Expansion of peripheral blood lymphocytes (PBLs) from peripheral blood

Abstract

To provide methods for conducting expansion culture of peripheral blood lymphocytes from peripheral blood.SOLUTION: A method disclosed herein for expanding peripheral blood lymphocytes (PBLs) from peripheral blood comprises: a. obtaining a sample of peripheral blood monocytes (PBMCs) from peripheral blood of a patient, where the patient is pretreated with ibrutinib or other insulin like tyrosine kinase (ITK) inhibitor and is unresponsive to the treatment with ibrutinib or other ITK inhibitors; b. culturing the PBMCs in a first cell culture medium comprising IL-2 and an anti-CD3 / anti-CD28 antibody for a period selected from the group consisting of about 9 days, about 10 days, about 11 days, about 12 days, about 13 days and about 14 days, thereby achieving expansion of peripheral blood lymphocytes (PBLs) from the PBMC; and c. collecting the PBLs from the culture in step b.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of International Patent Application No. PCT / US Patent Application Publication No. 18 / 032109, filed May 10, 2018, and is a continuation-in-part of U.S. Provisional Patent Application No. 62 / 504,337, filed May 10, 2017; U.S. Provisional Patent Application No. 62 / 530,681, filed July 10, 2017; U.S. Provisional Patent Application No. 62 / 550,398, filed August 25, 2017; U.S. Provisional Patent Application No. 62 / 550,398, filed November 2, 2017. This application claims priority to U.S. Provisional Patent Application No. 62 / 590,034, filed on January 2, 2018; U.S. Provisional Patent Application No. 62 / 621,462, filed on January 24, 2018; U.S. Provisional Patent Application No. 62 / 621,798, filed on January 25, 2018; and U.S. Provisional Patent Application No. 62 / 647,367, filed on March 23, 2018, all of which are incorporated by reference in their entirety.

[0002] FIELD OF THE INVENTION

[0002] Disclosed herein are methods for expanding tumor-infiltrating lymphocytes (TILs) derived from the blood and / or bone marrow of patients with hematological malignancies, such as liquid tumors, including lymphomas and leukemias, and compositions comprising populations of TILs obtained therefrom. Further disclosed herein are therapeutic uses of TILs expanded from the blood or bone marrow of patients with hematological malignancies, such as liquid tumors, including the treatment of such hematological malignancies. [Background technology]

[0003] Background of the Invention

[0003] Treatment of bulky, refractory cancers using adoptive autologous transplantation of tumor-infiltrating lymphocytes (TILs) offers a powerful therapeutic approach for patients with poor prognosis. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. TILs are predominantly T cells, and IL-2-based TIL expansion followed by the "rapid expansion process" (REP) has become the preferred method for TIL expansion due to its speed and efficiency. Dudley, et al., Science 2002, 298, 850-54; Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al., J. Immunother. 2003, 26, 332-42. Several approaches have been explored to improve the response to TIL therapy in melanoma and to expand TIL therapy to other tumor types, but with limited success, this field remains a challenge. Goff, et al., J. Clin. Oncol. 2016, 34, 2389-97; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Rosenberg, et al., Clin. Cancer Res. 2011, 17, 4550-57. Previous approaches to expanding TILs from B-cell lymphomas have yielded unsatisfactory results, with only two of 12 attempts at TIL expansion providing potential activity against tumors. 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). Summary of the Invention [Means for solving the problem]

[0004]

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

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

[0006] In one embodiment, the present invention provides a method of treating cancer in a patient with a population of tumor-infiltrating lymphocytes (TILs), comprising: (a) optionally pretreating the patient with a regimen comprising at least one kinase inhibitor; (b) obtaining a tumor from a patient by resection, biopsy, needle aspiration, or apheresis, the tumor comprising a first population of TILs; (c) optionally fragmenting or dissociating the tumor to obtain tumor fragments and contacting the tumor or tumor fragments with a first cell culture medium; (d) performing an initial expansion of the first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 5 times more numerous than the first population of TILs, the first cell culture medium comprising IL-2, and the initial expansion is performed for a period of 21 days or less; (e) performing a second expansion culture of the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein 7 days after the initiation of the second expansion culture, the number of the third population of TILs is at least 50 times greater than the number of the second population of TILs, the second cell culture medium comprising IL-2, OKT-3 (anti-CD3 antibody), and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the second expansion culture is performed for a period of 14 days or less; (f) recovering the third population of TILs; and (g) administering a therapeutically effective portion of the third population of TILs to the patient; and the tumor is a liquid tumor, and the cancer is acute myeloid leukemia (AML), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), activated B-cell (ABC) DLBCL, germinal center B-cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), small lymphocytic leukemia (SLL), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, relapsed and / or refractory Hodgkin's lymphoma, B-cell lymphoma, or leukemia. The hematological malignancy is selected from the group consisting of follicular acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt's lymphoma, Waldenstrom's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndrome, myelofibrosis, chronic myeloid leukemia, follicular central lymphoma, indolent NHL, human immunodeficiency virus (HIV)-associated B-cell lymphoma, and Epstein-Barr virus (EBV)-associated B-cell lymphoma.

[0007] In one embodiment of the present invention, the method further comprises adding an ITK inhibitor. In one embodiment, the ITK inhibitor is added to the cell culture medium during at least one of steps (d) and (e). In one embodiment of the present invention, the ITK inhibitor is a covalent ITK inhibitor that covalently and irreversibly binds to ITK. In one embodiment of the present invention, the ITK inhibitor is an allosteric ITK inhibitor that binds to ITK. In another embodiment, the ITK inhibitor is selected from the group consisting of aminothiazole ITK inhibitors, benzimidazole ITK inhibitors, aminopyrimidine ITK inhibitors, 3-aminopyrid-2-one ITK inhibitors, indolindazole ITK inhibitors, pyrazolyl-indole inhibitors, thienopyrazole inhibitors, and ITK inhibitors that target cysteine-442 within 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, also known as 1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-1-piperidinyl]-2-propen-1-one. 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, MN, USA), Selleckchem, Inc. (Houston, TX, USA), and AK Scientific, Inc. (Union City, CA, USA). In another embodiment, the ITK inhibitor is added at a concentration of about 0.1 nM to about 5 μM.In another embodiment, the ITK inhibitor is added at a concentration of about 0.1 nM to about 5 μM. In another embodiment, the ITK inhibitor is added at a concentration of about 0.1 nM to about 100 nM. In another embodiment, the ITK inhibitor is added at a concentration of about 0.5 nM to about 50 nM. In another embodiment, the ITK inhibitor is added at a concentration of about 1 nM to about 10 nM. In another embodiment, the ITK inhibitor is added at a concentration of about 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 2 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 150 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, and 50 μM.

[0008] In one embodiment of the present invention, a method for expanding peripheral blood lymphocytes (PBLs) from peripheral blood comprises: a. Obtaining a sample of peripheral blood mononuclear cells (PBMCs) from peripheral blood, the sample optionally being cryopreserved; b. isolating PBLs from the sample by selecting and removing CD19+ B cells; c. Optionally, co-culturing said PBLs with said CD19+ B cells; d. stimulating the PBLs in the first cell culture medium with IL-2 and anti-CD3 / anti-CD28 antibodies in a gas-permeable container for a period of about 2 to about 6 days; e. culturing the PBLs from step (d) with IL-2 and anti-CD3 / anti-CD28 antibodies for a period of about 2 to about 6 days; f. isolating antibody-bound PBLs from the culture of step (e); g. Recovering antibodies from the PBLs isolated in step (e); and h. Recovering PBL Includes:

[0009] In one embodiment of the present 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 cell culture medium. In one embodiment, the first cell culture medium, the second cell culture medium, or the third culture medium is selected from the group consisting of CM-2, CM-4, and AIM-V. In another embodiment, the first and second cell culture media are the same. In another embodiment, the first and second cell culture media are different. In one embodiment of the present invention, one or more of the first, second, and third cell culture media are the same. In another embodiment, the first, second, and third cell culture media are all different.

[0010] In one embodiment, the optional co-culture of said PBLs with said CD19+ B cells is carried out for a period of between 1 hour and 3 days.

[0011] 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 the group consisting of 0.1:1, 1:1, and 10:1 (B cells:T cells).

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

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

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

[0015] In one embodiment of the present invention, the method includes 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 present invention, the ITK inhibitor is selected from the group consisting of aminothiazole ITK inhibitors, benzimidazole ITK inhibitors, aminopyrimidine ITK inhibitors, 3-aminopyrid-2-one ITK inhibitors, indolindazole 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.

[0016] In one embodiment of the present invention, any of the aforementioned methods for preparing PBLs is carried out in a closed sterile system.

[0017] In one embodiment of the present invention, a method for expanding peripheral blood lymphocytes (PBLs) from peripheral blood comprises: a. Obtaining a sample of peripheral blood mononuclear cells (PBMCs) from peripheral blood, the sample optionally being cryopreserved; b. isolating PBLs from the sample by selecting and removing CD19+ B cells; c. co-culturing said PBLs with said CD19+ B cells for a period of 4 days; d. Approximately 2.5 x 10 cells in a gas-permeable container in CM-2 cell culture medium 5 ~Approx. 5×10 5 adding PBLs and stimulating said PBLs with 3000 IU / ml IL-2 and bead-immobilized anti-CD3 / anti-CD28 antibodies for a period of about 4 days; e. Replacing the CM-2 cell culture medium with AIM-V cell culture medium and approximately 3000 IU / ml of additional IL-2; f. Culturing the PBLs in AIM-V cell culture medium from step (e) with IL-2 and bead-immobilized anti-CD3 / anti-CD28 antibodies for an additional period of about 3 days; g. isolating antibody-bound PBLs from the culture of step (f); h. Recovering antibodies from the PBLs isolated in step (g); and i. Recovering PBL Includes:

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

[0019]

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

[0020] In one embodiment of the present invention, the hematological malignancy is selected from 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), CLL with Richter's transformation (Richter's syndrome), small lymphocytic leukemia (SLL), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma (HHL), and the like. The PBLs are selected from the group consisting of 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, follicular central 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 present invention, the PBLs are at a concentration of about 0.1 x 10 9 ~Approx. 15×10 9 The amount of PBLs administered is

[0021]

[0021] In one embodiment of the present invention, the method for expanding bone marrow infiltrating lymphocytes (MIL) from bone marrow comprises: a. obtaining a sample of peripheral blood mononuclear cells (PBMCs) from bone marrow, the sample optionally being cryopreserved; b. Selecting from a sample of PBMCs a CD3+, CD33+, CD20+, and CD14+ cell fraction containing MILs (MIL cell fraction) and a non-CD3+, non-CD33+, non-CD20+, non-CD14+ cell fraction (AML blast fraction); c. Optionally, disrupting the AML blast fraction; d. adding the optionally disrupted AML blast cell fraction to the MIL cell fraction at a cell number ratio of about 0.1:1 to about 10:1; e. culturing the AML cell fraction with or without the AML blast cell fraction in a gas permeable vessel in a first cell culture medium containing IL-2; f. Stimulating MILs with anti-CD3 / anti-CD28 antibodies to obtain expanded cultures of MILs; g. restimulating MILs with IL-2 and anti-CD3 / anti-CD28 antibodies for an additional period of about 2 to about 6 days; h. culturing the MILs with additional IL-2 for an additional period of about 1 to about 3 days; and i. Retrieving said MIL Includes:

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

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

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

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

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

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

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

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

[0030] In one embodiment of the present invention, the method of treating a hematological malignancy in a patient comprises: a. obtaining a sample of peripheral blood mononuclear cells (PBMCs) from the patient's bone marrow, said sample optionally being cryopreserved; b. Selecting from a sample of PBMCs a CD3+, CD33+, CD20+, and CD14+ cell fraction containing MILs (MIL cell fraction) and a non-CD3+, non-CD33+, non-CD20+, non-CD14+ cell fraction (AML blast fraction); c. Optionally, disrupting the AML blast fraction; d. adding the optionally disrupted AML blast cell fraction to the MIL cell fraction at a cell number ratio of about 0.1:1 to about 10:1; e. culturing the AML cell fraction with or without the AML blast cell fraction in a gas permeable vessel in a first cell culture medium containing IL-2; f. Stimulating MIL with anti-CD3 / anti-CD28 antibodies; g. restimulating the MILs with IL-2 and anti-CD3 / anti-CD28 antibodies for an additional period of at least about 4 days; h. culturing the MILs with additional IL-2 for an additional period of at least about 3 days; i. recovering said MIL; and j. Administering said MIL to a patient in a therapeutically effective amount to treat a hematological malignancy. Includes:

[0031] In one embodiment of the present invention, the hematological malignancy is selected from 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), CLL with Richter's transformation (Richter's syndrome), small lymphocytic leukemia (SLL), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma (HHL), and the like. The hematological malignancy is selected from the group consisting of acute myeloid leukemia (AML), 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, follicular central lymphoma, indolent NHL, human immunodeficiency virus (HIV)-associated B-cell lymphoma, and Epstein-Barr virus (EBV)-associated B-cell lymphoma. In another embodiment, the hematological malignancy is acute myeloid leukemia (AML). In one embodiment of the present invention, the MIL is about 4×10 8 ~Approx. 2.5×10 9 It is administered in an amount of 1 MIL.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. [Brief explanation of the drawings]

[0033] [Figure 1]

[0033] Pathological information of lymphoma tumors is shown. [Figure 2]

[0034] A comparison of the different subsets of lymphoma and melanoma TILs is shown, which shows that the effector memory (EM) subset of lymphoma TILs is significantly higher than the EM subset of melanoma TILs. [Figure 3]

[0035] A comparison of the different subsets of lymphoma and melanoma TILs is shown, which shows that the CD28+CD4+ subset of lymphoma TILs is significantly higher than these subsets of melanoma TILs. [Figure 4]

[0036] Comparison of CD4+ T cell subsets in non-Hodgkin's lymphoma and melanoma TILs, showing differentiation markers. The red line on the graph represents the median. CM refers to central memory T cells, EM refers to effector memory T cells, and TEMRA refers to effector memory CD45RA+ T cells. [Figure 5]

[0037] Comparison of CD8+ T cell subsets in non-Hodgkin's lymphoma and melanoma TILs, showing differentiation markers. The red line on the graph represents the median. CM refers to central memory T cells, EM refers to effector memory T cells, and TEMRA refers to effector memory CD45RA+ T cells. [Figure 6]

[0038] Comparison of CD4+ T cell subsets in non-Hodgkin's lymphoma TILs and melanoma TILs, showing exhaustion markers. The red line on the graph represents the median. LAG3 refers to lymphocyte activation gene 3, PD1 refers to programmed death 1, and TIGIT refers to T cell immunoreceptor with Ig and ITIM domains. [Figure 7]

[0039] Comparison of CD8+ T cell subsets in non-Hodgkin's lymphoma and melanoma TILs, showing exhaustion markers. The red line on the graph represents the median. LAG3 refers to lymphocyte activation gene 3, PD1 refers to programmed death 1, and TIGIT refers to T cell immunoreceptor with Ig and ITIM domains. [Figure 8]

