM and IL-2. After the 5th day, half of the medium was replaced every 2 to 3 days.

[0347] In one embodiment, the cell culture medium also comprises IL-2. In a preferred embodiment, the cell culture medium comprises about 3000 IU / mL of IL-2. In one embodiment, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL or about 8000 IU / mL of IL-2. In one embodiment, the cell culture medium comprises 1000 to 2000 IU / mL, 2000 to 3000 IU / mL, 3000 to 4000 IU / mL, 4000 to 5000 IU / mL, 5000 to 6000 IU / mL, 6000 to 7000 IU / mL, 7000 to 8000 IU / mL, or 8000 IU / mL of IL-2.

[0348] In some embodiments, as discussed in the Examples and Figures, the first amplification (including a process called pre-REP; step B) is shortened to 3 to 14 days. Figure 4 and Figure 5 In some embodiments, as discussed in the examples, the first amplification of step B is shortened to 7 to 14 days. In some embodiments, as discussed in the examples, the first amplification of step B is shortened to 10 to 14 days. In some embodiments, as discussed in the examples, the first amplification of step B is shortened to 11 days.

[0349] 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.

[0350] In some embodiments, step B is performed in a closed system bioreactor. In some embodiments, as described herein, a closed system is used to expand TILs. In some embodiments, a single bioreactor is used. In some embodiments, for example, the single bioreactor used is a G-REX-10 or G-REX-100.

[0351] C. Step C: Transition from the first amplification to the second amplification

[0352] In some embodiments, the bulk TIL population from step B can be immediately cryopreserved using methods known in the art and described herein. Alternatively, the bulk TIL population can be subjected to a second expansion (REP) and then cryopreserved as described below.

[0353] In some embodiments, the TIL from step B is not stored and the TIL from step B is directly subjected to step D. In some embodiments, the transforming occurs in a closed system, as further described herein.

[0354] D. Step D: Second Amplification

[0355] In some embodiments, after harvesting and initial bulk processes (i.e., after step A and step B), the TIL cell population is expanded in number. This is referred to herein as the second expansion, which may include an expansion process commonly referred to in the art as a rapid expansion process (REP). Typically, in a gas permeable container, a culture medium comprising several components (including feeder cells, a cytokine source, and an anti-CD3 antibody) is used to complete the second expansion. In some embodiments, the second expansion may include scaling-up to increase the number of TILs obtained from the second expansion.

[0356] In one embodiment, REP and / or secondary expansion can be performed in a gas permeable container using the methods of the present disclosure. For example, TILs can be rapidly expanded using non-specific T cell receptor stimulation in the presence of interleukin 2 (IL-2) or interleukin-15 (IL-15). Non-specific T cell receptor stimulation can include, for example, about 30 ng / mL of OKT-3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, New Jersey or Miltenyi Biotech, Auburn, California). TILs can be rapidly expanded by further stimulating TILs in vitro with one or more cancer antigens (including antigenic portions thereof, such as epitopes), optionally in the presence of T cell growth factors such as 300 IU / mL IL-2 or IL-15, which antigens can be optionally expressed by a vector, such as human leukocyte antigen A2 (HLA-A2) binding peptides, such as 0.3 μM MART-L26-35 (27L) or gp 100: 209-217 (210M). Other suitable antigens may include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigens, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. TILs can also be rapidly expanded by re-stimulating with the same cancer antigen pulsed with antigen presenting cells expressing HLA-A2. Alternatively, TILs can be further re-stimulated with, for example, irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0357] In one embodiment, the cell culture medium also comprises IL-2. In a preferred embodiment, the cell culture medium comprises about 3000 IU / mL of IL-2. In one embodiment, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In one embodiment, the cell culture medium comprises 1000 to 2000 IU / mL, 2000 to 3000 IU / mL, 3000 to 4000 IU / mL, 4000 to 5000 IU / mL, 5000 to 6000 IU / mL, 6000 to 7000 IU / mL, 7000 to 8000 IU / mL, or 8000 IU / mL of IL-2.

[0358] In one embodiment, the cell culture medium comprises the OKT3 antibody. In a preferred embodiment, the cell culture medium comprises about 30 ng / mL of the OKT3 antibody. In one embodiment, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, and about 1 μg / mL of the OKT3 antibody. In one embodiment, the cell culture medium comprises 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL, 50 ng / mL to 100 ng / mL of OKT3 antibody.

[0359] In some embodiments, IL-2, IL-7, IL-15, and IL-21, and combinations thereof, may be included during the second expansion of the Step D process as described herein.

[0360] In some embodiments, the second expansion can be performed in a supplemented cell culture medium comprising IL-2, OKT-3, and antigen presenting feeder cells.

[0361] In some embodiments, antigen presenting feeder cells (APC) are PBMCs. In one embodiment, in rapid expansion and / or the second expansion, the ratio of TIL to PBMC and / or antigen presenting cells 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, in rapid expansion and / or the second expansion, the ratio of TIL to PBMC is 1:50 to 1:300. In one embodiment, in rapid expansion and / or the second expansion, the ratio of TIL to PBMC is 1:100 to 1:200.

[0362] In one embodiment, REP and / or secondary expansion are performed in flasks, where a large number of TILs are mixed with 100 or 200-fold excess inactivated feeder cells, 30 mg / mL of OKT3 anti-CD3 antibodies, and 3000 IU / mL of IL-2 in 150 mL of culture medium. Culture medium replacement is performed (usually by replacing 2 / 3 of the culture medium with aspiration of fresh culture medium) until the cells are transferred to an alternative growth chamber. As discussed more fully below, the alternative growth chamber includes a G-REX flask and a gas permeable container.

[0363] In some embodiments, as discussed in the examples and figures, the second expansion (also referred to as the REP process) is shortened to 7 to 14 days. In some embodiments, the second expansion is shortened to 11 days.

[0364] In one embodiment, REP and / or secondary amplification can be performed using T-175 flasks and gas permeable bags (Tran et al., J Immunother., 2008, 31, 742-51; Dudley et al., J Immunother., 2003, 26, 332-42) or gas permeable culture vessels (G-REX flasks) as described above. For rapid amplification and / or secondary amplification in T-175 flasks, 1×10 6 TILs can be added to each T-175 flask. TILs can be cultured in a 1:1 mixture of CM and AFM-V medium supplemented with 3000 IU / mL IL-2 and 30 ng / mL anti-CD3. The T-175 flasks can be incubated at 37°C, 5% CO2. On day 5, half of the medium can be replaced with 50 / 50 medium containing 3000 IU / mL IL-2. On day 7, cells from two T-175 flasks can be combined in a 3L bag, and 300 mL of AIM V containing 5% human AB serum and 3000 IU / mL IL-2 can be added to the 300 mL TIL suspension. The number of cells in each bag is counted every day or every 2 days, and fresh medium is added to maintain the cell count at 0.5×10 6 Up to 2.0×10 6 cells / mL.

[0365] In one embodiment, the volume can be 500 mL and the air permeable silicon bottom is 100 cm 2 REP and / or secondary expansion were performed in gas-permeable flasks (G-REX 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA), 5 × 10 6 or 10×10 6Each TIL can be cultured with PBMC in 400 mL of 50 / 50 culture medium supplemented with 5% human AB serum, 3000 IU / mL IL-2, and 30 ng / mL anti-CD3 (OKT3). The G-REX100 flask can be incubated at 37°C, 5% CO2. On day 5, 250 mL of supernatant can be removed and placed in a centrifuge bottle and centrifuged at 1500 rpm (491 × g) for 10 minutes. The TIL pellet can be resuspended with 150 mL of fresh culture medium containing 5% human AB serum, 3000 IU / mL IL-2 and added back to the original G-REX 100 flask. When TIL is continuously expanded in G-REX 100 flasks, on the 7th day, the TIL in each G-REX 100 can be suspended in 300mL culture medium present in each flask, and the cell suspension can be divided into 3 100mL aliquots that can be used to inoculate 3 G-REX 100 flasks. Then 150mL of AIM-V containing 5% human AB serum and 3000IU / mL IL-2 can be added to each flask. G-REX 100 flasks can be incubated at 37°C, 5% CO2, and after 4 days, 150mL of AIM-V containing 3000IU / mL IL-2 can be added to each G-REX 100 flask. Cells can be harvested on the 14th day of culture.

[0366] In one embodiment, REP and / or secondary expansion are performed in flasks, where a large number of TILs are mixed with 100 or 200-fold excess inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibodies, and 3000 IU / mL IL-2 in 150 mL of culture medium. Culture medium replacement is performed (usually by replacing 2 / 3 of the culture medium with aspiration of fresh culture medium) until the cells are transferred to an alternative growth chamber. As discussed more fully below, alternative growth chambers include GRex flasks and gas permeable containers.

[0367] In one embodiment, performing REP and / or a second amplification further includes the step of selecting TILs with excellent tumor reactivity. Any selection method known in the art can be used. For example, the method described in U.S. Patent Application Publication No. 2016 / 0010058A1 (the disclosure of which is incorporated herein by reference) can be used to select TILs with excellent tumor reactivity.