[0040] Figure 1 shows a comparison of cell types between non-Hodgkin's lymphoma and melanoma TILs. NK refers to natural killer cells, and TCRab refers to cells expressing a T cell receptor with alpha and beta chains. [Figure 9]

[0041] 1 shows the results of a bioluminescent redirected lysis assay (BRLA). [Figure 10]

[0042] 1 shows interferon-γ (IFN-γ) enzyme-linked immunosorbent assay (ELISA) results for lymphoma versus melanoma TILs. [Figure 11]

[0043] 1 shows the results of an enzyme-linked immunospot (ELIspot) assay of lymphoma TILs. [Figure 12]

[0044] 1 shows the results of an ELIspot assay of melanoma TILs. [Figure 13]

[0045] NANOSTRING NCOUNTER analysis results are shown, which show that lymphoma TILs express higher levels of RORC IL17A (TH17 phenotype) and GATA3 (Th2 phenotype) compared to melanoma TILs. Each gene is highlighted in red in the heatmap. [Figure 14]

[0046] The TIL expansion and treatment process is shown. Step 1 refers to the addition of four tumor fragments to ten G-Rex 10 flasks. In step 2, approximately 40 x 10 TILs or more are obtained. In step 3, division into 36 G-Rex 100 flasks occurs for REP. In step 4, the TILs are harvested by centrifugation. A fresh TIL product is obtained in step 5 after a total process time of approximately 43 days, at which point the TILs can be infused into patients. [Figure 15]

[0047]

[0023] Figure 1 shows a treatment protocol for use with TILs obtained from lymphomas of the present disclosure. Surgery (and tumor resection) is performed at the outset, and lymphodepleting chemotherapy refers to non-myeloablative lymphodepletion with chemotherapy, as described elsewhere herein. [Figure 16A]

[0048] Figure 1 shows the results of flow cytometry analysis using the standard phenotypic panel DF2 described in Example 4 below. Lymphoma and melanoma TILs were stained using the standard phenotypic panel DF2 as described in Example 4. Data shown represent different subpopulations of total CD4 and CD8 T cells in TILs. Naive T cell subsets are shown. P values ​​were calculated using a two-tailed Mann-Whitney test (unpaired). The mean percentages of cell subsets are represented by horizontal bars. [Figure 16B]

[0048] Results of flow cytometry analysis using the standard phenotypic panel DF2 described in Example 4 below are shown. Lymphoma and melanoma TILs were stained using the standard phenotypic panel DF2 as described in Example 4. Data shown represent different subpopulations of total CD4 and CD8 T cells in TILs. Central memory T cell subsets (CM) are shown. P values ​​were calculated using a two-tailed Mann-Whitney test (unpaired). The mean percentages of cell subsets are represented by horizontal bars. [Figure 16C]

[0048] Results of flow cytometry analysis using the standard phenotypic panel DF2 described in Example 4 below are shown. Lymphoma and melanoma TILs were stained using the standard phenotypic panel DF2 as described in Example 4. Data shown represent different subpopulations of total CD4 and CD8 T cells in TILs. Effector memory T cell subsets (EM) are shown. P values ​​were calculated using a two-tailed Mann-Whitney test (unpaired). The mean percentages of cell subsets are represented by horizontal bars. [Figure 16D]

[0048] Results of flow cytometry analysis using the standard phenotypic panel DF2 described in Example 4 below are shown. Lymphoma and melanoma TILs were stained using the standard phenotypic panel DF2 as described in Example 4. Data shown represent different subpopulations of total CD4 and CD8 T cells in TILs. Percentages of CD4 and CD8 cells in terminally differentiated effector memory (TEMRA) T cell subsets are shown. P values ​​were calculated using a two-tailed Mann-Whitney test (unpaired). Mean percentages of cell subsets are represented by horizontal bars. [Figure 17A]

[0049] Results of flow cytometry analysis using standard phenotypic panel DF1, as described in Example 4 below, are shown. Lymphoma and melanoma TILs were stained using standard phenotypic panel DF1 as described in Example 4. Data shown represent distinct CD27+ subpopulations of total CD4 and CD9 T cells in TILs, indicating a higher proportion of CD4 T cells expressing the costimulatory molecule CD28 in lymphoma TILs. P values ​​were calculated using a two-tailed Mann-Whitney test (unpaired). [Figure 17B]

[0049] Results of flow cytometry analysis using the standard phenotypic panel DF1, as described in Example 4 below, are shown. Lymphoma and melanoma TILs were stained using the standard phenotypic panel DF1 as described in Example 4. The data shown represent distinct CD28+ subpopulations of total CD4 and CD9 T cells in TILs, indicating a higher proportion of CD4 T cells expressing the costimulatory molecule CD28 in lymphoma TILs. P values ​​were calculated using a two-tailed Mann-Whitney test (unpaired). [Figure 18A]

[0050]

[0033] Figure 1 shows the results of an interferon gamma (IFN-γ) test performed according to Example 4 below. Results using ELISA are shown. ELISA data are expressed as IFN-γ producing cells per 10 TILs. P values ​​were calculated using a two-tailed Mann-Whitney test (unpaired). [Figure 18B]

[0050] The results of an interferon gamma (IFN-γ) test performed according to Example 4 below are shown. Results using ELISA are shown. ELISA data are expressed as IFN-γ levels in the supernatants of TIL cultures of 5 x 105 TILs / well as measured by ELISA (logarithmic scale). P values ​​were calculated using a two-tailed Mann-Whitney test (unpaired). [Figure 19A]

[0051] Lytic capacity of TILs is shown. LU50 of target cells normalized to 10 TILs at 4 hours in co-culture (TIL effector cells and GFP+P815 target cells) is shown. [Figure 19B]

[0051] Lytic capacity of TILs. LU50 of target cells normalized to 106 TILs at 24 hours in co-culture (TIL effector cells and GFP+P815 target cells) is shown. [Figure 20A]

[0052] Figure 20A shows the cytolytic activity of different TILs against allogeneic and autologous tumor types. Figure 20B shows the cytolytic activity of melanoma TILs against allogeneic 526 target cells. Data in Figure 20A are presented as percent dead cells in co-cultures with an effector:target cell (E:T) ratio of 50:1. [Figure 20B]

[0052] Figure 20 shows the cytolytic activity of different TILs against allogeneic and autologous tumor types. Figure 20 shows the cytolytic activity of lymphoma TILs against autologous tumor cells as determined by 7-AAD uptake. Data in Figure 20B are presented as percent dead cells in co-cultures at an effector:target cell (E:T) ratio of 50:1. [Figure 20C]

[0052] Figure 1 shows the cytolytic activity of different TILs against allogeneic and autologous tumor types. Figure 2 shows the percentage killing of target cells induced by melanoma TILs. [Figure 20D]

[0052] Figure 1 shows the cytolytic activity of different TILs against allogeneic and autologous tumor types. Figure 2 shows the percentage killing of target cells induced by lymphoma TILs at different E:T ratios. [Figure 21]

[0053] This figure shows a heat map depicting the gene expression profiles of lymphoma and melanoma TILs. The expression profiles were determined using NanoString's 579-plex nCounter GX Human Immunology V2 CSO panel. The heat map demonstrates changes in the expression of specific gene sets in lymphoma TILs compared to melanoma TILs, suggesting higher expression of IL-17A and RORC from lymphoma-derived TILs. Cancers depicted in this figure include follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), and mantle cell lymphoma (MCL). [Figure 22]

[0054] FIG. 2A is a schematic diagram showing the 2A process of TIL preparation, harvesting and shipping schedule. [Figure 23]

[0055] 2A is a flowchart illustrating the 2A process for preparing TILs. [Figure 24A]

[0056] 1 is a flow chart showing three different methods for expanding peripheral blood lymphocytes (PBLs). [Figure 24B]

[0056] Figure 1 is a flow chart showing three different methods for expanding peripheral blood lymphocytes (PBLs). [Figure 24C]

[0056] Figure 1 is a flow chart showing three different methods for expanding peripheral blood lymphocytes (PBLs). [Figure 25A]

[0057] Three different methods for expanding bone marrow-infiltrating lymphocytes (MIL) from bone marrow are shown. [Figure 25B]

[0057] Three different methods for expanding bone marrow infiltrating lymphocytes (MIL) from bone marrow are presented. [Figure 25C]

[0057] Three different methods for expanding bone marrow infiltrating lymphocytes (MIL) from bone marrow are presented. [Figure 26]

[0058] Figures 26-34 depict graphs of fold expansion of PBLs isolated from fresh peripheral blood mononuclear cells (PBMCs) and cryopreserved PBMCs. Cryopreserved PBMCs were derived from patients with CLL who were either untreated (pre-Rx PBLs) or treated (post-Rx PBLs) with an ibrutinib regimen. For Figures 26-34, each point represents one patient. Shaded points represent patients whose PBLs were expanded using PBL Method 1; open points represent patients whose PBLs were expanded using PBL Method 2; and solid points represent patients whose PBLs were expanded using PBL Method 3. [Figure 27]

[0059] 1 shows a graph of IFN-γ producing cells in PBLs isolated from fresh and cryopreserved PBMCs. Pre-Rx and post-Rx PBLs are also shown in cryopreserved PBMCs. [Figure 28]

[0060] Percentages of CD4+ and CD8+ T cell subsets in pre-Rx and post-Rx PBLs are presented, using melanoma TILs as comparators. [Figure 29A]

[0061] Comparison between CD4 memory subsets of pre-Rx and post-Rx PBLs using melanoma TILs as a comparator. Data for naive (CCR7+ / CD45RA+) PBLs are shown. [Figure 29B]

[0061] Figure 1 shows a comparison between CD4 memory subsets of pre-Rx and post-Rx PBLs using melanoma TILs as a comparator. Data for central memory t cells (CM) (CCR7+ / CD45RA-) are shown. [Figure 29C]

[0061] Figure 1 shows a comparison between CD4 memory subsets of pre-Rx and post-Rx PBLs using melanoma TILs as a comparator. Data for effector memory T cells (EM) (CCR7- / CD45RA-) are shown. [Figure 29D]

[0061] Figure 1 shows a comparison between CD4 memory subsets of pre-Rx and post-Rx PBLs using melanoma TILs as a comparator. Data for terminally differentiated effector memory cells (TEMRA) (CCR7- / CD45RA+) are shown. [Figure 30A]

[0061] Comparison between CD8 memory subsets of pre-Rx and post-Rx PBLs using melanoma TILs as a comparator. Data for naive (CCR7+ / CD45RA+) is shown. [Figure 30B]

[0061] Figure 1 shows a comparison between CD8 memory subsets of pre-Rx and post-Rx PBLs using melanoma TILs as a comparator. Data for central memory t cells (CM) (CCR7+ / CD45RA-) are shown. [Figure 30C]

[0061] Comparison between CD8 memory subsets of pre-Rx and post-Rx PBLs using melanoma TILs as a comparator. Data for effector memory T cells (EM) (CCR7- / CD45RA-) are shown. [Figure 30D]

[0061] Figure 1 shows a comparison between CD8 memory subsets of pre-Rx and post-Rx PBLs using melanoma TILs as a comparator. Data for terminally differentiated effector memory cells (TEMRA) (CCR7- / CD45RA+) are shown. [Figure 31A]

[0062] A comparison between CD27 subsets of CD4 subsets in pre-Rx and post-Rx PBLs is shown using melanoma TILs as a comparator. [Figure 31B]

[0062] Comparison between CD27 subsets of CD8 subsets in pre-Rx and post-Rx PBLs using melanoma TILs as comparators. [Figure 32A]

[0063] A comparison between CD28 subsets of CD4 subsets in pre-Rx and post-Rx PBLs is shown using melanoma TILs as a comparator. [Figure 32B]

[0063] Comparison between CD28 subsets of CD8 subsets of pre-Rx PBLs and post-Rx PBLs using melanoma TILs as comparators. [Figure 33A]

[0064] A comparison of the LAG3+ subset within the CD4 population of both pre-Rx and post-Rx PBLs is shown. [Figure 33B]

[0064] Comparison of LAG3+ subsets within the CD8 population in both pre-Rx and post-Rx PBLs is shown. [Figure 34A]

[0065] A comparison of the PD1+ subset within the CD4 population of both pre-Rx and post-Rx PBLs is shown. [Figure 34B]

[0065] Comparison of PD1+ subsets within the CD8 population in both pre-Rx and post-Rx PBLs. [Figure 35A]

[0066] Figure 1 shows the results of the cytolytic activity of pre-Rx PBLs measured using an autologous tumor killing assay. Cytotoxicity is measured as LU50 (the number of PBLs required to kill 50% of the target cells). [Figure 35B]

[0066] Figure 1 shows the results of the cytolytic activity of post-Rx PBLs measured using an autologous tumor killing assay. Cytotoxicity is measured as LU50 (the number of PBLs required to kill 50% of the target cells). [Figure 36A]

[0067] This figure shows a graph of the fold expansion of MILs isolated from either bone marrow (MIL) or peripheral blood (PBL) of AML patients. MIL1.1 was expanded using MIL method 1, MIL1.2 was expanded using MIL method 2, and MIL1.3 was expanded using MIL method 3. MIL2 and MIL3 were expanded using MIL method 3. All 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, and the starting cell number for MIL3 was 28,000 cells. The starting cell number for PBL2 was 338,000, and the starting cell number for PBL3 was 336,000. [Figure 36B]

[0067] A graph of the fold expansion of PBLs isolated from either bone marrow (MIL) or peripheral blood (PBL) of AML patients is presented. MIL1.1 was expanded using MIL method 1, MIL1.2 was expanded using MIL method 2, and MIL1.3 was expanded using MIL method 3. MIL2 and MIL3 were expanded using MIL method 3. All 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, and the starting cell number for MIL3 was 28,000 cells. The starting cell number for PBL2 was 338,000, and the starting cell number for PBL3 was 336,000. [Figure 37A]

[0068] Each IFN-γ-producing cell in the MIL is shown. [Figure 37B]

[0068] The IFN-γ-producing cells of each PBL are shown. [Figure 38A]

[0069] Graphs depicting T cell memory subsets in MILs isolated from AML patients. TCRαβ+ subsets are shown. PBLs are shown on days 0 and 14. [Figure 38B]

[0069] Figure 1 shows graphs showing T cell memory subsets of MILs isolated from AML patients. CD4+ subsets are shown. PBLs are shown on days 0 and 14. [Figure 38C]

[0069] Figure 1 shows graphs showing T cell memory subsets of MILs isolated from AML patients. CD8 subsets are shown. PBLs are shown on days 0 and 14. [Figure 38D]

[0069] Figure 1 shows a graph depicting T cell memory subsets of PBL isolated from an AML patient. The TCRαβ+ subset is shown. PBL are shown on days 0 and 14. [Figure 38E]

[0069] Figure 1 shows graphs showing T cell memory subsets of PBLs isolated from AML patients. CD4+ subsets are shown. PBLs are shown on days 0 and 14. [Figure 38F]

[0069] Figure 1 shows graphs showing T cell memory subsets of PBLs isolated from AML patients. CD8 subsets are shown. PBLs are shown on days 0 and 14. [Figure 39A]

[0070] 1 shows a graph showing CD4 memory subsets of MILs isolated from AML patients. Data for naive (CCR7+ / CD45RA+) mice are shown. [Figure 39B]

[0070] Figure 1 shows a graph showing CD4 memory subsets of MILs isolated from AML patients. Data for central memory t cells (CM) (CCR7+ / CD45RA-) are shown. [Figure 39C]

[0070] Figure 1 shows a graph showing CD4 memory subsets of MILs isolated from AML patients. Data for effector memory T cells (EM) (CCR7- / CD45RA-) are shown. [Figure 39D]

[0070] Figure 1 shows a graph showing CD4 memory subsets of MILs isolated from AML patients. Data for terminally differentiated effector memory cells (TEMRA) (CCR7- / CD45RA+) are shown. [Figure 40A]

[0071] 1 depicts a graph showing CD4 memory subsets in PBL isolated from AML patients, with data shown for naive (CCR7+ / CD45RA+) PBLs. [Figure 40B]

[0071] Figure 1 shows a graph showing CD4 memory subsets of PBL isolated from AML patients. Data for central memory t cells (CM) (CCR7+ / CD45RA-) are shown. [Figure 40C]

[0071] Figure 1 shows a graph showing CD4 memory subsets of PBL isolated from AML patients. Data for effector memory T cells (EM) (CCR7- / CD45RA-) are shown. [Figure 40D]

[0071] Figure 1 shows a graph showing CD4 memory subsets of PBL isolated from AML patients. Data for terminally differentiated effector memory cells (TEMRA) (CCR7- / CD45RA+) are shown. [Figure 41A]

[0072] 1 shows a graph depicting the CD8 memory subset of MILs isolated from AML patients. Data for naive (CCR7+ / CD45RA+) mice are shown. [Figure 41B]

[0072] Figure 1 shows a graph showing CD8 memory subsets of MILs isolated from AML patients. Data for central memory t cells (CM) (CCR7+ / CD45RA-) are shown. [Figure 41C]

[0072] Figure 1 shows a graph showing CD8 memory subsets of MILs isolated from AML patients. Data for effector memory T cells (EM) (CCR7- / CD45RA-) are shown. [Figure 41D]

[0072] Figure 1 shows a graph showing CD8 memory subsets of MILs isolated from AML patients. Data for terminally differentiated effector memory cells (TEMRA) (CCR7- / CD45RA+) are shown. [Figure 42A]

[0073] 1 shows a graph depicting the CD8 memory subset of PBL isolated from AML patients, with data shown for naive (CCR7+ / CD45RA+) PBLs. [Figure 42B]

[0073] Figure 1 shows a graph showing CD8 memory subsets of PBL isolated from AML patients. Data for central memory t cells (CM) (CCR7+ / CD45RA-) are shown. [Figure 42C]

[0073] Figure 1 shows a graph showing CD8 memory subsets of PBL isolated from AML patients. Data for effector memory T cells (EM) (CCR7- / CD45RA-) are shown. [Figure 42D]

[0073] Figure 1 shows a graph showing CD8 memory subsets of PBL isolated from AML patients. Data for terminally differentiated effector memory cells (TEMRA) (CCR7- / CD45RA+) are shown. [Figure 43A]

[0074] 1 depicts a graph showing CD27 subsets in CD4 and CD8 cell populations of MILs. [Figure 43B]

[0074] Figure 1 shows a graph showing CD27 subsets in CD4 and CD8 cell populations of PBL. [Figure 44A]

[0075] 1 depicts a graph showing the CD28 subset of CD4 and CD8 cell populations in MILs. [Figure 44B]

[0075] Figure 1 shows a graph showing the CD28 subset of CD4 and CD8 cell populations of PBLs. [Figure 45A]

[0076] Graphs depicting PD1+ subsets of CD4 and CD8 cell populations in MILs. [Figure 45B]

[0076] Graph showing PD1+ subsets of CD4 and CD8 cell populations of PBLs. [Figure 46A]

[0077] 1 depicts a graph showing the LAG3+ subset of CD4 and CD8 cell populations in MILs. [Figure 46B]

[0077] Figure 1 shows a graph showing the LAG3+ subset of CD4 and CD8 cell populations in PBL. [Figure 47]

[0078] 1 is a timeline showing exemplary embodiments of PBL Method 1 and PBL Method 3. In this diagram, the addition of IL-2 can occur at any point during the process, in exemplary embodiments, above the bracketed area. [Figure 48]

[0079] 1 is a timeline illustrating an exemplary embodiment of MIL Method 3. In this figure, the addition of IL-2 can occur at any point during the process, in the exemplary embodiment, above the bracketed area. [Figure 49]

[0080] 1 is an exemplary embodiment of the present invention showing a method for expanding PBLs (including PBLs from patients pretreated with ibrutinib) useful for the treatment of hematological malignancies such as CLL, as described herein. DETAILED DESCRIPTION OF THE INVENTION

[0034] Brief Description of Sequence Listing

[0081] SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

[0035]

[0082] SEQ ID NO: 2 is the amino acid sequence of the light chain of muromonab.

[0036]

[0083] SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

[0037]

[0084] SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

[0038]

[0085] SEQ ID NO: 5 is the amino acid sequence of recombinant human IL-4 protein.

[0039]

[0086] SEQ ID NO: 6 is the amino acid sequence of recombinant human IL-7 protein.

[0040]

[0087] SEQ ID NO: 7 is the amino acid sequence of recombinant human IL-15 protein.

[0041]

[0088] SEQ ID NO: 8 is the amino acid sequence of recombinant human IL-21 protein.

[0042] Detailed Description of the Invention

[0089] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications mentioned herein are incorporated by reference in their entirety.

[0043] definition

[0090] As used herein, the terms "co-administration," "co-administering," "administered in combination with," "administering in combination with," "simultaneous," and "concurrent" encompass administration of two or more active pharmaceutical ingredients to a subject such that both active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Concurrent administration in separate compositions and administration in a composition in which both agents are present are preferred.

[0044]

[0091] The term "in vivo" refers to events that take place inside the body of a mammalian subject.

[0045]

[0092] The term "ex vivo" refers to events that take place outside the body of a mammalian subject in an artificial environment.

[0046]

[0093] The term "in vitro" refers to events that take place in a test system. In vitro assays include cell-based assays in which viable or dead cells can be used, and can also include cell-free assays, where no intact cells are used.

[0047]

[0094] The term "rapid expansion" refers to an increase in the number of antigen-specific TILs of at least about 3-fold (or 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold) over a one-week period, more preferably at least about 10-fold (or 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, or 90-fold) over a one-week period, or most preferably at least about 100-fold over a one-week period. Several rapid expansion protocols are described herein.

[0048]

[0095] The terms "fragmentation," "fragments," and "fragmented" as used herein to describe the process of disrupting tumors include mechanical fragmentation methods such as crushing, slicing, splitting, and mincing tumor tissue, as well as any other method that disrupts the physical structure of tumor tissue.

[0049]

[0096] 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, the peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells. PBMCs contain antigen-presenting cells. The term "PBLs" refers to peripheral blood lymphocytes, which are T cells expanded from peripheral blood. The terms PBLs and TILs are used interchangeably herein.

[0050]

[0097] The term "anti-CD3 antibody" refers to an antibody or variant thereof, e.g., a monoclonal antibody, including human, humanized, chimeric, or murine antibodies directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab, and UCHT-1. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0051]

[0098] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody, including a human, humanized, chimeric, or murine antibody, or a biosimilar or variant thereof, directed against the CD3 receptor in the T cell antigen receptor of mature T cells, including OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and commercially available forms such as muromonab or its variants, conservative amino acid substitutions, glycoforms, or biosimilars. The amino acid sequences of the heavy and light chains of muromonab are shown in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). A hybridoma capable of producing OKT-3 has been deposited with the American Type Culture Collection and assigned ATCC accession number CRL 8001. A hybridoma capable of producing OKT-3 has also been deposited with the European Collection of Authenticated Cell Cultures (ECACC) and assigned catalog number 86022706.

[0052] Table 1

[0053]

[0099] The term "IL-2" (also referred to herein as "IL2") refers to the T-cell growth factor known as interleukin-2 and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 3). For example, the term IL-2 encompasses human recombinant forms of IL-2, such as aldesleukin (PROLEUKIN, commercially available from multiple sources at 22 million IU per single-use vial) and commercially available forms of recombinant IL-2 from CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Catalog No. CYT-209-b), as well as other commercially available equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 4). The term IL-2, as described herein, also encompasses pegylated forms of IL-2, including the pegylated IL2 prodrug NKTR-214, available from Nektar Therapeutics, South San Francisco, CA, USA. NKTR-214 and pegylated IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication No. 2014 / 0328791A1 and WO 2012 / 065086A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 suitable for use in the present invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261, and 4,902,502, the disclosures of which are incorporated herein by reference.Formulations of IL-2 suitable for use in the present invention are described in US Pat. No. 6,706,289, the disclosure of which is incorporated herein by reference.

[0054] [Table 2]

[0055]

[0100] The term "IL-4" (also referred to herein as "IL4") refers to the cytokine known as interleukin 4, which is produced by Th2 T cells and by eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, inducing class switching from B cells to IgE and IgG1 expression. Recombinant human IL-4 suitable for use in the present invention is commercially available from several sources, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 5).

[0056]

[0101] The term "IL-7" (also referred to herein as "IL7") refers to a glycosylated tissue-derived cytokine known as interleukin-7, which is available from stromal and epithelial cells as well as dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate the development of T cells. IL-7 binds to the IL-7 receptor, a heterodimer consisting of the IL-7 receptor alpha and the common gamma chain receptor, in a series of signals important for T cell development in the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in the present invention is commercially available from several sources, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-7 recombinant protein, catalog number Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 6).

[0057]

[0102] The term "IL-15" (also referred to herein as "IL15") refers to the T-cell growth factor known as interleukin-15 and includes all forms of IL-15, including human and mammalian forms, 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 β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is commercially available from several sources, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 7).

[0058]

[0103] The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21, including human and mammalian forms, 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 primarily stimulates natural killer T cells and activated human CD4 +It is produced by T cells. Recombinant human IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular weight of 15.4 kDa. Recombinant human IL-21 is commercially available from several sources, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, catalog number 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 8).

[0059]

[0104] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inactive ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the present invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the compositions and methods described.

[0060]

[0105] The terms "antibody" and "antibodies" refer to whole immunoglobulins and any antigen-binding fragment ("antigen-binding portion") or single chains thereof. "Antibody" also refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or antigen-binding portions thereof. Each heavy chain comprises a heavy chain variable region (referred to herein as V H Each light chain is composed of a light chain variable region (abbreviated herein as V) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. L The light chain constant region is composed of one domain, CL. The V of an antibody H and V LThe regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs) or hypervariable regions (HVRs), which can be interspersed with more conserved regions, called framework regions (FRs). H and V L is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with one or more antigen epitopes. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0061]

[0106] The term "antigen" refers to a substance that induces an immune response. In some embodiments, an antigen is a molecule that can be bound by an antibody or TCR when presented by a major histocompatibility complex (MHC) molecule. As used herein, the term "antigen" also encompasses T cell epitopes. An antigen can be further recognized by the immune system. In some embodiments, an antigen can induce a humoral or cellular immune response that results in the activation of B and / or T lymphocytes. In some cases, this may require that the antigen contains or binds to a Th cell epitope. An antigen may also have one or more epitopes (e.g., a B epitope and a T epitope). In some embodiments, an antigen preferably reacts with its corresponding antibody or TCR, typically in a highly specific and selective manner, and does not react with many other antibodies or TCRs that may be induced by other antigens.

[0062]

[0107] The terms "monoclonal antibody," "mAb," "monoclonal antibody composition," or their plurals, refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Monoclonal antibodies specific for a particular receptor can be prepared using knowledge and techniques in the art by injecting a test subject with the appropriate antigen and then isolating hybridomas expressing antibodies with the desired sequence or functional characteristics. DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells such as Escherichia coli (E. coli) cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of the monoclonal antibody in the recombinant host cells. Recombinant production of antibodies is described in more detail below.

[0063]

[0108] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) V L , V H , C L (ii) a Fab fragment, which is a monovalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and an Fd fragment consisting of the CH1 domain; (iv) a V of a single arm of an antibody L and V H Fv fragment consisting of domains, (v) V H or VL (v) domain antibody (dAb) fragments, which may consist of two domains, V and V (Ward, et al., Nature, 1989, 341, 544-546); and (vi) isolated complementarity-determining regions (CDRs). In addition, the two domains of the Fv fragment, V, and V, may be isolated. L and V H are encoded by separate genes, which can be synthesized by recombinant methods. L and V H Pairs of domains can be linked by a synthetic linker, allowing them to be produced as a single protein chain, forming monovalent molecules known as single-chain Fvs (scFvs); see, e.g., Bird, et al., Science 1988, 242, 423-426; and Huston, et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883. Such scFv antibodies are also intended to be encompassed within 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 fragments are screened for utility in the same manner as intact antibodies.

[0064]

[0109] As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). 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, have been grafted onto human framework sequences.

[0065]

[0110] The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are obtained from a transgenic non-human animal, e.g., a transgenic mouse, and are produced by a hybridoma comprising B cells whose genome comprises human heavy chain and light chain transgenes fused to an immortalized cell.

[0066]

[0111] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom (described further below); (b) antibodies isolated from host cells, e.g., transfectomas, that have been 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, including splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework 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 in vivo somatic mutagenesis, when animals transgenic for human Ig sequences are used) to thereby modify the V and V sequences of the recombinant antibodies. H and V L The amino acid sequence of the region is similar to that of the human germline V H and V L These are sequences that, while derived from and related to sequences, may not naturally occur within the human antibody germline repertoire in vivo.

[0067]

[0112] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0068]

[0113] 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."

[0069]

[0114] The term "human antibody derivative" refers to any mutant form of a human antibody, including a conjugate of the antibody with another active pharmaceutical ingredient or antibody. The terms "conjugate," "antibody-drug conjugate," "ADC," or "immunoconjugate" refer 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.