[0368] REP and / or secondary expansion of TILs can be performed using T-175 flasks and gas-permeable bags as described previously (Tran KQ, Zhou J, Durflinger KH, et al., 2008, J. Immunother., 31:742-751 and Dudley ME, Wunderlich JR, Shelton TE, et al., 2003, J. Immunother., 26:332-342) or gas-permeable G-REX flasks. In some embodiments, REP and / or secondary expansion are performed using flasks. In some embodiments, REP and / or secondary expansion are performed using gas-permeable G-Rex flasks. For REP and / or secondary expansion of TILs in T-175 flasks, approximately 1×10 6 Each TIL is suspended in about 150mL culture medium, which is added to each T-175 flask. TIL is cultured with irradiated (50Gy) allogeneic PBMC (as "feeder" cells) at a ratio of 1: 100, and the cells are cultured in a 1: 1 mixture (50 / 50 culture medium) of CM and AIM-V culture medium, and the culture medium is supplemented with 3000IU / mL of IL-2 and 30ng / mL of anti-CD3. T-175 flasks are incubated at 37°C, 5% CO2. In some embodiments, at the 5th day, half of the culture medium is replaced with a 50 / 50 culture medium containing 3000IU / mL IL-2. In some embodiments, at the 7th day, cells from 2 T-175 flasks are merged in a 3L bag, and 300mL of AIM-V containing 5% human AB serum and 3000IU / mL IL-2 is added to 300mL TIL suspension. The number of cells in each bag can be counted daily or every 2 days, and fresh medium can be added to maintain the cell count at approximately 0.5 x 10 6 About 2.0×10 6 cells / mL.

[0369] For a 100cm 2 For TIL REP and / or secondary expansion, approximately 5×10 6 or 10×10 6Each TIL is cultured with irradiated allogeneic PBMC at a ratio of 1:100 in 400 mL of 50 / 50 culture medium supplemented with 3000 IU / mL of IL-2 and 30 ng / mL of anti-CD3. The G-REX 100 flask is incubated at 37°C, 5% CO2. In some embodiments, on day 5, 250 mL of supernatant is removed and placed in a centrifuge bottle and centrifuged at 1500 rpm (491 g) for 10 minutes. The TIL pellet can then be resuspended with 150 mL of fresh 50 / 50 culture medium containing 3000 IU / mL of IL-2 and added back to the original G-REX 100 flask. In the embodiment of TIL continuous expansion in G-REX 100 flasks, at the 7th day, the TIL in each G-REX 100 is suspended in the 300mL culture medium present in each flask, and the cell suspension is divided into 3 100mL aliquots for inoculating 3 G-REX 100 flasks. Then 150mL of AIM-V containing 5% human AB serum and 3000IU / mL IL-2 is added to each flask. The G-REX100 flask is incubated at 37°C, 5% CO2, and after 4 days, 150mL of AIM-V containing 3000IU / mL IL-2 is added to each G-REX 100 flask. Cells are harvested on the 14th day of culture.

[0370] 1. Feeder cells and antigen presenting cells

[0371] In one embodiment, during the REP TIL expansion and / or during the second expansion, the second expansion procedure described herein (step D, including REP) requires an excess of feeder cells. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of healthy donors. PBMCs are obtained using standard methods (such as Ficoll-Paque gradient separation).

[0372] Typically, allogeneic PBMCs are inactivated by irradiation or heat treatment and used in the REP procedure, as described in the Examples, particularly Example 14, which provides an exemplary protocol for assessing replication incompetence in irradiated allogeneic PBMCs.

[0373] In some embodiments, if the total number of viable cells on day 14 is less than the initial number of viable cells put into culture on day 0 of REP and / or day 0 of the second expansion (i.e., the start date of the second expansion), the PBMCs are considered to be replication incompetent and the PBMCs are accepted for the TIL expansion step described herein.

[0374] In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 does not increase on days 7 and 14 compared to the initial number of viable cells put into culture on day 0 of REP and / or day 0 of the second expansion (i.e., the start date of the second expansion), the PBMCs are considered to be replicatively incompetent and the PBMCs are accepted for the TIL expansion step described herein. In some embodiments, PBMCs are cultured in the presence of 30 ng / mL OKT3 antibody and 3000 IU / mL IL-2.

[0375] In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 does not increase on days 7 and 14 compared to the initial number of viable cells put into culture on day 0 of REP and / or day 0 of the second expansion (i.e., the start date of the second expansion), the PBMC replication function is considered to be incomplete, and the PBMC is accepted for the TIL expansion step described herein. In some embodiments, PBMCs are cultured in the presence of 5-60ng / mL OKT3 antibodies and 1000-6000IU / mL IL-2. In some embodiments, PBMCs are cultured in the presence of 10-50ng / mL OKT3 antibodies and 2000-5000IU / mL IL-2. In some embodiments, PBMCs are cultured in the presence of 20-40ng / mL OKT3 antibodies and 2000-4000IU / mL IL-2. In some embodiments, PBMCs are cultured in the presence of 25-35 ng / mL OKT3 antibody and 2500-3500 IU / mL IL-2.

[0376] In one embodiment, artificial antigen presenting cells are used as a substitute for or in combination with PBMCs during the REP phase.

[0377] 2. Cytokines

[0378] As is known in the art, the expansion methods described herein typically use culture medium containing high doses of cytokines, particularly IL-2.

[0379] Alternatively, a combination of cytokines can be used in addition to carry out rapid expansion and / or a second expansion of TIL;Wherein, the combination of two or more of IL-2, IL-15 and IL-21 is as summarized in U.S. Patent Application Publication No. US 2017 / 0107490 A1, International Publication No. WO 2015 / 189356, U.S. Patent Application Publication No. US 2017 / 0107490 A1 and International Publication No. WO2015 / 189357, and its respective full contents are expressly incorporated herein by reference. Therefore, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15 and IL-21, and it is found that the latter is particularly useful in many embodiments. The combination of cytokines is particularly beneficial to the production of lymphocytes (particularly T cells as described herein).

[0380] 3. Anti-CD3 Antibody

[0381] In some embodiments, the culture medium used in the expansion methods described herein (including REP) also contains anti-CD3 antibodies. Anti-CD3 antibodies combined with IL-2 induce T cell activation and cell division of TIL populations. This effect can be seen with full-length antibodies as well as Fab and F(ab')2 fragments, the former being generally preferred; for example, see Tsoukas et al., J Immunol., 1985, 135, 1719, the entire contents of which are incorporated herein by reference.

[0382] As will be appreciated by those skilled in the art, there are many suitable anti-human CD3 antibodies that can be used in the present invention, including anti-human CD3 polyclonal and monoclonal antibodies from various mammals, including but not limited to murine, human, primate, rat and canine antibodies. In a specific embodiment, the OKT3 anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, New Jersey or Miltenyi Biotech, Auburn, California) is used.

[0383] E. Step E: Harvesting TILs

[0384] After the second expansion step, cells can be harvested. In some embodiments, TILs are harvested after 1, 2, 3, 4 or more second expansion steps.

[0385] TIL can be harvested in any appropriate and aseptic manner, including, for example, by centrifugation. The method of harvesting TIL is well known in the art, and any such known method can be used together with the inventive method. In some embodiments, TIL is harvested using an automated system. In some embodiments, TIL is a harvest using a semi-automatic system. In some embodiments, TIL is harvested using a semi-automatic system. In some embodiments, TIL is harvested using a semi-automatic system. In some embodiments, the TIL amplified for the second time is harvested using a semi-automatic machine. In some embodiments, a LOVO system (for example, commercially available from Benchmark Electronics) is used. In some embodiments, the harvesting step includes washing TIL, preparing TIL and / or aliquoting TIL. In some embodiments, optionally, cells are frozen after harvesting or as a part of harvesting.

[0386] F. Step F: Final Formulation / Transfer to Infusion Bag

[0387] After steps A through E are completed, the cells are transferred to a container for administration to a patient.

[0388] In one embodiment, TILs expanded using APCs of the present disclosure are administered to patients as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded using PBMCs of the present disclosure can be administered by any suitable route known in the art. In one embodiment, T cells are administered in a single intra-arterial or intravenous infusion, and the infusion preferably lasts for about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic.

[0389] G. Additional Amplification Steps

[0390] It will be appreciated that any of the above steps A to F may be repeated any number of times and may additionally be performed in an order different from that described above.

[0391] In some embodiments, more than one amplification step can be repeated before the final formulation step F. These additional amplification steps can include elements of the first amplification and / or second amplification steps described above (e.g., including components described in the cell culture medium). Additional amplification steps can further include additional elements, including additional components in the cell culture medium, which are supplemented to the cell culture medium before and / or during the additional amplification steps.

[0392] In other embodiments, Fig.23 Any expansion step described in the above paragraphs may be preceded or followed by a cryopreservation step in which the cells produced in the expansion step are preserved using storage methods known in the art until needed for the remaining steps of the preparation / expansion process.

[0393] Pharmaceutical compositions, dosages and administration regimens of TIL, MIL and PBL

[0394] In one embodiment, the TIL amplified using the disclosed method is administered to the patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a TIL suspension in a sterile buffer. The TIL amplified using the disclosed method can be administered by any suitable route known in the art. Preferably, the TIL is administered in the form of a single intra-arterial infusion or intravenous infusion, and the infusion preferably lasts for about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

[0395] Any suitable dose of TILs may be administered. Preferably, particularly when the cancer is a hematological malignancy, about 2.3×10 10 to about 13.7×10 10 TILs, with an average of about 7.8×10 10 In one embodiment, about 1.2×10 10 to about 4.3×10 10 TILs.