[0070]

[0115] The terms "humanized antibody" or "humanized antibodies" and "humanization" are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, have been 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 containing minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the recipient's hypervariable region have been replaced by residues from 15 hypervariable regions of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, possessing the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin have been replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally 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, Curr. Op. Struct. Biol. 1992, 2, 593-596. The antibodies described herein can also be modified to use any Fc variants known to confer improved (e.g., decreased) effector function and / or FcR binding.Fc variants are described in, for example, WO 1988 / 07089A1, WO 1996 / 14339A1, WO 1998 / 05787A1, WO 1998 / 23289A1, WO 1999 / 51642A1, WO 99 / 58572A1, WO 2000 / 09560A2, WO 2000 / 32767A1, WO 2000 / 42072A2, WO 2002 / 44215A2, WO 2003 / 09560A2, WO 2004 / 09560A3, WO 2004 / 09560A4, WO 2004 / 09560A5, WO 2004 / 09560A6, WO 2004 / 09560A7, WO 2004 / 09560A8, WO 2004 / 09560A9, WO 2004 / 09560A1, WO 2004 / 09560A1, WO 2004 / 09560A1, WO 2004 / 09560A1, WO 2004 / 09560A2 ...3, WO 2004 / 09560A4, WO 2004 / 09560A5, WO 2004 / 09560A6, WO 2004 / 09560A7, WO 200 No. 2002 / 060919A2, No. 2003 / 074569A2, No. 2004 / 016750A2, No. 2004 / 029207A2, No. 2004 / 035752A2, No. 2004 / 063351A2, 2004 / 074455A2, 2004 / 099249A2, 2005 / 040217A2, 2005 / 070963A1, 20 Nos. 05 / 077981A2, 2005 / 092925A2, 2005 / 123780A2, 2006 / 019447A1, 2006 / 047350A2 and 2006 / 085967A2; and U.S. Patent Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6, 121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; and 7,083,784, the disclosures of which are incorporated herein by reference.

[0071]

[0116] The term "chimeric antibody" is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as antibodies in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0072]

[0117] A "diabody" is a small antibody fragment with two antigen-binding sites. The fragments are bound to the same polypeptide chain (V H -V L or V L -V H) in the light chain variable domain (V L ) connected to the heavy chain variable domain (V H ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains on another chain and create two antigen-binding sites. Diabodies are further described in, for example, EP 404,097; WO 93 / 11161; and Bolliger, et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.

[0073]

[0118] The term "glycosylation" refers to an engineered derivative of an antibody. An aglycosylated antibody lacks glycosylation. Glycosylation can be altered, for example, to increase the affinity of an antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the removal of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at those sites. As described in U.S. Pat. Nos. 5,714,350 and 6,350,861, aglycosylation can increase the affinity of an antibody for an antigen. Additionally or alternatively, antibodies can be generated with altered glycosylation types, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase antibody potency. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the invention, thereby producing antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (alpha(1,6)fucosyltransferase), and therefore antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were generated by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see, e.g., U.S. Patent Application Publication No. 2004 / 0110704 or Yamane-Ohnuki, et al., Biotechnol. Bioeng., 2004, 87, 614-622).As another example, European Patent No. 1,176,195 describes a cell line having a functionally disrupted FUT8 gene encoding fucosyltransferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating alpha-1,6 bond-related enzymes, and also describes a cell line with low or no enzymatic activity that adds fucose to N-acetylglucosamine attached to the Fc region of an antibody, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). WO 03 / 035835 describes a mutant CHO cell line, Lec13 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates and results in hypofucosylation of antibodies expressed in the host cells (see also Shields, et al., J. Biol Chem. 2002, 277, 26733-26740). WO 99 / 54342 describes cell lines that have been engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(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 (Umana, et al., Nat. Biotech. 1999, 17, (See also, e.g., 176-180.) Alternatively, the fucose residues of antibodies can be cleaved using a fucosidase enzyme. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies as described in Tarentino, et al., Biochem. 1975, 14, 5516-5523.

[0074]

[0119] "PEGylation" refers to a modified antibody or fragment thereof that is reacted with polyethylene glycol (PEG), typically a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups become attached to the antibody or antibody fragment. PEGylation can, for example, increase the biological (e.g., serum) half-life of the antibody. Preferably, PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to a mono(C1-C 10 PEG is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. The antibody to be pegylated may be an aglycosylated antibody. Methods of pegylation are known in the art and can be applied to the antibodies of the invention, for example, as described in European Patent Nos. 0154316 and 0401384 and U.S. Patent No. 5,824,778, the disclosures of each of which are incorporated herein by reference.

[0075]

[0120] The term "fusion protein" or "fusion polypeptide" refers to a protein that combines the properties of two or more individual proteins. Such proteins contain at least two heterologous polypeptides covalently linked, either directly or via an amino acid linker. The polypeptides forming the fusion protein are typically linked C-terminally to N-terminally, but can also be linked C-terminally to C-terminally, N-terminally to N-terminally, or N-terminally to C-terminally. The polypeptides of a fusion protein can be in any order and can include any two or more of the constituent polypeptides, or both. The term encompasses conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, interspecies homologs, and immunogenic fragments of the antigens that make up the fusion protein. Fusion proteins of the present disclosure can also contain additional copies of the component antigens or immunogenic fragments thereof. Fusion proteins can contain one or more binding domains linked to each other and further linked to an Fc domain, such as an IgG Fc domain. Fusion proteins can be further linked together to mimic monoclonal antibodies and provide six or more binding domains. Fusion proteins can be produced by recombinant methods, as known in the art. The preparation of fusion proteins is known in the art and is described, for example, in International Publication Nos. WO 1995 / 027735A1, WO 2005 / 103077A1, WO 2008 / 025516A1, WO 2009 / 007120A1, WO 2010 / 003766A1, WO 2010 / 010051A1, WO 2010 / 078966A1, U.S. Patent Application Publication Nos. 2015 / 0125419A1 and 2016 / 0272695A1, and U.S. Patent No. 8,921,519, the disclosures of each of which are incorporated herein by reference.

[0076]

[0121] The term "heterologous" when used with reference to portions of a nucleic acid or protein indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are typically produced recombinantly, with two or more sequences from unrelated genes, such as a promoter from one source and a coding region from another source, arranged to create a new functional nucleic acid. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0077]

[0122] The term "conservative amino acid substitution" refers to an amino acid sequence modification that does not abolish the binding of an antibody or fusion protein to an antigen. Conservative amino acid substitutions include the substitution of an amino acid of one class with an amino acid of the same class, where the class is defined by common physicochemical amino acid side chain properties and high substitution frequency in naturally occurring homologous proteins, as determined, for example, by standard Dayhoff frequency exchange matrices or BLOSUM matrices. Six general classes of amino acid side chains have been classified, including class I (Cys); class II (Ser, Thr, Pro, Ala, Gly); class III (Asn, Asp, Gln, Glu); class IV (His, Arg, Lys); class V (Ile, Leu, Val, Met); and class VI (Phe, Tyr, Trp). For example, substitution of Asp for another class III residue, such as Asn, Gln, or Glu, is a conservative substitution. Thus, predicted non-essential amino acid residues in an antibody are preferably substituted with another amino acid residue from the same class. Methods for identifying conservative amino acid substitutions that do not eliminate 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 (1999); and Burks, et al., Proc. Natl. Acad. Sci. USA 1997, 94, 412-417).

[0078]

[0123] The terms "sequence identity," "percent identity," and "sequence percent identity" (or their synonyms, e.g., "99% identical"), in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of the same nucleotides or amino acid residues when compared and aligned for maximum correspondence (introducing gaps as necessary), without considering conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to align 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 U.S. government's National Center for Biotechnology Information. Comparisons between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign available from DNASTAR are additional publicly available software programs that can be used to align sequences. Those skilled in the art can determine appropriate parameters for maximal alignment depending on the particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0079]

[0124] The term "variant" as used herein includes, but is not limited to, antibodies or fusion proteins that contain an amino acid sequence that differs from that of a reference antibody by one or more substitutions, deletions, and / or additions at specific positions within or adjacent to the amino acid sequence 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 include, for example, substitutions of amino acids that are similarly charged or uncharged. A variant retains the ability of the reference antibody to specifically bind to an antigen. The term variant also includes pegylated antibodies or proteins.

[0080]

[0125] Nucleic acid sequences implicitly encompass not only the explicitly set forth sequence, but also its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base 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 is used interchangeably with cDNA, mRNA, oligonucleotide, and polynucleotide.

[0081]

[0126] The term "biosimilar" refers to a biological product, including a monoclonal antibody or protein, that is highly similar to a U.S.-approved reference biological product, despite minor differences in clinically inactive components, and there are no clinically meaningful differences between the biological product and the reference product in terms of product safety, purity, and potency. Furthermore, a similar biological or "biosimilar" drug is a biological drug similar to another biological drug already approved for use by the European Medicines Agency. The term "biosimilar" is also used interchangeably by regulatory agencies in other countries and regions. Biological products or biological medicines are medicines made by 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, a protein approved by a drug regulatory agency for aldesleukin is a "biosimilar to" or "biosimilar of" aldesleukin. In Europe, a similar biological or "biosimilar" medicinal product is a biological product similar to another biological product already authorized for use by the European Medicines Agency (EMA). The relevant legal basis for similar biological uses in Europe is Article 6 of Regulation (EC) No. 726 / 2004, as amended, and Article 10(4) of Directive 2001 / 83 / EC. Therefore, in Europe, a biosimilar may be authorized, granted authorization, or the subject of an authorization application under Article 6 of Regulation (EC) No. 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. In Europe, the original biological product already authorized is sometimes referred to as the "reference medicinal product." Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP guidelines on similar biological medicinal products. Additionally, product-specific guidelines, including those for monoclonal antibody biosimilars, are provided by the EMA on a product-by-product basis and are published on its website.Biosimilars described herein may be similar to the reference medicinal product in terms of quality attributes, biological activity, mechanism of action, safety profile, and / or efficacy. Furthermore, biosimilars may be used or intended for use to treat the same condition as the reference medicinal product. Thus, biosimilars described herein may be considered to have similar or very similar quality attributes to the reference medicinal product. Alternatively or additionally, biosimilars described herein may be considered to have similar or very similar biological activity to the reference medicinal product. Alternatively or additionally, biosimilars described herein may be considered to have a similar or very similar safety profile to the reference medicinal product. Alternatively or additionally, biosimilars described herein may be considered to have similar or very similar efficacy to the reference medicinal product. As described herein, biosimilars in Europe are compared to reference medicinal products authorized by the EMA. However, in some cases, biosimilars may be compared to biological medicinal products authorized outside the European Economic Area (non-EEA "comparators") in specific studies. Such studies include, for example, specific clinical trials and in vivo non-clinical studies. As used herein, the term "biosimilar" also refers to a biological drug that has been or can be compared to a non-EEA-approved comparator. Particular biosimilars are proteins, such as antibodies, antibody fragments (e.g., antigen-binding portions), and fusion proteins. Protein biosimilars may have amino acid sequences with minor modifications to the amino acid structure (e.g., including amino acid deletions, additions, and / or substitutions) that do not significantly affect the function of the polypeptide. A biosimilar may include an amino acid sequence with 97% or greater sequence identity, e.g., 97%, 98%, 99%, or 100%, to the amino acid sequence of its reference drug. A biosimilar may include one or more post-translational modifications that differ from those of the reference drug, such as, but not limited to, glycosylation, oxidation, deamidation, and / or cleavage, provided that the differences do not result in changes in the safety and / or efficacy of the drug. A biosimilar may have the same or a different glycosylation pattern as the reference drug.Although not exclusively, biosimilars may have different glycosylation patterns, particularly if the differences address or are intended to address safety concerns related to the reference drug. Furthermore, biosimilars may deviate from the reference drug, for example, in its strength, pharmaceutical form, formulation, excipients, and / or presentation, provided that the drug's safety and efficacy are not compromised. Biosimilars may contain differences, for example, in their pharmacokinetic (PK) and / or pharmacodynamic (PD) profiles compared to the reference drug, but still be deemed sufficiently similar to the reference drug to be approved or considered suitable for approval. In certain circumstances, biosimilars exhibit different binding properties compared to the reference drug, where the different binding properties are not considered by regulatory authorities, such as the EMA, to be a barrier to approval as a similar biological product. The term "biosimilar" is also used interchangeably by regulatory agencies in other countries and regions.

[0082]

[0127] The term "hematologic malignancies" refers to mammalian cancers and tumors of hematopoietic and lymphoid tissues, including, but not limited to, blood, bone marrow, lymph nodes, and lymphatic tissues. Hematologic malignancies can result in the formation of "liquid tumors." Hematologic malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematologic malignancies" refers to hematologic malignancies affecting B cells.

[0083]

[0128] The term "liquid tumor" refers to an abnormal mass of cells that is fluid in nature. Liquid tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematological malignancies. TILs obtained from liquid tumors, including those present in the bone marrow, may also be referred to herein as bone marrow-infiltrating lymphocytes (MILs). TILs obtained from liquid tumors, including those circulating in the peripheral blood, may also be referred to herein as PBLs. The terms MILs, TILs, and PBLs are used interchangeably herein and differ only by the tissue type from which the cells are derived.

[0084]

[0129] The term "biopsy" refers to any medical procedure used to obtain cancer cells, including a bone marrow biopsy.

[0085]

[0130] The term "acute myeloid leukemia" or "AML" refers to a cancer of the myeloid blood cell lineage, also known in the art as acute myelogenous leukemia and acute nonlymphocytic leukemia. AML is a liquid tumor; however, some manifestations of AML, including extramedullary findings such as chloroma, exhibit characteristics of solid tumors but are classified herein as liquid tumors.

[0086]

[0131] As used herein, the term "microenvironment" may refer to the solid or hematologic tumor microenvironment as a whole or to individual subsets of cells within the microenvironment. As used herein, the tumor microenvironment refers to the complex mixture of cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect tumors from host immunity, cultivate therapeutic resistance, and provide a niche for successful and dominant metastasis, as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Tumors express antigens that are recognized by T cells, but tumor elimination by the immune system is rare due to immunosuppression by the microenvironment.

[0087]

[0132] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds described herein sufficient to achieve the intended use, including, but not limited to, disease treatment. A therapeutically effective amount may vary depending on the intended use (in vitro or in vivo) or the subject and disease state being treated (e.g., the subject's weight, age, and sex), the severity of the disease state, or the method of administration. The term also applies to a dose that induces a specific response in target cells (e.g., decreased platelet adhesion and / or cell migration). The specific dose will vary depending on the particular compound selected, the administration regimen to be followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system by which the compound is delivered.

[0088]

[0133] A "therapeutic effect," as that term is used herein, encompasses a therapeutic benefit and / or a prophylactic benefit. A prophylactic benefit includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

[0089]

[0134] The terms "treatment," "treating," "treating," and the like refer to achieving a desired pharmacological and / or physiological effect. The effect can be prophylactic, meaning that a disease or its symptoms are completely or partially prevented, and / or therapeutic, meaning that the disease and / or adverse effects resulting from the disease are partially or completely cured. "Treatment," as used herein, encompasses any treatment of disease in a mammal, particularly a human, and includes (a) preventing the occurrence of the disease in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., halting its development or progression; and (c) palliating the disease, i.e., causing regression of the disease and / or alleviating one or more disease symptoms. "Treatment" is also intended to encompass the delivery of an agent to provide a pharmacological effect even in the absence of a disease or condition. For example, "treatment" encompasses the delivery of a composition capable of eliciting an immune response or conferring immunity in the absence of a disease state, e.g., in the case of a vaccine.