[0396] 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 , 3×109 , 4×10 9 , 5×10 9 , 6×10 9 ,7×10 9 , 8×10 9 ,9×10 9 , 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 ,7×10 10 , 8×10 10 ,9×10 10 , 1×10 11 , 2×10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 ,7×10 11 , 8×10 11 ,9×10 11 , 1×10 12 , 2×10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 ,7×10 12 , 8×10 12 ,9×10 12 , 1×10 13 , 2×10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 ,7×10 13 , 8×10 13 , and 9×10 13 In one embodiment, the number of TILs provided in the pharmaceutical composition of the present invention is 1×10 6 Up to 5×10 6 , 5×10 6 Up to 1×10 7 , 1×10 7 Up to 5×10 7 , 5×10 7 Up to 1×10 8 , 1×10 8 Up to 5×10 8 , 5×10 8 Up to 1×109 , 1×10 9 Up to 5×10 9 , 5×10 9 Up to 1×10 10 , 1×10 10 Up to 5×10 10 , 5×10 10 Up to 1×10 11 , 5×10 11 Up to 1×10 12 , 1×10 12 Up to 5×10 12 , and 5×10 12 Up to 1×10 13 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 9 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 .

[0397] In one embodiment of the present invention, the amount of TIL provided in the pharmaceutical composition of the present invention is about 0.1×10 9 About 15×10 9 TIL, about 0.1×10 9 About 15×10 9 TIL, about 0.12×10 9 About 12×10 9 TIL, about 0.15×10 9 About 11×10 9 TIL, about 0.2×10 9 to about 10×10 9 TIL, about 0.3×10 9 About 9×10 9 TIL, about 0.4×10 9 About 8×10 9 TIL, about 0.5×10 9 About 7×10 9 TIL, about 0.6×10 9 About 6×10 9 TIL, about 0.7×10 9 About 5×10 9TIL, about 0.8×10 9 About 4×10 9 TIL, about 0.9×10 9 About 3×10 9 TIL or about 1×10 9 About 2×10 9 TILs.

[0398] In some embodiments, the concentration of TIL provided in the pharmaceutical composition of the present invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 0.10%, 0.11%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.30%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.30%, 0.31%, 0.32%, 0.33%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.30%, 0.31 ...3%, 0.33%, 0.33%, 0.33%, 0.33%, 0.33%, 0.33%, 0.33%, 0.33%, 0.33%, 0.33%, 0.33%, %. %, 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.

[0399] In some embodiments, the concentration of TIL provided in the pharmaceutical composition of the present invention is greater 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 ... 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.

[0400] In some embodiments, the concentration of TIL provided in the pharmaceutical composition of the present invention ranges from 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 26%, about 0.08% to about 27%, about 0.09% to about 28%, about 0.10% to about 29%, about 0.11% to about 29%, about 0.12% to about 29%, about 0.13% to about 29%, about 0.14% to about 29%, about 0.15% to about 29%, about 0.16% to about 29%, about 0.17% to about 29%, about 0.18% to about 29%, about 0.19% to about 29%, about 0.19% to about 29%, about 0.11% to about 29%, about 0.12% to about 29%, about 0.13% to about 29%, about 0.14% to about 29%, 4%, 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.

[0401] In some embodiments, the concentration of TIL provided in the pharmaceutical composition of the present invention ranges from 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.

[0402] In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is equal to or less than 10g, 9.5g, 9.0g, 8.5g, 8.0g, 7.5g, 7.0g, 6.5g, 6.0g, 5.5g, 5.0g, 4.5g, 4.0g, 3.5g, 3.0g, 2.5g, 2.0g, 1.5g, 1.0g, 0.95g, 0.9g, 0.85g, 0.8g, 0.75g, 0.7g, 0.65g, 0.6g, 0.55g, 0.5g, 0.45g, 0.4g, 0.35g, 0.3g, 0.25 g, 0.2g, 0.15g, 0.1g, 0.09g, 0.08g, 0.07g, 0.06g, 0.05g, 0.04g, 0.03g, 0.02g, 0.01g, 0.009g, 0.008g, 0.007g, 0.006g, 0.0 05g, 0.004g, 0.003g, 0.002g, 0.001g, 0.0009g, 0.0008g, 0.0007g, 0.0006g, 0.0005g, 0.0004g, 0.0003g, 0.0002g or 0.0001g.

[0403] In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is greater 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, 0.0036 g, 0.0037 g, 0.0038 g, 0.0039 g, 0.010 g, 0.011 g, 0.012 g, 0.013 g, 0.014 g, 0.015 g, 0.016 g, 0.017 g, 0.018 g, 0.019 g, 0. .003g, 0.0035g, 0.004g, 0.0045g, 0.005g, 0.0055g, 0.006g, 0.0065g, 0.007g, 0.0075g, 0.008g, 0.0085g, 0.009g, 0.0095g, 0.01g, 0.015g, 0.02g, 0.025g, 0. 03g, 0.035g, 0.04g, 0.045g, 0.05g, 0.055g, 0.06g, 0.065g, 0.07g, 0.075g, 0.0 8g, 0.085g, 0.09g, 0.095g, 0.1g, 0.15g, 0.2g, 0.25g, 0.3g, 0.35g, 0.4g, 0.45g , 0.5g, 0.55g, 0.6g, 0.65g, 0.7g, 0.75g, 0.8g, 0.85g, 0.9g, 0.95g, 1g, 1.5g, 2g , 2.5g, 3g, 3.5g, 4g, 4.5g, 5g, 5.5g, 6g, 6.5g, 7g, 7.5g, 8g, 8.5g, 9g, 9.5g or 10g.

[0404] The TIL provided in the pharmaceutical composition of the present invention is effective in a wide dosage range. The exact dosage depends on the route of administration, the form of the compound to be administered, the sex and age of the subject to be treated, the weight of the subject to be treated, and the preference and experience of the attending physician. If appropriate, clinically determined TIL dosages may also be used. The amount of the pharmaceutical composition administered using the methods herein, such as the dosage of TIL, will depend on the severity of the person or mammal being treated, the condition or condition, the rate of administration, the configuration of the active pharmaceutical ingredient, and the discretion of the prescribing physician.

[0405] In some embodiments, TIL can be administered in a single dose. Such administration can be by injection, such as intravenous injection. In some embodiments, TIL can be administered in multiple doses. The dosage can be once, twice, three times, four times, five times, six times, or more than six times per year. The dosage can be once a month, once every two weeks, once a week, or once every 2 days. As long as necessary, the administration of TIL can continue.

[0406] In some embodiments, the effective dose of TIL is about 1×10 6 , 2×10 6 , 3×10 6 , 4×106 、5×10 6 、6×10 6 、7×10 6 、8×10 6 、9×10 6 、1×10 7 、2×10 7 、3×10 7 、4×10 7 、5×10 7 、6×10 7 、7×10 7 、8×10 7 、9×10 7 、1×10 8 、2×10 8 、3×10 8 、4×10 8 、5×10 8 、6×10 8 、7×10 8 、8×10 8 、9×10 8 、1×10 9 、2×10 9 、3×10 9 、4×10 9 、5×10 9 、6×10 9 、7×10 9 、8×10 9 、9×10 9 、1×10 10 、2×10 10 、3×10 10 、4×10 10 、5×10 10 、6×10 10 、7×10 10 、8×10 10 、9×10 10 、1×10 11 、2×10 11 、3×10 11 、4×10 11 、5×10 11 、6×10 11 、7×10 11 、8×10 11 、9×10 11 、1×10 12 、2×10 12 、3×10 12 、4×10 12 、5×10 12, 6×10 12 ,7×10 12 , 8×10 12 ,9×10 12 , 1×10 13 , 2×10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 ,7×10 13 , 8×10 13 , and 9×10 13 In some embodiments, the effective dose of TIL is in the range of 1×10 6 Up to 5×10 6 , 5×10 6 Up to 1×10 7 , 1×10 7 Up to 5×10 7 , 5×10 7 Up to 1×10 8 , 1×10 8 Up to 5×10 8 , 5×10 8 Up to 1×10 9 , 1×10 9 Up to 5×10 9 , 5×10 9 Up to 1×10 10 , 1×10 10 Up to 5×10 10 , 5×10 10 Up to 1×10 11 , 5×10 11 Up to 1×10 12 , 1×10 12 Up to 5×10 12 , and 5×10 12 Up to 1×10 13 .

[0407] In one embodiment of the present invention, the clinical dose of MIL useful for acute myeloid leukemia (AML) patients is about 4×10 8 About 2.5×10 9 In another embodiment, the number of MILs provided in the pharmaceutical composition of the present invention is 9.5×10 8 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 amount of MIL provided in the pharmaceutical composition of the present invention is 2.2×10 9 .

[0408] In some embodiments, the effective dose of TIL ranges from about 0.01 mg / kg to about 4.3 mg / kg, about 0.15 mg / kg to about 3.6 mg / kg, about 0.3 mg / kg to about 3.2 mg / kg, about 0.35 mg / kg to about 2.85 mg / kg, about 0.15 mg / kg to about 2.85 mg / kg, about 0.3 mg to about 2.15 mg / kg, about 0.45 mg / kg to about 1.7 mg / kg, about 0.15 mg / kg to about 1.3 mg / kg, about 0.3 mg / kg to about 1.15 mg / kg, about 0.45 mg / kg to about 1 mg / kg, about 0.55 mg / kg to about 0.85 mg / kg, about 0.65 mg / kg to about 0.8 mg / kg, about 0.75 mg / kg to about 1.6 mg / kg. / kg to about 0.75mg / kg, about 0.7mg / kg to about 2.15mg / kg, about 0.85mg / kg to about 2mg / kg, about 1mg / kg to about 1.85mg / kg, about 1.15mg / kg to about 1.7mg / kg, about 1.3mg / kg to about 1.6mg / kg, about 1.35mg / kg to about 1.5mg / kg, about 2.15mg / kg to about 3.6mg / kg, about 2.3mg / kg to about 3.4mg / kg, about 2.4mg / kg to about 3.3mg / kg, about 2.6mg / kg to about 3.15mg / kg, about 2.7mg / kg to about 3mg / kg, about 2.8mg / kg to about 3mg / kg, or about 2.85mg / kg to about 2.95mg / kg.