[0090]

[0135] The terms "QD", "qd" or "qd" mean once a day, once a day, or once daily. The terms "BID", "bid" or "bid" mean twice a day, twice a day, or twice daily. The terms "TID", "tid" or "tid" mean three times a day, three times a day, or three times daily. The terms "QID", "qid" or "qid" mean four times a day, four times a day, or four times daily.

[0091]

[0136] As used herein, "tumor-infiltrating lymphocytes" or "TILs" refer to a population of cells originally obtained as white blood cells that have left a subject's bloodstream 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. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any TIL cell population that has been expanded or grown as discussed herein, including, but not limited to, bulk TILs, expanded TILs ("REP TILs"), and "reREP TILs" as discussed herein.

[0092]

[0137] 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, TCR αβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients. TILs can be further characterized by potency—for example, TILs can be considered potent if, for example, interferon (IFN) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL.

[0093]

[0138] As used herein, "cryopreserved TILs" (or cryopreserved MILs or PBLs) refers to TILs, either primary, bulk, or expanded culture (REP TILs), that are treated and stored at temperatures ranging from about -150°C to -60°C. General methods for cryopreservation are also described elsewhere herein, including in the Examples. For clarity, "cryopreserved TILs" are distinguishable from frozen tissue samples that may be used as a source of primary TILs.

[0094]

[0139] As used herein, "thawed cryopreserved TILs" (or thawed MILs or PBLs) refers to a population of TILs that have previously been cryopreserved and then treated to return to room temperature or above, including, but not limited to, cell culture temperature or a temperature at which the TILs can be administered to a patient.

[0095]

[0140] As used herein, a "population of cells" (including TILs) refers to several cells that share a common trait. Generally, a population is typically 1 x 10 6 ~1×10 10 For example, the initial proliferation of primary TILs in the presence of IL-2 ranges from approximately 1 x 10 8 Repeat expansion cultures typically yield a bulk TIL population of 1.5 x 10 cells for injection. 9 ~1.5×10 10 This is done to provide a population of individual cells.

[0096]

[0141] Generally, TILs are initially obtained from a patient tumor sample ("primary TILs"), then expanded into larger populations for further manipulation as described herein, optionally cryopreserved, restimulated as outlined herein, and optionally have phenotypic and metabolic parameters determined as indicators of TIL health.

[0097]

[0142] The collected cell suspension is generally referred to as a "primary cell population" or a "freshly harvested" cell population.

[0098]

[0143] Generally, as discussed herein, TILs are prepared by first obtaining a primary TIL population from a tumor resected from a patient as discussed herein (the "primary cell population" or "first cell population"). This is followed by an initial bulk expansion using culturing the cells with IL-2 to form a second cell population (sometimes referred to herein as the "bulk TIL population" or "second population").

[0099]

[0144] The term "cytotoxic lymphocytes" includes cytotoxic T (CTL) cells (CD8 + Cytotoxic T lymphocytes and CD4 + Cytotoxic lymphocytes include T-helper lymphocytes, natural killer T (NKT) cells, and natural killer (NK) cells. Cytotoxic lymphocytes can include, for example, peripheral blood-derived αβ TCR-positive T cells or γδ TCR-positive T cells activated by tumor-associated antigens and / or transduced with tumor-specific chimeric antigen receptors or T cell receptors, as well as tumor-infiltrating lymphocytes (TILs).

[0100]

[0145] The term "central memory T cells" refers to a subset of T cells that are CD45RO+ and constitutively express CCR7 (CCR7hi) and CD62L (CD62hi) in humans. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Central memory T cells primarily secrete IL-2 and CD40L as effector molecules after TCR triggering. Central memory T cells predominate in the CD4 compartment in the blood and are proportionally enriched in lymph nodes and tonsils in humans.

[0101]

[0146] The term "effector memory T cells" refers to a subset of human or mammalian T cells that, like central memory T cells, are CD45R0+ but lack constitutive expression of CCR7 (CCR7lo) and have heterogeneous or low CD62L expression (CD62Llo). 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. After antigen stimulation, effector memory T cells rapidly secrete high levels of inflammatory cytokines, including interferon-γ, IL-4, and IL-5. Effector memory T cells are predominant in the CD8 compartment of the blood and are proportionally enriched in the lungs, liver, and intestine in humans. CD8+ effector memory T cells possess large amounts of perforin. The term "closed system" refers to a system closed to the external environment. Any closed system suitable for cell culture methods can be used with the methods of the present invention. A closed system includes, but is not limited to, a sealed G-container, in which tumor segments are added and the system is not opened to the outside environment until the TILs are ready to be administered to a patient.

[0102]

[0147] In some embodiments, the disclosed methods further include a "pre-REP" stage in which tumor tissue or cells from tumor tissue are grown in standard laboratory medium (including, without limitation, RPMI) and treated with reagents such as irradiated feeder cells and anti-CD3 antibodies to achieve a desired effect, such as an increase in TIL numbers and / or enrichment of the population for cells containing desired cell surface markers or other structural, biochemical, or functional characteristics. The pre-REP stage may utilize laboratory-grade reagents (with the understanding that the laboratory-grade reagents will be diluted during the subsequent REP stage) to facilitate the incorporation of alternative strategies for improving TIL production. Thus, in some embodiments, the culture medium during the pre-REP stage may include the disclosed TLR agonists and / or peptides or peptidomimetics. The pre-REP culture may, in some embodiments, include IL-2. In preferred embodiments, the present invention relates to novel methods of enhancing restimulation TILs (also referred to herein as "reTILs") with one or more additional restimulation protocols, also referred to herein as "reREP," which unexpectedly lead to an expansion of memory T cell subsets, including memory effector T cell subsets, and / or lead to a significant enhancement of glycolytic respiration, when compared to freshly harvested TILs or thawed cryopreserved TILs for restimulation (also referred to herein as "reTILs"). That is, by using the reREP procedure on cryopreserved TILs, patients can receive highly metabolically active and healthy TILs, which may lead to better outcomes.

[0103]

[0148] When an "antitumor effective amount," "tumor suppression effective amount," or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject). Generally, pharmaceutical compositions comprising the genetically modified cytotoxic lymphocytes described herein are administered in an amount of 100 mg / kg or more. 4 ~10 11 cells / kg body weight (e.g., 10 5 ~10 6 , 10 5 ~10 10 , 105 ~10 11 , 10 6 ~10 10 , 10 6 ~10 11 ,10 7 ~10 11 , 10 7 ~10 10 , 10 8 ~10 11 , 10 8 ~10 10 , 10 9 ~10 11 or 10 9 ~10 10 It can be said that the genetically modified cytotoxic lymphocyte compositions can be administered at a dosage of 1000 to 10000 cells / kg body weight (including all integer values ​​within these ranges). The genetically modified cytotoxic lymphocyte compositions can also be administered multiple times at these dosages. The genetically modified cytotoxic lymphocytes can be administered by using injection techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by one skilled in the art of medicine by monitoring the patient for symptoms of disease and adjusting treatment accordingly.

[0104]

[0149] For the avoidance of doubt, it is intended that a particular feature (e.g., an integer, property, value, use, disease, formula, compound, or group) described herein in connection 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. Accordingly, such features may be used, where appropriate, with any of the definitions, claims, or embodiments defined herein. All features disclosed herein (including any accompanying claims, abstract, and drawings) and / or all steps of any method or process so disclosed may be combined in any combination, except for combinations in which at least some features and / or steps are mutually exclusive. The invention is not limited to any details of any disclosed embodiment. The invention extends to any novel or novel combination of features disclosed herein (including any accompanying claims, abstract, and drawings) or any novel or novel combination of steps of any method or process so disclosed.

[0105]

[0150] The terms "about" and "approximately" refer to a statistically significant range of values. Such a range may be within one order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The allowable variation encompassed by the terms "about" or "approximately" depends on the particular system under study and can be readily understood by one of ordinary skill in the art. Furthermore, as used herein, the terms "about" and "approximately" mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as appropriate, reflecting tolerances, conversion factors, rounding, measurement errors, etc., and other factors known to those of ordinary skill in the art. Generally, a dimension, size, formulation, parameter, shape, or other quantity or characteristic is "about" or "approximately" regardless of whether it is explicitly stated to be so. It should be noted that embodiments of widely different sizes, shapes, and dimensions may employ the described features.

[0106]

[0151] When used in the appended claims, in their original and amended forms, the transitional terms "comprising," "consisting essentially of," and "consisting of" define the subject matter of the claim with respect to what, if any, additional unrecited claim elements or steps would be excluded from the subject matter of the claim. 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" excludes any elements, steps, or materials other than those explicitly recited in the claim and, in the case of materials, normal impurities associated with the explicitly recited 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. All compositions, methods, and kits described herein embodying the present invention can be more specifically defined in alternative embodiments by any of the transitional terms "comprising," "essentially consisting of," and "consisting of."

[0107] Embodiments of methods for expanding therapeutic T cells containing peripheral blood (PBL) and / or bone marrow (MIL) A method for expanding peripheral blood lymphocytes (PBL) from peripheral blood

[0152] PBL Method 1. In one embodiment of the present invention, PBLs are expanded using the processes described herein. In one embodiment of the present invention, the method involves obtaining a PBMC sample from whole blood. In one embodiment, the method involves enriching T cells by isolating pure T cells from PBMCs using negative selection of the non-CD19+ fraction. On day 0, pure T cells are cultured with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a 1:1 ratio (beads:cells) and 3000 IU / ml of IL-2. On day 4, additional IL-2 is added to the culture at 3000 IU / ml. On day 7, the culture is again stimulated with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a 1:1 ratio (beads:cells), and 3000 IU / ml of additional IL-2 is added to the culture. On day 14, PBLs are harvested, the beads removed, and the PBLs are counted and phenotyped. In one embodiment, the method comprises enriching for T cells by separating pure T cells from PBMCs using magnetic bead-based negative selection of the non-CD19+ fraction.

[0108]

[0153] In one embodiment of the present invention, PBL Method 1 is performed as follows: On day 0, a cryopreserved PBMC sample is thawed and PBMCs are counted. T cells are isolated using a Human Pan T-cell Isolation Kit and LS columns (Miltenyi Biotec). Isolated T cells are counted and plated at 5 x 10 per well of a GRex 24-well plate. 5 Cells are seeded and co-cultured with DynaBeads® (anti-CD3 / anti-CD28) at a 1:1 ratio with 3000 IU / ml of IL-2 in a total of 8 ml of CM2 medium per well. On day 4, the medium in each well is replaced from CM2 with AIM-V containing 3000 IU / ml of fresh IL-2. On day 7, the expanded cells are harvested, counted, and then plated at 15 x 10 cells per flask in a GRex 10M flask in a total of 100 ml of AIM-V medium with 3000 IU / ml of IL-2 and DynaBeads® at a 1:1 ratio (beads:cells). 6On day 11, the medium is replaced with CM-4 medium supplemented with 3000 IU / ml of fresh IL-2. On day 14, the DynaBeads are removed using a DynaMag Magnet (DynaMag™-15) and the cells are counted.

[0109]

[0154] In one embodiment of the present invention, PBL Method 1 is performed as follows: On day 0, a cryopreserved PBMC sample is thawed and PBMCs are counted. T cells are isolated using a Human Pan T-cell Isolation Kit and LS columns (Miltenyi Biotec). Isolated T cells are counted and plated at 5 x 10 per well of a GRex 24-well plate. 5 Cells are seeded and co-cultured with DynaBeads® (anti-CD3 / anti-CD28) at a 1:1 ratio with 3000 IU / ml of IL-2 in a total of 8 ml of CM2 medium per well. On day 4, the medium in each well is changed from CM2 to AIM-V containing 3000 IU / ml of fresh IL-2. On day 7, PBLs are harvested, counted, and then plated at 1 x 10 cells per well of a new GRex-24-well plate in a total of 8 ml of AIM-V medium with 3000 IU / ml of IL-2 and DynaBeads® at a 1:1 ratio (beads:cells). 6 On day 11, the medium is replaced with CM-4 medium supplemented with fresh IL-2 at 3000 IU / ml. On day 14, the DynaBeads are removed using a DynaMag Magnet (DynaMag™-15) and the cells are counted.

[0110]

[0155] PBL Method 2. In one embodiment of the present invention, PBLs are expanded using PBL Method 2, which involves obtaining a PBMC sample from whole blood. T cells from the PBMCs are enriched by incubating the PBMCs at 37°C for at least 3 hours and then separating the non-adherent cells. The non-adherent cells are expanded as in PBL Method 1, i.e., on day 0, the non-adherent cells are cultured with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a 1:1 ratio (beads:cells) and 3000 IU / ml of IL-2. On day 4, additional IL-2 is added to the culture at 3000 IU / ml. On day 7, the cultures are again stimulated with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a 1:1 ratio (beads:cells), and 3000 IU / ml of additional IL-2 is added to the culture. On day 14, PBLs are harvested, the beads removed, and the PBLs counted and phenotyped.

[0111]

[0156] In one embodiment of the present invention, PBL method 2 is performed as follows: On day 0, cryopreserved PBMC samples are thawed, and PBMC cells are seeded into 6-well plates in CM-2 medium at 6 million cells per well and incubated at 37°C for 3 hours. After 3 hours, non-adherent cells, which are PBLs, are removed and counted. PBLs are cultured at 1 x 10 per well. 6 10 cells per flask in a total of 7 ml of CM-2 medium in each well of a GRex 24-well plate with anti-CD3 / anti-CD28 DynaBeads® at a 1:1 bead:cell ratio and 3000 IU / ml of IL-2. On day 4, the medium in each well is replaced with AIM-V medium and 3000 IU / ml of fresh IL-2. On day 7, the expanded cells are harvested, counted, and then plated at 15 x 10 cells per flask in a GRex 10M flask in a total of 100 ml of AIM-V medium with 3000 IU / ml of IL-2 and a 1:1 ratio (T cells:beads) of DynaBeads®. 6On day 11, the medium is changed to CM-4 medium and supplemented with fresh IL-2 (3000 IU / ml). On day 14, the DynaBeads are removed and the cells are counted using a DynaMag™ Magnet (DynaMag™-15).

[0112]

[0157] In one embodiment of the present invention, PBL method 2 is performed as follows: On day 0, cryopreserved PBMC samples are thawed, and PBMC cells are seeded into 6-well plates in CM-2 medium at 6 million cells per well and incubated at 37°C for 3 hours. After 3 hours, non-adherent cells, which are PBLs, are removed and counted. PBLs are cultured at 1 x 10 per well. 6 1 x 10 cells are cultured in each well of a GRex 24-well plate in a total of 7 ml of CM-2 medium with anti-CD3 / anti-CD28 DynaBeads® at a 1:1 bead:cell ratio and 3000 IU / ml of IL-2. On day 4, the medium in each well is replaced with AIM-V medium and 3000 IU / ml of fresh IL-2. On day 7, the expanded cells are harvested, counted, and then cultured at 1 x 10 cells per well of a new GRex 24-well plate in a total of 8 ml of AIM-V medium with 3000 IU / ml of IL-2 and DynaBeads® at a 1:1 ratio (T cells:beads). 6 On day 11, the medium is changed to CM-4 medium and supplemented with fresh IL-2 (3000 IU / ml). On day 14, the DynaBeads are removed and the cells are counted using a DynaMag™ Magnet (DynaMag™-15).