[0409] In some embodiments, the effective dose of TIL ranges from about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg, about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 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 From about 150 mg to about 250 mg, from about 160 mg to about 240 mg, from about 170 mg to about 230 mg, from about 180 mg to about 220 mg, from about 190 mg to about 210 mg, from about 195 mg to about 205 mg, or from about 198 to about 207 mg.

[0410] An effective amount of TILs can be administered in single or multiple doses by any accepted mode of administration for agents with similar utilities, including intranasal and transdermal routes, by intraarterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, topically, by implantation or injection directly into a tumor or by inhalation.

[0411] Methods of treating cancer

[0412] The compositions and combinations of the above TILs, PBLs and / or MILs (and their groups) can be used in methods for treating hyperproliferative diseases. In a preferred embodiment, they are used to treat cancer. In a preferred embodiment, the 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 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's lymphoma (NHL), Hodgkin's lymphoma, relapsed and / or refractory Hodgkin's lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt's lymphoma, Waldenstrom's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndrome, myelofibrosis, chronic myeloid leukemia, follicle-centered lymphoma, indolent NHL, human immunodeficiency virus (HIV)-associated B-cell lymphoma, and Epstein-Barr virus (EBV)-associated B-cell lymphoma.

[0413] In one embodiment, the present invention provides a method of treating cancer, wherein the cancer is a hematological malignancy responsive to treatment with a PD-1 and / or PD-L1 inhibitor, including pembrolizumab, nivolumab, durvalumab, avelumab, or atezolizumab.

[0414] In one embodiment, the present invention provides a method for treating cancer using a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

[0415] (a) obtaining a tumor from a patient by resection, biopsy, needle aspiration or apheresis, wherein the tumor comprises a first TIL population;

[0416] (b) optionally, fragmenting or dissociating the tumor, obtaining tumor fragments, and contacting the tumor fragments with a first cell culture medium;

[0417] (c) initially expanding the first TIL population in a first cell culture medium to obtain a second TIL population; wherein the number of the second TIL population is at least 5 times higher than the number of the first TIL population; wherein the first cell culture medium contains IL-2;

[0418] (d) amplifying the second TIL population for a second time in a second cell culture medium to obtain a third TIL population; wherein, 7 days after the start of the second amplification, the number of the third TIL population is at least 50 times higher than the number of the second TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC); and wherein the second amplification is performed for less than 14 days;

[0419] (e) harvesting a third TIL population; and

[0420] (f) administering a therapeutically effective amount of the third TIL population to the cancer patient;

[0421] wherein the tumor is a liquid tumor; and wherein the cancer is a hematological malignancy.

[0422] In one embodiment, the present invention provides a method for treating cancer using a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

[0423] (a) obtaining a tumor from a patient by resection, biopsy, needle aspiration or apheresis, wherein the tumor comprises a first TIL population;

[0424] (b) optionally, fragmenting or dissociating the tumor, obtaining tumor fragments, and contacting the tumor fragments with a first cell culture medium;

[0425] (c) initially expanding the first TIL population in a first cell culture medium to obtain a second TIL population; wherein the number of the second TIL population is at least 5 times higher than the number of the first TIL population; wherein the first cell culture medium contains IL-2;

[0426] (d) amplifying the second TIL population for a second time in a second cell culture medium to obtain a third TIL population; wherein, 7 days after the start of the second amplification, the number of the third TIL population is at least 50 times higher than the number of the second TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC); and wherein the second amplification is performed for less than 14 days;

[0427] (e) harvesting a third TIL population; and

[0428] (f) administering a therapeutically effective amount of the third TIL population to the cancer patient;

[0429] wherein the tumor is a liquid tumor; and wherein the cancer 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's lymphoma, Waldenstrom's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndrome, myelofibrosis, chronic myeloid leukemia, follicle-center lymphoma, indolent NHL, human immunodeficiency virus (HIV)-associated B-cell lymphoma, and Epstein-Barr virus (EBV)-associated B-cell lymphoma.

[0430] In one embodiment, the present invention provides a method for treating cancer using a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

[0431] (a) the patient was pretreated with a regimen containing a kinase inhibitor or an ITK inhibitor;

[0432] (b) obtaining a tumor from a patient by resection, biopsy, needle aspiration or apheresis, wherein the tumor comprises a first TIL population;

[0433] (c) optionally, fragmenting or dissociating the tumor, obtaining tumor fragments, and contacting the tumor fragments with a first cell culture medium;

[0434] (d) initially expanding the first TIL population in a first cell culture medium to obtain a second TIL population; wherein the number of the second TIL population is at least 5 times higher than the number of the first TIL population; wherein the first cell culture medium contains IL-2;

[0435] (e) amplifying the second TIL population for a second time in a second cell culture medium to obtain a third TIL population; wherein, 7 days after the start of the second expansion, the number of the third TIL population is at least 50 times higher than the number of the second TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC); and wherein the second expansion is performed for less than 14 days;

[0436] (f) harvesting a third TIL population; and

[0437] (g) administering a therapeutically effective amount of the third TIL population to the cancer patient;

[0438] wherein the tumor is a liquid tumor; and wherein the cancer is a hematological malignancy.

[0439] In one embodiment, the present invention provides a method for treating cancer using a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:

[0440] (a) the patient was pretreated with a regimen containing a kinase inhibitor or an ITK inhibitor;

[0441] (b) obtaining a tumor from a patient by resection, biopsy, needle aspiration or apheresis, wherein the tumor comprises a first TIL population;

[0442] (c) optionally, fragmenting or dissociating the tumor, obtaining tumor fragments, and contacting the tumor fragments with a first cell culture medium;

[0443] (d) initially expanding the first TIL population in a first cell culture medium to obtain a second TIL population; wherein the number of the second TIL population is at least 5 times higher than the number of the first TIL population; wherein the first cell culture medium contains IL-2;

[0444] (e) amplifying the second TIL population for a second time in a second cell culture medium to obtain a third TIL population; wherein, 7 days after the start of the second expansion, the number of the third TIL population is at least 50 times higher than the number of the second TIL population; wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC); and wherein the second expansion is performed for less than 14 days;

[0445] (f) harvesting a third TIL population; and

[0446] (g) administering a therapeutically effective amount of the third TIL population to the cancer patient;

[0447] wherein the tumor is a liquid tumor; and wherein the cancer 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's lymphoma, Waldenstrom's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndrome, myelofibrosis, chronic myeloid leukemia, follicle-center lymphoma, indolent NHL, human immunodeficiency virus (HIV)-associated B-cell lymphoma, and Epstein-Barr virus (EBV)-associated B-cell lymphoma.

[0448] In one embodiment of the invention, TILs are expanded using MIL method 1 and administered to a patient according to the present invention.

[0449] In one embodiment of the invention, TILs are expanded using MIL method 2 and administered to a patient to treat cancer according to the present invention.

[0450] In one embodiment of the invention, TILs are expanded using MIL method 3 and administered to a patient to treat cancer according to the present invention.

[0451] In one embodiment of the invention, according to the present invention, TILs expanded using MIL Method 1, MIL Method 2 or MIL Method 3 are administered to a patient to treat AML.

[0452] In one embodiment of the invention, TILs are expanded using PBL method 2 and administered to a patient to treat cancer according to the present invention.

[0453] In one embodiment of the invention, TILs are expanded using PBL method 2 and administered to a patient to treat cancer according to the present invention.

[0454] In one embodiment of the invention, TILs are expanded using PBL method 2 and administered to a patient to treat cancer according to the present invention.

[0455] In one embodiment of the invention, according to the present invention, TILs expanded using PBL method 1, PBL method 2 or PBL method 3 are administered to a patient to treat CLL.

[0456] In any of the foregoing embodiments of the invention, pretreatment with a kinase inhibitor is described. In one embodiment, the kinase inhibitor is selected from: imatinib, dasatinib, ibrutinib, bosutinib, nilotinib, erlotinib or other kinase inhibitors known in the art, tyrosine kinase inhibitors or serine / threonine kinase inhibitors. In one embodiment, the pretreatment regimen with a kinase inhibitor is known in the art and / or prescribed by a physician.