[0113]

[0158] PBL Method 3. In one embodiment of the present invention, PBLs are expanded using PBL Method 3, which involves obtaining a PBMC sample from peripheral blood. B cells are isolated using CD19+ selection, and T cells are selected using negative selection of the non-CD19+ fraction of the PBMC sample. On day 0, T cells and B cells are co-cultured with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a 1:1 ratio (beads:cells) and 3000 IU / ml of IL-2. On day 4, additional IL-2 is added to the culture at 3000 IU / ml. On day 7, the culture is again stimulated with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a 1:1 ratio (beads:cells), and 3000 IU / ml of additional IL-2 is added to the culture. On day 14, PBLs are harvested, the beads removed, and the PBLs are counted and phenotyped.

[0114]

[0159] In one embodiment of the present invention, PBL method 3 is performed as follows: On day 0, cryopreserved PBMCs derived from peripheral blood are thawed and counted. CD19+ B cells are sorted using a CD19 Multisort Kit, Human (Miltenyi Biotec). From the non-CD19+ cell fraction, T cells are purified using a Human Pan T-cell Isolation Kit and an LS column (Miltenyi Biotec). T cells (PBLs) and B cells are co-cultured at different ratios in a Grex 24-well plate in approximately 8 ml of CM2 medium in the presence of approximately 3000 IU / ml of IL-2. The B cell:T cell ratios are 0.1:1, 1:1, and 10:1. T cell / B cell co-cultures are stimulated with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a 1:1 ratio (beads:cells). On day 4, the medium is changed from CM2 to AIM-V medium and additional IL-2 is added to the cultures at 3000 IU / ml. On day 7, cells are harvested, counted, and plated at approximately 1.5 x 10 cells per well in a new Grex 24-well plate in AIM-V medium. 5 ~Approx. 4×10 5The cells are replated at 100x the cell lineage and stimulated with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a 1:1 ratio (beads:cells) with 3000 IU / ml of additional IL-2. On day 14, the DynaBeads are removed and the cells are counted using a DynaMag™ Magnet (DynaMag™-15).

[0115]

[0160] In one embodiment, PBMCs are isolated from a whole blood sample. In one embodiment, the PBMC sample is used as starting material for expanding PBLs. In one embodiment, the sample is cryopreserved before the expansion process. In another embodiment, a fresh sample is used as starting material for expanding PBLs. In one embodiment of the present invention, T cells are isolated from PBMCs using methods known in the art. In one embodiment, T cells are isolated using a Human Pan T-cell Isolation Kit and an LS column. In one embodiment of the present invention, T cells are isolated from PBMCs using antibody selection methods known in the art, such as CD19 negative selection.

[0116]

[0161] 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.

[0117]

[0162] In one embodiment of the present invention, PBMCs are cultured with anti-CD3 / anti-CD28 antibodies. In one embodiment, any available anti-CD3 / anti-CD28 product is useful in the present invention. In one embodiment, the commercially available product used is DynaBeads®. In one embodiment, DynaBeads® are cultured with PBMCs at a 1:1 ratio (beads:cells). In another embodiment, the antibody is DynaBeads® cultured with PBMCs at a 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1 ratio (beads:cells). In one embodiment of the present invention, the antibody culture step and / or the step of restimulating the cells with the antibody are carried out 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 present invention, the antibody culture step is carried out for a period of about 2, 3, 4, 5, or 6 days.

[0118]

[0163] In one embodiment, the PBMC sample is cultured with IL-2. In one embodiment of the present invention, the cell culture medium used for expansion of PBLs from PBMCs contains about 100 IU / mL, about 200 IU / mL, about 300 IU / mL, about 400 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 500 IU / mL, about 600 IU / mL, about 700 IU / mL, about 800 IU / mL, about 900 IU / mL, about 1,000 IU / mL, about 1,100 IU / mL, about 1,200 IU / mL, about 1,300 IU / mL mL, approximately 1,400IU / mL, approximately 1,500IU / mL, approximately 1,600IU / mL, approximately 1,700IU / mL, approximately 1,800IU / mL, approximately 1,900IU / mL, approximately 2,000IU / mL, approximately 2,100IU / mL, approximately 2,200IU / mL, approximately 2, 300IU / mL, approximately 2,400IU / mL, approximately 2,500IU / mL, approximately 2,600IU / mL, approximately 2,700IU / mL, approximately 2,800IU / mL, approximately 2,900IU / mL, approximately 3,000IU / mL, approximately 3,100IU / mL, approximately 3,200IU / m L, approximately 3,300IU / mL, approximately 3,400IU / mL, approximately 3,500IU / mL, approximately 3,600IU / mL, approximately 3,700IU / mL, approximately 3,800IU / mL, approximately 3,900IU / mL, approximately 4,000IU / mL, approximately 4,100IU / mL, approximately 4,2 00IU / mL, approximately 4,300IU / mL, approximately 4,400IU / mL, approximately 4,500IU / mL, approximately 4,600IU / mL, approximately 4,700IU / mL, approximately 4,800IU / mL, approximately 4,900IU / mL, approximately 5,000IU / mL, approximately 5,100IU / mL , about 5,200 IU / mL, about 5,300 IU / mL, about 5,400 IU / mL, about 5,500 IU / mL, about 5,600 IU / mL, about 5,700 IU / mL, about 5,800 IU / mL, about 5,900 IU / mL, about 6,000 IU / mL, about 6,500 IU / mL, about 7,000 IU / mL, about 7,500 IU / mL, about 8,000 IU / mL, about 8,500 IU / mL, about 9,000 IU / mL, about 9,500 IU / mL and about 10,000 IU / mL.

[0119]

[0164] In an embodiment of the present invention, the starting cell number of PBMCs for the expansion culture process is about 25,000 to about 1,000,000, about 30,000 to about 900,000, about 35,000 to about 850,000, about 40,000 to about 800,000, about 45,000 to about 800,000, about 50,000 to about 750,000, about 55,000 to about 700,000, about 60,000 to about 650,000, about 65,000 to about 700,000, about 75,000 to about 800,000, about 80,000 to about 900,000, about 90,000 to about 1000,000, about 100,000 to about 1200,000, about 130,000 to about 1400,000, about 150,000 to about 1600,000, about 160,000 to about 1700,000, about 170,000 to about 1800,000, about 180,000 to about 2000,000, about 210,000 to about 2200,000, about 230,000 to about 2400,000, about 250,000 to about 2600,000, about 260,000 to about 2700,000, about 280,000 to about 2900,000, about 300,000 to about 3200,000, about 330 The initial cell number of PBMCs is about 138,000 to about 600,000, about 70,000 to about 550,000, preferably about 75,000 to about 500,000, about 80,000 to about 450,000, about 85,000 to about 400,000, about 90,000 to about 350,000, about 95,000 to about 300,000, about 100,000 to about 250,000, about 105,000 to about 200,000, or about 110,000 to about 150,000. In one embodiment of the present invention, the initial cell number of PBMCs is about 138,000, 140,000, 145,000, or more. In another embodiment, the initial cell number of PBMCs is about 28,000. In another embodiment, the starting cell number of PBMCs is about 62,000. In another embodiment, the starting cell number of PBMCs is about 338,000. In another embodiment, the starting cell number of PBMCs is about 336,000. In another embodiment, the starting cell number of PBMCs is 1 million, 2 million, 3 million, 4 million, 5 million, 6 million, 7 million, 8 million, 9 million, 10 million, or more. In another embodiment, the starting cell number of PBMCs is 1 million to 10 million, 2 million to 9 million, 3 million to 8 million, 4 million to 7 million, or 5 million to 6 million. In another embodiment, the starting cell number of PBMCs is about 4 million. In yet another embodiment, the starting cell number of PBMCs is at least about 4 million, at least about 5 million, or at least about 6 million, or more.

[0120]

[0165] In one embodiment of the invention, cells are grown in GRex 24-well plates. In one embodiment of the invention, equivalent well plates are used. In one embodiment, the starting material for expansion is approximately 5 x 10 cells per well. 5In one embodiment of the present invention, 1 x 10 T cells per well 6 In an embodiment of the invention, the number of cells per well is sufficient to seed the well and expand the T cells.

[0121]

[0166] In one embodiment of the present invention, the expansion factor 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 present invention, the expansion factor is about 25%. In another embodiment of the present invention, the expansion factor is about 50%. In another embodiment, the expansion factor is about 75%.

[0122]

[0167] 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 other embodiments, additional IL-2 is added on days 4, 7, and / or 11. In one embodiment of the invention, the cell culture medium may be changed on one or more days throughout the cell culture process. In one embodiment, the cell culture medium is changed on days 4, 7, and / or 11 of the process. In one embodiment of the invention, 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, PBLs are cultured for a period of 3 days after each addition of IL-2.

[0123]

[0168] In one embodiment, the cell culture medium is changed at least once during the method. In one embodiment, the cell culture medium is changed at the same time that additional IL-2 is added. In another embodiment, the cell culture medium is changed on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In one embodiment of the invention, the cell culture medium used throughout the method can be the same or different. In one embodiment of the invention, the cell culture medium is CM-2, CM-4, or AIM-V.

[0124]

[0169] In one embodiment of the present invention, T cells may be restimulated with anti-CD3 / anti-CD28 antibodies for 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, comparable flasks are used.

[0125]

[0170] In one embodiment of the invention, the DynaBeads® are removed using a DynaMag™ Magnet, the cells are counted, and the cells are analyzed using phenotypic and functional assays further described in the Examples below. In one embodiment of the invention, antibodies are isolated from PBLs or MILs using methods known in the art. In any of the foregoing embodiments, magnetic bead-based selection of TILs, PBLs, or MILs is used.

[0126]

[0171] In one embodiment of the invention, the PBMC sample is incubated for a period of time at a desired temperature effective to identify non-adherent cells. In one embodiment of the invention, the incubation time is about 3 hours. In one embodiment of the invention, the temperature is about 37°C. The non-adherent cells are then expanded using the process described above.

[0127]

[0172] In one embodiment of the present invention, PBMCs are obtained from a patient treated with ibrutinib or another ITK or kinase inhibitor, such as an ITK or kinase inhibitor described elsewhere herein. In one embodiment of the present invention, the ITK inhibitor is a covalent ITK inhibitor that covalently and irreversibly binds to ITK. In one embodiment of the present invention, the ITK inhibitor is an allosteric ITK inhibitor that binds to ITK. In one embodiment of the present invention, PBMCs are obtained from a patient treated with ibrutinib or another ITK inhibitor, including an ITK inhibitor described elsewhere herein, prior to obtaining a PBMC sample for use in any of the aforementioned methods, including PBL Method 1, PBL Method 2, or PBL Method 3. In one embodiment of the present invention, the ITK inhibitor treatment has been administered at least once, at least twice, or at least three times or more. In one embodiment of the present invention, PBLs expanded from patients pretreated with ibrutinib or another ITK inhibitor contain fewer LAG3+, PD-1+ cells than those expanded from patients not pretreated with ibrutinib or another ITK inhibitor. In one embodiment of the present invention, PBLs expanded from patients pretreated with ibrutinib or other ITK inhibitors contain increased levels of IFNγ production compared to those expanded from patients not pretreated with ibrutinib or other ITK inhibitors. In one embodiment of the present invention, PBLs expanded from patients pretreated with ibrutinib or other ITK inhibitors contain increased lytic activity at a lower effector:target cell ratio compared to those expanded from patients not pretreated with ibrutinib or other ITK inhibitors. In one embodiment of the present invention, patients pretreated with ibrutinib or other ITK inhibitors have a higher fold expansion compared to untreated patients.

[0128]

[0173] In one embodiment of the present invention, the method includes adding an ITK inhibitor to the cell culture. In one embodiment, the ITK inhibitor is added on one or more of days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the process. In one embodiment, the ITK inhibitor is added on days during the method when the cell culture medium is changed. In one embodiment, the ITK inhibitor is added on day 0 and when the cell culture medium is changed. In one embodiment, the ITK inhibitor is added during the method when IL-2 is added. In one embodiment, the ITK inhibitor is added on days 0, 4, 7, and optionally day 11 of the method. In one embodiment, the ITK inhibitor is added on days 0 and 7 of the method. In one embodiment, the ITK inhibitor is known in the art. In one embodiment, the ITK inhibitor is described elsewhere herein.

[0129]

[0174] In one embodiment of the present invention, the ITK inhibitor is used in the present methods at a concentration of about 0.1 nM to about 5 uM. In one embodiment, the ITK inhibitor is used in the present methods at a concentration of about 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1 uM, 2 uM, 3 uM, 4 uM, or 5 uM.

[0130]

[0175] In one embodiment of the invention, the method comprises the step of adding an ITK inhibitor when the PBMCs are from a patient with no previous exposure to ITK inhibitor treatment, such as ibrutinib.

[0131]

[0176] In some embodiments, the PBMC sample is from a subject or patient who has been optionally pretreated with a regimen comprising a kinase inhibitor or an ITK inhibitor. In some embodiments, the tumor sample is from a subject or patient who has been pretreated with a regimen comprising a kinase inhibitor or an ITK inhibitor. In some embodiments, the PBMC sample is from a subject or patient who has been pretreated with a regimen comprising a kinase inhibitor or an ITK inhibitor and has 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 1 year or more. In another embodiment, the PBMCs are from a patient currently receiving an ITK inhibitor regimen, such as ibrutinib.

[0132]

[0177] In some embodiments, the PBMC sample is from a subject or patient who has been pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor and is refractory to treatment with a kinase inhibitor or an ITK inhibitor, such as ibrutinib.

[0133]

[0178] In some embodiments, the PBMC sample is from a subject or patient who has been pretreated with a regimen comprising a kinase inhibitor or an ITK inhibitor but 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 an ITK inhibitor but 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 PBMCs are from a patient who has had previous exposure 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.

[0134]

[0179] In one embodiment of the invention, on day 0, cells are selected for CD19+ and sorted accordingly. In one embodiment of the invention, selection is performed using antibody-coupled beads. In one embodiment of the invention, pure T cells are isolated from PBMCs on day 0. In one embodiment of the invention, CD19+ B cells and pure T cells are co-cultured with anti-CD3 / anti-CD28 antibodies on day 0 for a minimum of 4 days. In one embodiment of the invention, IL-2 is added to the culture on day 4. In one embodiment of the invention, the culture is restimulated with anti-CD3 / anti-CD28 antibodies and additional IL-2 on day 7. In one embodiment of the invention, PBLs are harvested on day 14.