[0457] In any of the foregoing embodiments of the present invention, pretreatment with an IL-2 inducible T cell kinase (ITK) inhibitor is described. Interleukin-2 inducible T cell kinase (ITK) is a non-receptor tyrosine kinase expressed in T cells that regulates multiple pathways. Any ITK inhibitor known in the art can be used in embodiments of the present invention (see, e.g., Lo et al., Expert Opinion on Therapeutic Patents, 20: 459-469 (2010); Vargas et al., Scandinavian Journal of Immunology, 78 (2): 130-139 (2013); WO2015112847; WO2016118951; WO2007136790; US20120058984A1, and U.S. Pat. Nos. 9,531,689 and 9,695,200; the entire contents of which are incorporated herein by reference). In one embodiment of the invention, the ITK inhibitor is a covalent ITK inhibitor that covalently and irreversibly binds to ITK. In one embodiment of the invention, the ITK inhibitor is an allosteric ITK inhibitor that binds to ITK. In one embodiment of the invention, the ITK inhibitor is selected from the group consisting of aminothiazole ITK inhibitors, 5-aminomethylbenzimidazole ITK inhibitors, 3-aminopyridin-2-one ITK inhibitors, (4- or 5-aryl)pyrazolyl indole ITK inhibitors, benzimidazole ITK inhibitors, aminobenzimidazole ITK inhibitors, aminopyrimidine ITK inhibitors, aminopyridine ITK inhibitors, diazolodiazine ITK inhibitors, triazole ITK inhibitors, 3-aminopyridin-2-one ITK inhibitors, indolyl indazoles ITK inhibitors, indole ITK inhibitors, azaindole TK inhibitors, pyrazolyl indole inhibitors, thienopyrazole ITK inhibitors, heterocyclic ITK inhibitors, and ITK inhibitors targeting cysteine ​​442 in the ATP pocket (e.g., ibrutinib), azabenzimidazole ITK inhibitors, benzothiazole ITK inhibitors, indole ITK inhibitors, pyridone ITK inhibitors, sulfoximine-substituted pyrimidine ITK inhibitors, arylpyridone ITK inhibitors, and any other ITK inhibitors known in the art. In one embodiment of the invention, the pretreatment regimen with the ITK inhibitor is known in the art and / or as prescribed by a physician. In one embodiment of the invention, the ITK inhibitor is selected from:

[0458] Ibrutinib,

[0459] BMS509744, CTA056、 GSK2250665A、 PF06465469、

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469] And combinations thereof. In one embodiment of the present invention, the ITK inhibitor is selected from: imatinib, dasatinib (BMS-354825), Sprycel (N-(2-chloro-6-methylphenyl)-2-(6-(4-(2-hydroxyethyl)-piperazine-1-yl)-2-methyl-pyrimidin-4-ylamino)thiazole-5-carboxamide), ibrutinib (1-{(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin =Boehringer Ingelheim, Moriarty et al., Bioorg Med Chem Lett., 18:5537-40 (2008)), compound 19 (Boehringer Ingelheim, Moriarty et al., Bioorg Med Chem Lett., 18:5537-40 (2008)), compound 27 (Boehringer Ingelheim, Moriarty et al., Bioorg Med Chem Lett., 18:5537-40 (2008)), compound 26 (Boehringer Ingelheim, Winters et al., Bioorg Med Chem Lett., 18:5541-40 (2008)), compound 37 (Boehringer Ingelheim, Cook et al., Bioorg Med Chem Lett., 19:773-7 (2009)), compound 41 (Boehringer Ingelheim, Cook et al., Bioorg Med Chem Lett., 19: 773-7 (2009)), compound 48 (Boehringer Ingelheim, Cook et al., Bioorg Med Chem Lett., 19: 773-7 (2009)), compound 51 (Boehringer Ingelheim, Cook et al., Bioorg Med ChemLett., 19: 773-7 (2009)), compound 10n (Boehringer Ingelheim, Riethe et al., Bioorg Med ChemLett., 19: 1588-91 (2009)), compound 10o (Boehringer Ingelheim, Riethe et al., Bioorg Med ChemLett., 19: 1588-91 (2009)), compound 7v (Vertex, Charrier et al., J Med Chem., 54:2341-50 (2011)), compound 7w (Vertex, Charrier et al., J Med Chem., 54:2341-50 (2011)), compound 7x (Vertex, Charrier et al., J Med Chem., 54:2341-50 (2011)), compound 7y (Verrier, Charrier et al., J Med Chem., 54:2341-50 (2011)), compound 44 (Bayer Schering Pharma, von Bonin et al., Exp Dermatol., 20:41-7 (2011)), compound 13 (Nycomed, Velankar et al., Bioorg Med Chem., 18:4547-59 (2010)), compound 24 (Nycomed, Velankar et al., Bioorg Med Chem., 18: 4547-59 (2010)), compound 34 (Nycomed, Velankar et al., Bioorg Med Chem., 18: 4547-59 (2010)), compound 10o (Nycomed, Herdemann et al., Bioorg Med Chem Lett., 21: 1852-6 (2011)), compound 3 (Sanofi, McLean et al., Bioorg Med Chem Lett., 22: 3296-300 (2012)), compound 7 (Sanofi, McLean et al., Bioorg Med Chem Lett., 22: 3296-300 (2012)), and / or other kinase inhibitors, tyrosine kinase inhibitors or serine / threonine kinase inhibitors known in the art, and any combination thereof. .

[0470] In any of the foregoing embodiments, the pretreatment regimen comprising ibrutinib (commercially available as IMBRUVICA, whose chemical name is 1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-1-piperidinyl]-2-propen-1-one) may comprise: 1 140 mg capsule orally administered once a day (qd), 2 140 mg capsules orally administered once a day (qd), 3 140 mg capsules orally administered once a day (qd), or 4 140 mg capsules orally administered once a day (qd) for a duration 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. In the foregoing embodiment, the pretreatment regimen comprising ibrutinib can also include oral administration of ibrutinib at a dose 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 frequency of administration is once a day, twice a day, three times a day, or four times a day; and wherein the duration of administration is selected from 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.

[0471] In any of the preceding 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's lymphoma, Waldenstrom's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndrome, myelofibrosis, chronic myeloid leukemia, follicle-centered lymphoma, indolent NHL, human immunodeficiency virus (HIV)-associated B-cell lymphoma, and Epstein-Barr virus (EBV)-associated B-cell lymphoma.

[0472] Various animal models known in the art can be used to test the efficacy of the methods and compositions described herein in treating, preventing and / or managing the indicated diseases or conditions.

[0473] Nonmyeloablative lymphocyte depletion with chemotherapy

[0474] In one embodiment, the present invention provides a method for treating cancer with a population of TILs, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of the TILs in accordance with the present disclosure. In one embodiment, the non-myeloablative chemotherapy is one or more chemotherapeutic agents. In one embodiment, the non-myeloablative chemotherapy is administration of cyclophosphamide 60 mg / kg / day for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg / m 2 / day for 5 days (Days 27 to 23 before TIL infusion). In one embodiment, after non-myeloablative chemotherapy and TIL infusion (on Day 0) according to the present disclosure, the patient receives an intravenous infusion of IL-2 at 720,000 IU / kg every 8 hours until physiological tolerance.

[0475] Experimental results show that lymphocyte depletion before adoptive transfer of tumor-specific T lymphocytes plays a key role in improving the therapeutic efficacy by eliminating regulatory T cells and competing components of the immune system ("cytokine sinks"). Therefore, some embodiments of the present invention use a lymphocyte depletion step (sometimes also referred to as "immunosuppressive conditioning") to patients before introducing the TILs of the present invention.

[0476] Typically, lymphocyte depletion is achieved by administering fludarabine or cyclophosphamide (the active form is called mafosfamide) and combinations thereof. This approach is 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 incorporated herein by reference in their entirety.

[0477] 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 administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or longer. 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 administered for 2 to 7 days at a dosage of 35 mg / kg / day. In some embodiments, fludarabine treatment is administered for 4 to 5 days at a dosage of 35 mg / kg / day. In some embodiments, fludarabine treatment is administered for 4 to 5 days at a dosage of 25 mg / kg / day.

[0478] In some embodiments, cyclophosphamide is administered to achieve a concentration of 0.5 μg / mL to 10 μg / mL of mafosfamide (active form of cyclophosphamide). In some embodiments, cyclophosphamide is administered to achieve a concentration of 1 μg / mL of mafosfamide (active form of cyclophosphamide). In some embodiments, cyclophosphamide treatment is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or longer. In some embodiments, cyclophosphamide is administered at 100 mg / mL. 2 / day, 150mg / m 2 / day, 175mg / m 2 / day, 200mg / m 2 / day, 225mg / m 2 / day, 250mg / m 2 / day, 275mg / m 2 / day or 300mg / m 2 In some embodiments, cyclophosphamide is administered intravenously (i.e., iv). In some embodiments, cyclophosphamide treatment is administered at a dose of 35 mg / kg / day for 2 to 7 days. In some embodiments, cyclophosphamide treatment is administered at a dose of 250 mg / m 2 / day intravenously for 4 to 5 days. In some embodiments, cyclophosphamide treatment is at 250 mg / m 2 The dose was administered intravenously for 4 days.

[0479] In some embodiments, lymphocyte depletion is performed by administering fludarabine and cyclophosphamide together to the patient. In some embodiments, fludarabine is administered at 25 mg / m 2 / day intravenously and cyclophosphamide at 250 mg / m 2 / day dose administered intravenously for 4 days.

[0480] In one embodiment, by 60 mg / m 2 Cyclophosphamide was administered at a dose of 25 mg / m 2 Lymphocyte depletion is performed by administering fludarabine at a dose of 60 mg / m 2 Cyclophosphamide was administered at a dose of 25 mg / m 2 Lymphocyte depletion was performed by administering fludarabine at a dose of 100 mg / day for 5 days. Several methods for expanding TILs obtained from bone marrow or peripheral blood are described herein.

[0481] Example

[0482] The embodiments included herein are now described with reference to the following examples. These examples are provided for illustrative purposes only, and the disclosure included herein should in no way be construed as being limited to these examples, but rather should be construed to include any and all variations that become apparent based on the teachings provided herein.

[0483] Example 1. Expansion of TILs from non-Hodgkin's lymphoma

[0484] From having Figure 1 TILs were expanded from 5 non-Hodgkin lymphoma tumors of the indicated pathology (1 mantle cell lymphoma tumor, 3 follicular lymphoma tumors, and 1 ABC type diffuse large B-cell lymphoma tumor) using IL-2 for 11 to 14 days in the pre-REP phase, followed by IL-2, mitogenic anti-CD3 antibodies, and irradiated allogeneic peripheral blood mononuclear cell (PBMC) feeder cells for a subsequent REP of 14 days. TILs were successfully generated from all 5 lymphoma tumors, with a maximum expansion index of 680-fold, which is significantly higher than that previously observed using other methods. Schwartzentruber et al., Blood 1993, 82, 1204-1211. In addition, the average CD3 + The T cell population was 95% (compared to 75% using the method of Schwartzentruber et al., Blood 1993, 82, 1204-1211).