[0135]

[0180] In one embodiment of the present invention, for patients not pretreated with ibrutinib or other ITK inhibitors, 10-15 ml of buffy coat contains approximately 5 x 10 9 Generate approximately 5.5 x 10 PBMCs. 7 pieces of starting cell material and approximately 11 x 10 cells at the end of the expansion culture process 9 In one embodiment of the present invention, approximately 54×10 PBLs are generated. 6 Approximately 6 x 10 PBMCs 5 pieces of starting material and approximately 1.2 x 10 8 MIL (approximately 205x magnification).

[0136]

[0181] In one embodiment of the present invention, for patients pretreated with ibrutinib or other ITK inhibitors, the expansion process involves approximately 20 x 10 9 In one embodiment of the present invention, 40.3×10 PBLs are generated. 6 Approximately 4.7 x 10 PBMCs 5 pieces of starting cell material and approximately 1.6 x 10 8 PBLs (approximately 338x magnification) are generated.

[0137]

[0182] In one embodiment of the present invention, the clinical dose of PBL useful in the present invention for patients with chronic lymphocytic leukemia (CLL) is about 0.1 x 10 9 ~Approx. 15×109 PBL, approximately 0.1 x 10 9 ~Approx. 15×10 9 PBL, approximately 0.12 x 10 9 ~Approx. 12×10 9 PBL, approximately 0.15 x 10 9 ~Approx. 11×10 9 PBL, approximately 0.2 x 10 9 ~About 10×10 9 PBL, approximately 0.3 x 10 9 ~Approx. 9×10 9 PBL, approximately 0.4 x 10 9 ~Approx. 8×10 9 PBL, approximately 0.5 x 10 9 ~Approx. 7×10 9 PBL, approximately 0.6 x 10 9 ~about 6×10 9 PBL, approximately 0.7 x 10 9 ~Approx. 5×10 9 PBL, approximately 0.8 x 10 9 ~Approx. 4×10 9 PBL, approximately 0.9 x 10 9 ~Approx. 3×10 9 PBL or approximately 1 x 10 9 ~about 2×10 9 It is an individual PBL.

[0138]

[0183] In any of the foregoing embodiments, the PBMCs may be obtained from a whole blood sample, by apheresis, from a buffy coat, or any other method known in the art for obtaining PBMCs.

[0139] A method for expanding bone marrow-infiltrating lymphocytes (MIL) from bone marrow-derived PBMCs

[0184] MIL Method 1. In one embodiment of the present invention, a method for expanding MILs from bone marrow-derived PBMCs is described. In one embodiment of the present invention, the method is carried out over a 14-day period. In one embodiment, the method includes obtaining bone marrow PBMCs and cryopreserving the PBMCs. On day 0, the PBMCs are cultured with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a 1:1 ratio (beads:cells) and 3000 IU / ml of IL-2. On day 4, additional IL-2 is added to the culture at 3000 IU / ml. On day 7, the culture is again stimulated with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a 1:1 ratio (beads:cells), and 3000 IU / ml of additional IL-2 is added to the culture. On day 14, the MILs are harvested, the beads removed, and the MILs are optionally counted and phenotyped.

[0140]

[0185] In one embodiment of the present invention, MIL method 1 is performed as follows: On day 0, bone marrow-derived cryopreserved PBMC samples are thawed and PBMCs are counted. PBMCs are plated at 5x10 cells per well in a GRex 24-well plate in approximately 8 ml per well of CM-2 cell culture medium (composed of RPMI-1640, human AB serum, l-glutamine, 2-mercaptoethanol, gentamicin sulfate, AIM-V medium) in the presence of 3000 IU / ml of IL-2. 5 1 x 10 cells per well are co-cultured with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a 1:1 ratio. On day 4, the cell culture medium is replaced with AIM-V supplemented with 3000 IU / ml of additional IL-2. On day 7, the expanded MILs are counted. 1 x 10 cells per well are co-cultured with anti-CD3 / anti-CD28 antibodies (DynaBeads®). 6The cells are transferred to a new GRex 24-well plate and cultured with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a 1:1 ratio in approximately 8 ml per well of AIM-V medium in the presence of 3000 IU / ml of IL-2. On day 11, the cell culture medium is changed from AIM-V to CM-4 (composed of AIM-V medium, 2 mM Glutamax, and 3000 IU / ml of IL-2). On day 14, the DynaBeads® are removed using a DynaMag Magnet (DynaMag™ 15) and the ILs are counted.

[0141]

[0186] MIL Method 2. In one embodiment of the present invention, the method is carried out over a 7-day period. In one embodiment, the method involves obtaining PBMCs from bone marrow and cryopreserving the PBMCs. On day 0, the PBMCs are cultured with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a 3:1 ratio (beads:cells) and 3000 IU / ml of IL-2. On day 7, the MILs are harvested, the beads removed, and the MILs are optionally counted and phenotyped.

[0142]

[0187] In one embodiment of the present invention, MIL method 2 is performed as follows: On day 0, cryopreserved PBMC samples are thawed and PBMCs are counted. PBMCs are cultured at 5x10 per well in a GRex 24-well plate in approximately 8 ml per well of CM-2 cell culture medium (composed of RPMI-1640, human AB serum, l-glutamine, 2-mercaptoethanol, gentamicin sulfate, AIM-V medium) in the presence of 3000 IU / ml of IL-2. 5 The cells are co-cultured with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a 1:1 ratio. On day 7, the DynaBeads® are removed using a DynaMag Magnet (DynaMag™ 15) and the MILs are counted.

[0143]

[0188] MIL Method 3. In one embodiment of the present invention, the method involves obtaining PBMCs from bone marrow. On day 0, PBMCs are selected and sorted for CD3+ / CD33+ / CD20+ / CD14+, the non-CD3+ / CD33+ / CD20+ / CD14+ cell fraction is sonicated, and a portion of the sonicated cell fraction is added back to the selected cell fraction. IL-2 is added to the cell culture at 3000 IU / ml. On day 3, PBMCs are cultured with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a 1:1 ratio (beads:cells) and 3000 IU / ml of IL-2. On day 4, additional IL-2 is added to the culture at 3000 IU / ml. On day 7, the cultures are again stimulated with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a 1:1 ratio (beads:cells), and 3000 IU / ml of additional IL-2 is added to the cultures. On day 11, IL-2 is added to the cultures at 3000 IU / ml. On day 14, the MILs are harvested, the beads removed, and the MILs are optionally counted and phenotyped.

[0144]

[0189] In one embodiment of the present invention, MIL method 3 is performed as follows: on day 0, PBMCs are selected for CD45hiCD3+ cells (immune cell fraction) by a sorting method, the CD45lowCD3- fraction (AML blast fraction) is sonicated, and a portion of the sonicated cell fraction is returned to the selected cell fraction. IL-2 is added to the cell culture at 3000 IU / ml. On day 3, PBMCs are cultured with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a 1:1 ratio (beads:cells) and 3000 IU / ml of IL-2. On day 4, additional IL-2 is added to the culture at 3000 IU / ml. On day 7, the culture is again stimulated with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a 1:1 ratio (beads:cells), and 3000 IU / ml of additional IL-2 is added to the culture. IL-2 is added to the cultures at 3000 IU / ml on day 11. On day 14, MILs are harvested, the beads removed, and optionally the MILs are counted and phenotyped.

[0145]

[0190] In one embodiment of the present invention, MIL method 3 is performed as follows: On day 0, a sample of cryopreserved PBMCs is thawed and the PBMCs are counted. Cells are stained with CD3, CD33, CD20, and CD14 antibodies and sorted using S3e cell sorting (Bio-Rad). Cells are sorted into two fractions: the immune cell fraction (or MIL fraction) (CD3+CD33+CD20+CD14+) and the AML blast fraction (non-CD3+CD33+CD20+CD14+). A number of cells from the AML blast fraction approximately equal to the number of cells from the immune cell fraction (or MIL fraction) seeded into a Grex 24-well plate are suspended in 100 ul of medium and sonicated. In this example, approximately 2.8 x 10 cells from the AML blast fraction are selected. 4 ~Approx. 3.38×10 5 10 cells were taken, suspended in 100 μl of CM2 medium, and 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. Immune cells were cultured at approximately 2.8 × 10 cells per well in approximately 8 ml of CM-2 cell culture medium per well in the presence of 6000 IU / ml of IL-2. 4 ~Approx. 3.38×10 5 The cells are present in an amount of approximately 1.5 x 10 cells per well and cultured with a portion of the AML blast fraction for approximately 3 days. On day 3, anti-CD3 / anti-CD28 antibodies (DynaBeads®) are added in a 1:1 ratio to each well and cultured for approximately 1 day. On day 4, the cell culture medium is replaced with AIM-V supplemented with 3000 IU / ml of additional IL-2. On day 7, the expanded ILs are counted. Approximately 1.5 x 10 cells per well are cultured. 5 ~4×10 5 The cells are transferred to a new GRex 24-well plate and cultured with a 1:1 ratio of anti-CD3 / anti-CD28 antibodies (DynaBeads®) in approximately 8 ml per well of AIM-V medium in the presence of 3000 IU / ml of IL-2. On day 11, the cell culture medium is changed from AIM-V to CM-4 (supplemented with 3000 IU / ml of IL-2). On day 14, the DynaBeads® are removed using a DynaMag Magnet (DynaMag™ 15) and, optionally, the ILs are counted.

[0146]

[0191] In one embodiment of the present invention, PBMCs are obtained from bone marrow. In one embodiment, PBMCs are obtained from bone marrow through apheresis, aspiration, needle biopsy, or other similar means known in the art. In one embodiment, the PBMCs are fresh. In another embodiment, the PBMCs are cryopreserved.

[0147]

[0192] In one embodiment of the invention, the method 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 method is carried out for about 7 days. In another embodiment, the method is carried out for about 14 days.

[0148]

[0193] In one embodiment of the present invention, PBMCs are cultured with anti-CD3 / anti-CD28 antibodies. In one embodiment, any available anti-CD3 / anti-CD28 product is useful in the present invention. In one embodiment, the commercially available product used is DynaBeads®. In one embodiment, DynaBeads® are cultured with PBMCs at a 1:1 ratio (beads:cells). In another embodiment, the antibody is DynaBeads® cultured with PBMCs at a 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1 ratio (beads:cells). In any of the foregoing embodiments, magnetic bead-based selection of immune cell fractions (or MIL fractions) (CD3+CD33+CD20+CD14+) or AML blast fractions (non-CD3+CD33+CD20+CD14+) is used. In one embodiment of the present invention, the antibody culture step and / or the step of restimulating the cells with the antibody is carried out 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 present invention, the antibody culture step is carried out for a period of about 2, 3, 4, 5, or 6 days.

[0149]

[0194] In one embodiment of the present invention, the ratio of the number of cells from the AML blast fraction to the number of cells from the immune cell fraction (or MIL fraction) is about 0.1:1 to about 10:1. In other embodiments, 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 present invention, the AML blast fraction is optionally disrupted to break up cell aggregates. In one embodiment, the AML blast fraction is disrupted using sonication, homogenization, cell lysis, vortexing, or vibration. In another embodiment, the AML blast fraction is disrupted using sonication. In one embodiment of the present invention, the non-CD3+, non-CD33+, non-CD20+, non-CD14+ cell fraction (AML blast fraction) is lysed using a suitable lysis method, including high-temperature lysis, chemical lysis (e.g., organic alcohols), enzymatic lysis, and other cell lysis methods known in the art.

[0150]

[0195] In one embodiment of the invention, cells from the AML blast fraction are about 0.2 x 10 cells per 100 uL. 5 ~about 2×10 5 In another embodiment, the concentration is about 0.5 x 10 cells per 100 uL. 5 ~about 2×10 5 Approximately 0.7 x 10 cells per 100uL 5 ~about 2×10 5 Approximately 1 x 10 cells per 100uL 5 ~about 2×10 5 cells or approximately 1.5 x 10 per 100uL 5 ~about 2×10 5 Each cell is an individual cell.

[0151]

[0196] In one embodiment, the PBMC sample is cultured with IL-2. In one embodiment of the present invention, the cell culture medium used for expansion of MILs contains about 100 IU / mL, about 200 IU / mL, about 300 IU / mL, about 400 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 500 IU / mL, about 600 IU / mL, about 700 IU / mL, about 800 IU / mL, about 900 IU / mL, about 1,000 IU / mL, about 1,100 IU / mL, about 1,200 IU / mL, about 1,300 IU / mL, about 1, 400IU / mL, approximately 1,500IU / mL, approximately 1,600IU / mL, approximately 1,700IU / mL, approximately 1,800IU / mL, approximately 1,900IU / mL, approximately 2,000IU / mL, approximately 2,100IU / mL, approximately 2,200IU / mL, approximately 2,300IU / mL, approximately 2,400IU / mL, approximately 2,500IU / mL, approximately 2,600IU / mL, approximately 2,700IU / mL, approximately 2,800IU / mL, approximately 2,900IU / mL, approximately 3,000IU / mL, approximately 3,100IU / mL, approximately 3,200IU / mL, approximately 3 ,300IU / mL, approximately 3,400IU / mL, approximately 3,500IU / mL, approximately 3,600IU / mL, approximately 3,700IU / mL, approximately 3,800IU / mL, approximately 3,900IU / mL, approximately 4,000IU / mL, approximately 4,100IU / mL, approximately 4,200I U / mL, approximately 4,300IU / mL, approximately 4,400IU / mL, approximately 4,500IU / mL, approximately 4,600IU / mL, approximately 4,700IU / mL, approximately 4,800IU / mL, approximately 4,900IU / mL, approximately 5,000IU / mL, approximately 5,100IU / mL, approximately The present invention comprises IL-2 at a concentration selected from the group consisting of about 5,200 IU / mL, about 5,300 IU / mL, about 5,400 IU / mL, about 5,500 IU / mL, about 5,600 IU / mL, about 5,700 IU / mL, about 5,800 IU / mL, about 5,900 IU / mL, about 6,000 IU / mL, about 6,500 IU / mL, about 7,000 IU / mL, about 7,500 IU / mL, about 8,000 IU / mL, about 8,500 IU / mL, about 9,000 IU / mL, about 9,500 IU / mL and about 10,000 IU / mL.

[0152]

[0197] In one embodiment of the invention, additional IL-2 may be added to the culture for 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 days 4, 7, and / or 11. In one embodiment of the invention, MILs are cultured with additional IL-2 for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In one embodiment of the invention, MILs are cultured for a period of 3 days after each addition of IL-2.