[0485] Cell sorting and flow cytometry were performed using the Becton, Dickinson & Co. (BD) FACS CANTO II system. A significant relative increase in effector memory cells comparable to those in melanoma TILs was observed by flow cytometry analysis ( Figure 2 ). Effector memory CD45RA was observed in lymphoma compared to melanoma TIL cultures+ There was a significant increase in the CD4+CD8+ (TEMRA) cell (p=0.0013; CD4+CD8) and CD28+CD4+ (p=0.008) subsets ( Figure 3 ).

[0486] Figure 4 and Figure 5 Comparison of phenotypic markers of T cell differentiation in CD4+ and CD8+ subsets, respectively, is shown. Figure 6 and Figure 7 Comparison of phenotypic markers of T cell exhaustion in CD4+ and CD8+ subsets, respectively, is shown.

[0487] Figure 8 A comparison of cell types of non-Hodgkin lymphoma TILs and melanoma TILs is shown. It is shown that CD4 + The number of T cells showed an increasing trend.

[0488] Fig. 9 Results of bioluminescent redirected lysis assay (BRLA) are shown. Minimal cytolytic activity LU of TILs measured by BRLA 50 / 10 6 :With melanoma TIL (11-75LU 50 , 4 hours), compared with lymphoma TILs <1-6LU at 4 hours 50 , 1-39LU at 24 hours 50 .

[0489] Fig.10 Results of interferon-γ (IFN-γ) enzyme-linked immunosorbent assay (ELISA) of lymphoma TILs and melanoma TILs are shown. Comparable results are shown. Results of ELIspot assay of lymphoma TILs are shown in Fig.11 , and with Fig.12 In the ELIspot assay, lymphoma TILs were observed to produce significant amounts of IFN-γ after stimulation with phorbol 12-myristate 13-acetate / ionomycin, anti-CD3 antibodies, or CD3 / CD28 / 4-1BB beads; under these conditions, IFN-γ production by some lymphoma TILs was comparable to that by melanoma TILs; in some cases, IFN-γ production by lymphoma TILs was much higher.

[0490] Fig.13Results from NANOSTRING NCOUNTER analysis (Nanostring Technologies, Inc., Seattle, WA) were shown, showing that lymphoma TILs expressed 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 primarily TH2 and TH17.

[0491] Taken together, these results provide evidence that TIL cell therapy can be used to treat patients with lymphoma.

[0492] Example 2. Bone marrow infiltrating lymphocytes (MIL) grown from the bone marrow of AML patients and peripheral blood of AML patients Phenotypic and functional characterization of outgrown peripheral blood lymphocytes (PBL)

[0493] Bone marrow samples and associated blood samples (if available) were obtained from patients with acute myeloid leukemia (AML), including patients who had undergone at least three cycles of pretreatment with 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), with information on the patient's age, sex, stage, tumor type, cancer site, treatment history, de-identified pathology report, and any molecular testing performed (e.g., MSI expression and Raf / Ras expression). MILs and PBLs were expanded using MIL Method 1, MIL Method 2, or MIL Method 3, or one of PBL Method 1, PBL Method 2, or PBL Method 3, and the MILs and PBLs were phenotypically and functionally characterized.

[0494] Fig.36A and 36B Fold expansion of MILs and PBLs is shown. Fig.36A The fold expansion of 3 patients (MIL1, MIL2, MIL3) is shown. Fig.36BFold expansion of matched PBLs from patients 2 and 3 (PBL2, PBL3) is shown. MIL1.1 was amplified using MIL Method 1, MIL1.2 was amplified using MIL Method 2, MIL1.3 was amplified using MIL Method 3, and PBLs were amplified using PBL Method 3. MIL1 fold expansion shows that individual samples in MIL1 increased: 25-fold (MIL1.1), 50-fold (MIL1.2), and 75-fold (MIL1.3). This provides preliminary evidence that MIL Method 3 may be the preferred expansion method. MIL2 and MIL3 fold expansion data appear to be inferior, perhaps due to low starting cell numbers. For comparison, sample 3 from patient MIL1 (MIL1.3) had a starting cell number of 138,000 cells, while the starting cell numbers for MIL2 and MIL3 were 62,000 and 28,000, respectively. As Fig.36B As shown, the fold expansion of PBLs for MIL2 and MIL3 was approximately 10-fold and 40-fold, respectively, with similar starting cell numbers (338,000 for PBL2 and 336,000 for PBL3).

[0495] Fig.37A and 37B Shows MIL( Fig.37A ) and the matching PBL ( Fig.37B ) of IFN-γ producing cells. IFN-γ secretion was significantly increased by MIL1.3, MIL2, and MIL3, indicating that MIL method 3 is the preferred expansion method. Data for PBL are inconclusive.

[0496] Figures 38A-38F TCRαβ+, CD4+, and CD8+ subsets of MILs and PBLs are shown. Fig.38A and 38D MILs amplified using all three methods (MIL1.1, MIL1.2, MIL1.3) are shown. Fig.38A ) and PBLs expanded using PBL method 3 ( Fig.38D The data showed that the TCRαβ+ subset of all MILs and PBLs accounted for almost 100%, indicating that the expansion process successfully expanded almost all T cells. Fig.38B and 38E showed that the CD4 subset of MIL expanded by MIL method 3 was reduced (which is consistent with Fig.38C The increase of CD8 subset in Fig.38E and 38F The PBL data in the figure are consistent with the MIL1.3 data.

[0497] Fig.39A -D and 40A-D show data for CD4 subsets in MIL (Figure 39) and PBL (Figure 40). Fig.39A and 40A Data for naive (CCR7+ / CD45RA+) are shown; Fig.39B and 40B Data are shown for central memory T cells (CM) (CCR7+ / CD45RA-); Fig.39C and 40C Data are shown for effector memory T cells (EM) (CCR7- / CD45RA-); and Fig.39D and 40D Data for terminally differentiated effector memory cells (TEMRA) (CCR7- / CD45RA+) are shown. All samples expanded using MIL method 3 (MIL1.3) and PBL method 3 (PBL2 and PBL3) were consistent with the CD4 subset of the comparator (melanoma TIL).

[0498] Fig.41A -D and 42A-D show data for CD8 subsets in MIL (Figure 41) and PBL (Figure 42). Fig.41A and 42A Data for naive (CCR7+ / CD45RA+) are shown; Fig.41B and Fig.42B Data are shown for central memory T cells (CM) (CCR7+ / CD45RA-); Fig.41C and Fig.42C Data are shown for effector memory T cells (EM) (CCR7- / CD45RA-); and Fig.41D and 42D Data for terminally differentiated effector memory cells (TEMRA) (CCR7- / CD45RA+) are shown. Samples expanded using MIL method 3 (MIL1.3) were consistent with the CD4 subset of the comparator (melanoma TIL). Data for PBL2 and PBL3 were used as controls.

[0499] Fig.43A and 43B Shows MIL( Fig.43A ) and PBL( Fig.43B ) data for CD4CD27 and CD8CD27 subsets. Fig.44A and 44B Shows MIL( Fig.44A ) and PBL( Fig.44B ). Data for the CD4CD28 and CD8CD28 subsets of MIL1.3 and MIL1.3 are shown for each sample at day 0 and day 14 of the expansion process compared to melanoma TIL. Only MIL data for MIL1.3 at day 0 and day 14 compared to melanoma TIL are shown. The CD28 subsets of MIL and PBL are similar to melanoma TIL.

[0500] Fig.45A and 45B Indicates MIL( Fig.45A ) and PBL( Fig.45B )Comparison of PD1+ cells in various CD4 and CD8 subsets. Fig.46A and 46B Indicates MIL( Fig.46A ) and PBL( Fig.46B ) Comparison of LAG3+ cells of various CD4 and CD8 subsets of PD1+ and LAG3+ data showed that MIL1.3 samples were significantly reduced in the measurement on day 0; while MIL1.1 and MIL1.2 showed a trend towards an increase in both PD1 and LAG3 on day 0. PBL data were used as a control.

[0501] The experiments in this example show that MILs expanded using MIL method 3 have higher fold expansion, high functionality, a higher proportion of CD8 subsets, and fewer LAG3+ and PD1+ T cell subsets. The data also show that the memory subsets are similar to melanoma TILs. The data also show that cryopreserved samples show higher fold expansion compared to fresh samples. Much of the data for PBL samples seems inconclusive, probably due to small sample sizes.

[0502] Example 3. Methods of expanding TIL and treating cancer using expanded TIL

[0503] Bone marrow was obtained using needle aspiration. Bone marrow samples were aspirated into syringes containing heparin and stored overnight at room temperature. After storage, the contents of the syringes were combined into a sterile container for quality control. Mononuclear cells (MNCs) in the bone marrow were enriched using lymphocyte separation medium (LSM) and centrifuged using a COBE Spectra. Cells in the gradient were collected until the red blood cells and washed with HBSS. MNCs were cryopreserved using a hydroxyethyl starch-based cryoprotectant supplemented with 2% HSA and 5% DMSO, and some MNCs were retained for quality control. QC vials were thawed and the CD3+ and CD38+ / 138+ cell content of the MNC product was determined.

[0504] The bone marrow is aspirated and fractionated in a density gradient of "lymphocyte separation fluid" to collect cells that are almost at the red blood cell sedimentation layer. This fractionation method substantially removes red blood cells and neutrophils, providing an almost complete bone marrow. The resulting fractionated material is T cells and tumor cells. The bone marrow is subjected to Ficoll, and TILs are expanded using methods known in the art and any method described herein. For example, Fig.14 An exemplary method for expanding TILs is described in . Fig.15 Exemplary methods of expanding TILs and treating cancer patients with the expanded TILs are shown.

[0505] Example 4. Phenotypic and functional characterization of tumor infiltrating lymphocytes (TILs) grown from non-Hodgkin lymphoma tumors Levy

[0506] The objectives of the experiments described in this example include determining whether TILs with therapeutic potential can be isolated and cultured from NHL tumors, and comparing the characteristics of NHL-derived TILs and melanoma-derived TILs.

[0507] The materials and methods for extracting and expanding TILs from patients are as described herein. The patient's TILs are extracted from the suppressive tumor microenvironment by surgical resection of the lesion (in this case, lymphoid tissue). The TILs are expanded using the expansion methods disclosed herein to produce 10 9 Up to 10 11 TILs.

[0508] Flow cytometry was used to analyze differentiation markers of NHL-derived TILs (1 mantle cell lymphoma (MCL), 3 follicular lymphomas (FL), 3 diffuse large B-cell lymphomas (DLBCL)) relative to melanoma-derived TILs. Anti-CD56, anti-TCRab, anti-CD3, anti-CD4, anti-CD8, anti-CD27, and anti-CD28 antibodies were analyzed for TILs. These antibodies were used as Differentiation Panel 1 (DF1). Anti-CD3, anti-CD4, anti-CD9, anti-CD38, as well as 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+).

[0509] Figure 16 shows CD4 and CD8 T cells of different cell subsets in different cancer types. Melanoma (black), mantle cell (red), diffuse large B-cell lymphoma (blue), and follicular lymphoma (purple) cancer types were examined. Figures 16A-16D Overall, lymphoma TILs tend to be more proliferative and therefore have higher antitumor activity compared to melanoma TILs. Fig. 17B showed that lymphoma T cells expressing CD4 / CD28 had a higher proliferation capacity than melanoma T cells expressing CD4 / CD28.

[0510] Following the manufacturer's instructions, Dynabeads coated with mAb were used to TM (CD3, CD28 and CD137) to stimulate TILs and then use ELIspot TM(Immunospot CTL) stimulation, using Immunospot TM S6 Entry Analyzer counts and also uses DuoSet TM ELISA kit (R&D Systems) was used to detect interferon (IFNγ) produced by TILs.

[0511] Fig.18A and 18B demonstrated that IFNγ production by NHL TILs and melanoma TILs was similar, suggesting that both TIL types have similar cytotoxic functions.

[0512] The lytic potential of TIL was detected using the bioluminescence redirected lysis assay (BRLA). P815 cells transduced with lentiviral vectors encoding eGFP and firefly luciferase were used as target cells. TIL and target cells were co-cultured for 4 hours / 24 hours in the presence of OKT3. Luciferin was then added and the cells were incubated for 5 minutes. Bioluminescence was detected using a luminometer. The survival percentage and cytotoxicity percentage were calculated as follows:

[0513] Survival rate % = (experimental survival rate - minimum) / (maximum signal - minimum signal) × 100

[0514] Cytotoxicity % = 100 – (survival rate %)

[0515] The lytic potential of TILs is expressed as lytic units (LU). 50 , which represents 50% cytotoxicity of target cells induced by effector cells.

[0516] TILs were analyzed to determine their tumoricidal capacity against autologous and allogeneic tumors. TILs were mixed with autologous lymphoma cells or allogeneic melanoma cell lines (526 melanoma cell line) at different effector cell to target cell ratios (E:T ratios) - 10:1, 20:1, 50:1, or 100:1. Tumor cells were labeled with CellTrace Violet dye (Thermo Fisher) before co-culture. After 24 hours, cells were stained with 7-AAD to determine cell death. The proportion of tumor cells killed by TILs is expressed as: 7-AAD-positive tumor cells gated by CellTrace Violet dye vs CD19 of lymphoma cells, and CellTrace Violet vs MCSP of melanoma cells.

[0517] Figure 19 shows that at 4 hours ( Fig.19A ) and 24 hours ( Fig.19B), NHL TILs and melanoma TILs had similar cytotoxic functions against allogeneic and autologous tumors.

[0518] The nCounter GX Human Immunology V2 panel (NanoString, Seattle) was also used to analyze gene expression in TILs. Following the manufacturer's instructions, 100 ng of total RNA was measured. The data were normalized by converting the geometric mean of the built-in control gene probes of each sample. The data were plotted for melanoma gene expression and compared with melanoma gene expression.

[0519] Fig.21 Results of gene expression analysis are shown. This heat map shows the fold change in gene expression relative to melanoma TILs. Lymphoma-derived TILs expressed higher IL17A and RORC compared to melanoma-derived TILs.

[0520] In summary, the results of this experiment show that the functional characteristics of lymphoma-derived TILs are similar to those of melanoma-derived TILs, which suggests that the use of lymphoma-derived TILs will successfully treat lymphoma cancers.

[0521] Example 5. Peripheral blood lymphocytes grown from peripheral blood of patients with chronic lymphocytic leukemia (CLL) Phenotypic and functional characterization of PBL

[0522] PBMCs were collected from CLL patients before and after three cycles of treatment with ibrutinib.

[0523] As described in Figure 24 and elsewhere herein, three different methods, PBL Method 1, PBL Method 2, and PBL Method 3, were used to expand T cells. Some samples were from fresh PBMCs and some samples were from cryopreserved PBMCs. Once expanded and harvested, the cells were phenotypically identified and functionally characterized using the methods described in Example 4 above and elsewhere herein. The purpose of this example was to determine the optimal expansion process for PBLs and to determine whether PBLs expanded from samples treated with ibrutinib were more effective than PBLs expanded from untreated samples.

[0524] Fig.26Fold expansion of PBL is shown. Results are shown for PBL expanded using PBL Method 1, PBL Method 2, and PBL Method 3. Untreated PBL (PreRx PBL) showed an average of 179-fold expansion, and PBL treated with ibrutinib (PostRx PBL) showed an average of 306-fold expansion. PBL from fresh PBMC (PBL) only showed an average of 82-fold expansion. Between PBL and PostRx PBL, p=0.006. Between PBL and PreRx PBL, p=0.3; between PreRx PBL and PostRx PBL, p=0.1. In summary, all PostRx PBL groups had an average fold increase compared to all groups of PBL and PreRx PBL.

[0525] Fig. 27 Interferon-γ (IFN-γ) producing cells in PBL, PreRx PBL, and PostRx PBL are shown. For PBL, the average number of IFN-γ producing cells was about 1864. For PreRx PBL, the average number of IFN-g producing cells was about 7530; for PostRx PBL, the average number of IFN-γ producing cells was about 11984. Between PBL and PostRxPBL, p=0.006. Between PBL and PreRx PBL, p=0.006; between PreRx PBL and PostRx PBL, p=0.01. In summary, the average number of IFN-γ producing cells was significantly increased in all PostRx PBL groups compared to all groups of PBL and PreRx PBL.

[0526] Each sample was phenotypically characterized. Fig.28 Represents the ratio of CD4+ and CD8+ T cell subsets in PreRx PBL and PostRx PBL, using melanoma TIL as a comparator. Here, the data shows that the CD4 subset of PreRx PBL and PostRx PBL (left panel) is comparable regardless of the method used to expand the cells. It is shown that the CD4 subset of PreRx PBL and PostRxPBL is higher than that of melanoma TIL (p=0.0006 for each). Regardless of the method used to expand the cells, the CD8 subset of PreRXPBL and PostRX PBL (as shown in the right panel) is lower. It is shown that the CD8 subset of PreRx PBL and PostRx PBL is lower than that of melanoma TIL (p=0.0006 for each). As a hypothesis, the lower CD8 subset is simply due to the cancer type (i.e., the CD4 subset of CLL is usually expanded).

[0527] Figures 29A-29DFigure 5. Comparison of CD4 memory subsets of PreRx PBL and PostRx PBL using melanoma TILs as comparator. Fig.29A Data for naive (CCR7+ / CD45RA+) are shown. Fig.29B Data for central memory T cells (CM) (CCR7+ / CD45RA-) are shown. Fig.29C Data for effector memory T cells (EM) (CCR7- / CD45RA-) are shown. Fig.29D Data for terminally differentiated effector memory cells (TEMRA) (CCR7- / CD45RA+) are shown. Figure 29 shows that the CD4 memory subsets of PreRx PBL and PostRx PBL are comparable to those of melanoma TIL.

[0528] Figures 30A-30D Figure 5. Comparison of CD8 memory subsets of PreRx PBL and PostRx PBL using melanoma TILs as comparator. Fig. 30A Data for naive (CCR7+ / CD45RA+) are shown. Fig. 30B Data for central memory T cells (CM) (CCR7+ / CD45RA-) are shown. Fig. 30C Data for effector memory T cells (EM) (CCR7- / CD45RA-) are shown. Fig.30D Data for terminally differentiated effector memory cells (TEMRA) (CCR7- / CD45RA+) are shown. Figure 30 demonstrates that the CD8 memory subset of PreRx PBL and PostRx PBL is comparable to that of melanoma TIL.

[0529] Fig.31A and 31B CD4 cells ( Fig.31A ) and CD8 cells ( Fig.31B ) using melanoma TILs as comparators. The CD4CD27 cell subset was significantly higher in PreRx PBLs (p=0.03) and PostRx PBLs (p=0.02) compared to melanoma TILs. The CD8CD27 cell subset was significantly higher in PreRx PBLs (p=0.002) and PostRx PBLs (p=0.001) compared to melanoma TILs.

[0530] Fig.32A and 32B CD4 cells ( Fig. 30A ) and CD8 cells ( Fig. 30B) using melanoma TIL as a comparator. It was shown that both the CD4CD28 cell subset and the CD8CD28 cell subset of PreRx PBL and PostRx PBL were comparable to those of melanoma TIL.

[0531] Fig.33A and 33B Indicates the CD4+( Fig.33A ) and CD8+( Fig.33B The data showed that the mean values ​​of LAG3+ subpopulation in CD4+ (p=0.06) and CD8+ (p=0.01) populations were significantly decreased in PostRx PBL.

[0532] Fig.34A and 34B Indicates the CD4+( Fig.34A ) and CD8+( Fig.34B The data showed that the mean values ​​of the PD1+ subpopulation in the CD4+ and CD8+ populations in PostRx PBL were decreased, but the decrease was not significant.

[0533] Fig.35A and -35B show PreRx PBL ( Fig.35A ) and PostRx PBL( Fig.35B ) using CellTrace TM The Violet cell proliferation kit (Invitrogen) was used to perform the assay as follows: effector cells (i.e., PBLs) were labeled with carboxyfluorescein succinimidyl ester (CFSE). Target cells (autologous CD19+ tumor cells) were incubated with Mitocyin C and then assayed according to the CellTrace TM Violet Cell Proliferation Kit Instructions for use with CellTrace TM Violet (CTV) was used to label target cells. Effector and target cells were incubated for 24 h at a ratio of 2:1, 5:1, and 20:1 (E:T cells). CountBright magnetic beads were added, cells were stained with Annexin V-PI, and then CTV+ / Annexin-V PI+ cells (which provide the number of dead cells) were analyzed. PostRx PBL appeared to be more effective because fewer PostRx PBL cells were required to kill 50% of target tumor cells (i.e., LU of PostRx PBL was 2.000 cells / mL compared to PreRx PB). 50 lower).

[0534] The experiments performed in this example demonstrated the following results: PBL expanded from fresh CLL PBMCs showed lower expansion folds and significantly lower IFN-γ production compared with PBL expanded from cryopreserved PBMCs (PreRx PBL and PostRx PBL); PostRx PBL consistently showed higher expansion folds and significantly increased IFN-γ production compared with PreRx PBL; although the LU of PostRx PBL was 50 Lower than PreRx PBL, both PreRx PBL and PostRx PBL showed lytic activity against autologous (CD19+) tumor cells.

[0535] The above examples are provided to provide those of ordinary skill in the art with a complete disclosure and description of embodiments of how to make and use the compositions, systems and methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above modes of carrying out the present invention that are obvious to those skilled in the art are intended to fall within the scope of the appended claims. All patents and publications mentioned in the specification are indicative of the technical levels of those skilled in the art to which the present invention pertains.

[0536] All titles and section names are for clarification and reference purposes only and should not be considered limiting in any way. For example, those skilled in the art will appreciate that it is useful to appropriately combine various aspects of different titles and subsections according to the spirit and scope of the invention described herein.

[0537] All references cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0538] As will be apparent to those skilled in the art, many modifications and variations may be made to the present application without departing from the spirit and scope of the present invention. The specific embodiments and examples described herein are provided by way of example only, and the present application is limited only by the terms of the appended claims and the full scope of equivalents to which the claims are entitled. Sequence Listing <110> IOVANCE BIOTHERAPEUTICS, INC. <120> Expansion of tumor-infiltrating lymphocytes from liquid tumors and their therapeutic use <130> 116983-5030-WO <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> muromonab heavy chain <400> 1 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Val Cys Gly Gly Thr Thr Gly Ser Ser Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser 180 185 190 Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 2 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Muromonab light chain <400> 2 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn Arg Ala Asp Thr Ala Pro 100 105 110 Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly 115 120 125 Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn 130 135 140 Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn 145 150 155 160 Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser 165 170 175 Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr 180 185 190 Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe 195 200 205 Asn Arg Asn Glu Cys 210 <210> 3 <211> 134 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Human IL-2 <400> 3 Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu 1 5 10 15 His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr 20 25 30 Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro 35 40 45 Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu 50 55 60 Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His 65 70 75 80 Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu 85 90 95 Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr 100 105 110 Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser 115 120 125 Ile Ile Ser Thr Leu Thr 130 <210> 4 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> Aldesleukin <400> 4 Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu 1 5 10 15 Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn 20 25 30 Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys 35 40 45 Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro 50 55 60 Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg 65 70 75 80 Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys 85 90 95 Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr 100 105 110 Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile 115 120 125 Ser Thr Leu Thr 130 <210> 5 <211> 130 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Human IL-4 <400> 5 Met His Lys Cys Asp Ile Thr Leu Gln Glu Ile Ile Lys Thr Leu Asn 1 5 10 15 Ser Leu Thr Glu Gln Lys Thr Leu Cys Thr Glu Leu Thr Val Thr Asp 20 25 30 Ile Phe Ala Ala Ser Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg 35 40 45 Ala Ala Thr Val Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr 50 55 60 Arg Cys Leu Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu 65 70 75 80 Ile Arg Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly 85 90 95 Leu Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn 100 105 110 Phe Leu Glu Arg Leu Lys Thr Ile Met Arg Glu Lys Tyr Ser Lys Cys 115 120 125 Ser Ser 130 <210> 6 <211> 153 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Human IL-7 <400> 6 Met Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser Val 1 5 10 15 Leu Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu Ile Gly 20 25 30 Ser Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His Ile Cys 35 40 45 Asp Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg Lys Leu 50 55 60 Arg Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu His Leu 65 70 75 80 Leu Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly Gln 85 90 95 Val Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr Lys 100 105 110 Ser Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp 115 120 125 Leu Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn 130 135 140 Lys Ile Leu Met Gly Thr Lys Glu His 145 150 <210> 7 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Human IL-15 <400> 7 Met Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu 1 5 10 15 Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val 20 25 30 His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu 35 40 45 Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val 50 55 60 Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn 65 70 75 80 Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn 85 90 95 Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile 100 105 110 Asn Thr Ser 115 <210> 8 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> IL-21 <400> 8 Met Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val 1 5 10 15 Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro 20 25 30 Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys 35 40 45 Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg 50 55 60 Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr 65 70 75 80 Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp 85 90 95 Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser 100 105 110 Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly 115 120 125 Ser Glu Asp Ser 130

Claims

1. A method for amplifying peripheral blood lymphocytes (PBL) from peripheral blood, wherein: The method comprises the following steps: (a) culturing a sample of peripheral blood mononuclear cells (PBMCs) obtained from peripheral blood of a patient with a hematological malignancy in a culture comprising a first cell culture medium with IL-2 and anti-CD3 / anti-CD28 antibodies, thereby achieving expansion of peripheral blood lymphocytes (PBLs) from the PBMCs, wherein the patient has been pre-treated with ibrutinib or other interleukin-2-inducible T-cell kinase, ITK, inhibitor, and the patient is refractory to treatment with ibrutinib or other ITK inhibitor, wherein the ITK inhibitor is selected from the group consisting of aminothiazole ITK inhibitors, benzimidazole ITK inhibitors, aminopyrimidine ITK inhibitors, 3-aminopyridin-2-one ITK inhibitors, indolyl indazole ITK inhibitors, pyrazolyl indole inhibitors, thienopyrazole inhibitors, and ITK inhibitors targeting cysteine ​​442 in the ATP pocket; and (b) harvesting PBLs from the culture of step (a); the method being performed for a period of time selected from: 9 days, 10 days, 11 days, 12 days, 13 days and 14 days, and the expanded PBLs comprise increased levels of IFNγ production compared to those expanded from patients not pre-treated with ibrutinib or other ITK inhibitors.

2. The method according to claim 1, wherein: Prior to step (a), the PBMCs are subjected to CD19+ screening to separate the PBMCs into CD19+PBMCs and non-CD19+PBMCs, and step (a) is performed by culturing non-CD19+PBMCs.

3. The method according to claim 1 or 2, wherein: In step (a), the anti-CD3 / anti-CD28 antibodies are bound to magnetic beads, and the ratio of magnetic beads to cells in culture is 3:

1.

4. The method according to claim 1 or 2, wherein: In step (a), on day 4 of culturing the PBMCs, additional IL-2 is added to the culture, and the first cell culture medium in the culture is replaced.

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

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

7. The method according to claim 6, wherein: The first cell culture medium comprises RPMI-1640, human AB serum, 1-glutamine, 2-mercaptoethanol, gentamicin sulfate, AIM-V medium, and the second cell culture medium comprises AIM-V medium, 2 mM Glutamax, and 3000 IU / mL IL2.

8. The method according to claim 3, wherein: The magnetic beads in step (a) are 9. The method according to claim 1, wherein: The hematological malignancy is a liquid tumor.

10. The method according to claim 9, wherein: The liquid tumor is a leukemia.

11. The method according to claim 9, wherein: The liquid tumor is chronic lymphocytic leukemia.

12. The method according to claim 1 or 2, wherein: The first cell culture medium comprises 3000 IU / mL of IL-2, and / or the culture is incubated at 37° C. and 5% CO 2 .

13. The method according to claim 1 or 2, wherein: The method is performed for 9 days, alternatively, the method is performed for 11 days.

14. The method according to claim 1 or 2, wherein: The patient was pretreated with ibrutinib and was refractory to treatment with ibrutinib.

15. The method according to claim 1 or 2, wherein: The patients had not received treatment with ibrutinib or other ITK inhibitors for at least 1 month prior to pretreatment with ibrutinib or other ITK inhibitors.

16. The method according to claim 1 or 2, wherein: The patients were pretreated with ibrutinib for at least 3 months.

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