[0153]

[0198] In one embodiment, the cell culture medium is changed at least once during the method. In one embodiment, the cell culture medium is changed at the same time that additional IL-2 is added. In another embodiment, the cell culture medium is changed on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In one embodiment of the present invention, the cell culture medium used throughout the method can be the same or different. In one embodiment of the present invention, the cell culture medium is CM-2, CM-4, or AIM-V. In one embodiment of the present invention, the cell culture medium change step on day 11 is optional. In one embodiment of the present invention, the starting cell number of PBMCs for the expansion culture process is about 25,000 to about 1,000,000, about 30,000 to about 900,000, about 35,000 to about 850,000, about 40,000 to about 800,000, about 45,000 to about 800,000, about 50,000 to about 750,000, about 55,000 to about 700,000, about 60,000 to about 650,000, about 65 The initial cell number of PBMCs is about 138,000 to about 600,000, about 70,000 to about 550,000, preferably about 75,000 to about 500,000, about 80,000 to about 450,000, about 85,000 to about 400,000, about 90,000 to about 350,000, about 95,000 to about 300,000, about 100,000 to about 250,000, about 105,000 to about 200,000, or about 110,000 to about 150,000. In one embodiment of the present invention, the initial cell number of PBMCs is about 138,000, 140,000, 145,000, or more. In another embodiment, the initial cell number of PBMCs is about 28,000. In another embodiment, the starting cell number of PBMCs is about 62,000. In another embodiment, the starting cell number of PBMCs is about 338,000. In another embodiment, the starting cell number of PBMCs is about 336,000.

[0154]

[0199] In one embodiment of the present invention, the expansion factor 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 present invention, the expansion factor is about 25%. In another embodiment of the present invention, the expansion factor is about 50%. In another embodiment, the expansion factor is about 75%.

[0155]

[0200] In one embodiment of the present invention, MIL is expanded from 10-50 ml of bone marrow aspirate. In one embodiment of the present invention, 10 ml of bone marrow aspirate is obtained from a patient. In another embodiment, 20 ml of bone marrow aspirate is obtained from a patient. In another embodiment, 30 ml of bone marrow aspirate is obtained from a patient. In another embodiment, 40 ml of bone marrow aspirate is obtained from a patient. In another embodiment, 50 ml of bone marrow aspirate is obtained from a patient.

[0156]

[0201] In one embodiment of the present invention, the number of PBMCs obtained from about 10 to 50 ml of bone marrow aspirate is about 5 x 10 7 ~About 10×10 7 In another embodiment, the number of PBMCs obtained is about 7 x 10 7 PBMCs.

[0157]

[0202] In one embodiment of the present invention, about 5×10 7 ~About 10×10 7 From PBMCs, approximately 0.5 x 10 6 ~Approx. 1.5×10 6 In one embodiment of the present invention, approximately 1 x 10 expansion starting cell material is obtained. 6 1000 pieces of expansion culture starting cell material are obtained.

[0158]

[0203] In one embodiment of the present invention, the total number of MILs harvested at the end of the expansion period is about 0.01 x 10 9 ~Approx. 1×10 9 pieces, approximately 0.05×10 9 ~Approx. 0.9×109 pieces, approximately 0.1×10 9 ~Approx. 0.85×10 9 pieces, approximately 0.15×10 9 ~Approx. 0.7×10 9 pieces, approximately 0.2×10 9 ~Approx. 0.65×10 9 pieces, approximately 0.25×10 9 ~Approx. 0.6×10 9 pieces, approximately 0.3×10 9 ~Approx. 0.55×10 9 pieces, approximately 0.35×10 9 ~about 0.5×10 9 or approximately 0.4 x 10 9 ~about 0.45×10 9 There are individuals.

[0159]

[0204] In one embodiment of the present invention, 12 x 10 cells derived from bone marrow aspirate 6 PBMCs are approximately 1.4 x 10 5 This produced approximately 1.1 x 10 starting cell material at the end of the expansion process. 7 Generate MILs.

[0160]

[0205] In one embodiment of the invention, MILs expanded from bone marrow PBMCs using MIL method 3 above contain a higher percentage of CD8+ cells and lower numbers of LAG3+ and PD1+ cells compared to MILs expanded using MIL method 1 or MIL method 2. In one embodiment of the invention, PBLs expanded from blood PBMCs using MIL method 3 above contain a higher percentage of CD8+ cells and increased levels of IFNγ production compared to PBLs expanded using MIL method 1 or MIL method 2.

[0161]

[0206] In one embodiment of the present invention, the clinical dose of MIL useful for patients with acute myeloid leukemia (AML) is about 4×10 8 ~Approx. 2.5×10 9 In another embodiment, the number of MILs provided in the pharmaceutical composition of the present invention is within the range of 9.5 x 10 8In another embodiment, the number of MILs provided in the pharmaceutical composition of the present invention is 4.1 x 10 8 In another embodiment, the number of MILs provided in the pharmaceutical composition of the present invention is 2.2 x 10 9 There are individuals.

[0162]

[0207] In any of the foregoing embodiments, the PBMCs may be obtained from a whole blood sample, from bone marrow, by apheresis, from buffy coat, or any other method known in the art for obtaining PBMCs.

[0163] TIL expansion culture method using the "2A process"

[0208] In one embodiment of the present invention, the present invention provides an apparatus and method for expanding T cells derived from bone marrow and / or peripheral blood. In one embodiment of the present invention, the T cells are polyclonal but in a highly tumor-specific manner, with enhanced tumor specificity from the bone marrow microenvironment. In one embodiment, a bone marrow microenvironment is used to maintain and expand the T cells. In one embodiment of the present invention, a 7-day or 14-day expansion process results in an approximately 25-100 fold expansion of TILs. In one embodiment, the expansion of TILs is about 30-90 fold. In one embodiment, the expansion is about 35-85 fold. In one embodiment, the expansion is about 40-80 fold. In one embodiment, the expansion is about 45-75 fold. In another embodiment, the expansion is about 40-70 fold. In another embodiment, the expansion is about 45-65 fold. In another embodiment, the expansion factor is about 25x, about 30x, about 35x, about 40x, about 45x, 50x, about 55x, about 60x, about 65x, about 70x, about 75x, about 80x, about 85x, about 90x, about 95x, or about 100x.

[0164]

[0209] In one embodiment of the present invention, the T cell manufacturing process does not require any intervention to select for tumor specificity. In one embodiment of the present invention, the T cell manufacturing process does not require the presence of tumor in the bone marrow and / or peripheral blood during T cell expansion. In one embodiment, T cells are expanded in the presence of nearly complete bone marrow.

[0165]

[0210] 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 of WO 2010 / 062742, which is incorporated herein by reference, particularly Example 21. In one embodiment, the present invention provides a method for extracting T cells from bone marrow and / or peripheral blood, e.g., as described in Noonan, et al., 2005, Cancer Res. 65:2026-2034, which is incorporated herein by reference.

[0166]

[0211] In one embodiment, methods for obtaining bone marrow and / or peripheral blood known to those skilled in the art are useful in the present invention. In one embodiment, bone marrow and / or peripheral blood are obtained using needle aspiration. In one embodiment, bone marrow from a patient is aspirated into a heparin-containing syringe and stored overnight at room temperature. In one embodiment, after storage, the contents of the syringes are pooled together in a sterile container and tested for quality. The bone marrow is enriched for mononuclear cells (MNCs) using lymphocyte separation medium (LSM) and centrifugation on a COBE Spectra. The cells in the gradient are collected down to red blood cells and washed using HBSS. The MNCs are cryopreserved using a hetastarch-based cryoprotectant supplemented with 2% HSA and 5% DMSO, and a portion of the MNCs is reserved for quality control. The QC vial is thawed and the CD3 of the MNC product is analyzed. + and CD38 + / 138 + It is important to note that the collection of bone marrow is not a limitation of the present invention.

[0167]

[0212] In one embodiment of the present invention, bone marrow is aspirated and fractionated on a lymphocyte separation medium density gradient to collect cells approximately to the level of the red blood cell pellet. In one embodiment, this fractionation method substantially removes red blood cells and neutrophils, providing a nearly intact bone marrow. In one embodiment, the resulting fractionated material is T cells and tumor cells. In one embodiment of the present invention, the method can be 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 labels, and without sorting using fluorescence-activated cell sorting (FACS).

[0168]

[0213] In one embodiment of the present invention, the obtained bone marrow is ficolled or peripheral blood is diluted to 1×10 in 200 μL / well of AIM-V medium. 6 cells / mL and suspended in serum-free conditions.

[0169]

[0214] In one embodiment of the invention, bone marrow is harvested from a subject not in complete remission. In one embodiment of the invention, bone marrow is harvested from a subject in complete remission.

[0170]

[0215] In one embodiment of the present invention, bone marrow may be obtained and frozen, hi one embodiment, bone marrow may be obtained and used immediately to extract T cells.

[0171]

[0216] In a further embodiment and in accordance with any of the above, the present invention provides a method of expanding TILs, the method comprising contacting a population of TILs comprising at least one TIL obtained from a liquid tumor. All discussion herein regarding the expansion of TILs is applicable to the expansion of TILs obtained from bone marrow, peripheral blood, and / or hematological malignancies, including liquid tumors.

[0172]

[0217] In one embodiment, the present invention provides a process for preparing a population of tumor infiltrating lymphocytes (TILs) from a tumor obtained from a cancer, comprising: (a) contacting a fragmented tumor comprising a first population of TILs with a first cell culture medium; (b) performing an initial expansion (pre-REP) of the first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 5 times more numerous than the first population of TILs, and the first cell culture medium comprises IL-2; (c) performing a second expansion culture of the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein 7 days after the initiation of the second expansion culture, the third population of TILs is at least 50 times more numerous than the second population of TILs, the second cell culture medium comprising IL-2, OKT-3 (anti-CD3 antibody), and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the second expansion culture is performed for a period of 14 days or less; (d) recovering the third population of TILs wherein the tumor is a liquid tumor and the cancer is a hematological malignancy.

[0173]

[0218] In one embodiment, the present invention provides a process for expanding a population of TILs, comprising a first pre-rapid expansion (pre-REP) process followed by a second expansion process (which may be a rapid expansion process - REP), wherein the cell culture medium used for expansion is selected from the group consisting of 100 IU / mL to 10,000 IU / mL, 200 IU / mL to 5,000 IU / mL, 300 IU / mL to 4,800 IU / mL, 400 IU / mL to 4,600 IU / mL, 500 IU / mL to 4,400 IU / mL, 600 IU / mL to 4,200 IU / mL, 700 IU / mL to 4,000 IU / mL, 800 IU / mL to 3,800 IU / mL, 900 IU / mL to 3,600 IU / mL, 1,000 IU / mL to 1,000 IU / mL. mL~3,400IU / mL, 1,100IU / mL~3,200IU / mL, 1,200IU / mL~3,000IU / mL, 1,300IU / mL~2,800IU / mL, 1,400IU / mL~2,600IU / mL, 1,500IU / mL~2,400IU / mL, 1,600IU / mL~2,200IU / mL, 1,700IU / m and 7,500 IU / mL to 8,000 IU / mL.

[0174]

[0219] In one embodiment, the present invention provides a process for expanding a population of TILs, comprising a pre-rapid expansion (pre-REP) process and a rapid expansion process (REP), wherein the cell culture medium used for expansion is at least one of about 100 IU / mL, about 200 IU / mL, about 300 IU / mL, about 400 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 500 IU / mL, about 600 IU / mL, about 700 IU / mL, about 800 IU / mL, about 900 IU / mL, about 1,0 ... IU / mL, approximately 1,100IU / mL, approximately 1,200IU / mL, approximately 1,300IU / mL, approximately 1,400IU / mL, approximately 1,500IU / mL, approximately 1,600IU / mL, approximately 1,700IU / mL, approximately 1,800IU / mL, approximately 1,900IU / mL, approximately 2,000IU / mL, approximately 2,100IU / mL, approximately 2,200IU / mL, approximately 2,300IU / mL, approximately 2,400IU / mL, approximately 2,500IU / mL, approximately 2,600IU / mL, approximately 2,700IU / mL, approximately 2,800IU / mL, approximately 2,900IU / mL, approximately 3,000IU / mL , about 3,100IU / mL, about 3,200IU / mL, about 3,300IU / mL, about 3,400IU / mL, about 3,500IU / mL, about 3,600IU / mL, about 3,700IU / mL, about 3,800IU / mL, about 3,900IU / mL, about 4,000IU / mL, about 4,100IU / mL, approximately 4,200IU / mL, approximately 4,300IU / mL, approximately 4,400IU / mL, approximately 4,500IU / mL, approximately 4,600IU / mL, approximately 4,700IU / mL, approximately 4,800IU / mL, approximately 4,900IU / mL, approximately 5,000IU / mL, approximately 5,1 In one embodiment, the method comprises administering to a subject a therapeutically effective amount of IL-2 containing IL-2 at a concentration selected from the group consisting of about 5,000 IU / mL, about 5,200 IU / mL, about 5,300 IU / mL, about 5,400 IU / mL, about 5,500 IU / mL, about 5,600 IU / mL, about 5,700 IU / mL, about 5,800 IU / mL, about 5,900 IU / mL, about 6,000 IU / mL, about 6,500 IU / mL, about 7,000 IU / mL, about 7,500 IU / mL, about 8,000 IU / mL, about 8,500 IU / mL, about 9,000 IU / mL, about 9,500 IU / mL and about 10,000 IU / mL.

[0175]

[0220] In one embodiment, the present invention provides a process for expanding a population of TILs, comprising a pre-rapid expansion (pre-REP) process. In one embodiment, the present invention provides a pre-REP process for expanding a population of TILs, comprising contacting a population of TILs 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.

[0176]

[0221] In one embodiment, the present invention provides a pre-REP process for expanding a population of TILs, comprising contacting a population of TILs obtained from a liquid tumor with cell culture medium, wherein the cell culture medium further comprises IL-2 at an initial concentration of about 6000 IU / mL.

[0177]

[0222] In one embodiment, REP can be performed in a gas-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 nonspecific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Nonspecific T cell receptor stimulation can include, for example, about 30 ng / mL OKT-3, a monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA). TILs can be rapidly expanded by further stimulating them in vitro with one or more antigens, including cancer epitopes and antigenic portions thereof, optionally expressed from vectors such as human leukocyte antigen A2 (HLA-A2)-binding peptides, e.g., 0.3 μM MART-1:26-35(27L) or gpl 00:209-217(210M), optionally in the presence of a T cell growth factor such as 300 IU / mL IL-2 or IL-15. 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. TILs can also be rapidly expanded by restimulation with the same cancer antigen pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, TILs can be further restimulated, for example, with irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0178]

[0223] In one embodiment, the 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, a method for expanding TILs may include using about 1000 mL to about 2000 mL of cell culture medium, about 2000 mL to about 3000 mL of cell culture medium, about 3000 mL to about 4000 mL of cell culture medium, about 4000 mL to about 5000 mL of cell culture medium, about 5000 mL to about 6000 mL of cell culture medium, about 6000 mL to about 7000 mL of cell culture medium, about 7000 mL to about 8000 mL of cell culture medium, about 8000 mL to about 9000 mL of cell culture medium, about 9000 mL to about 10000 mL of cell culture medium, about 10000 mL to about 15000 mL of cell culture medium, about 15000 mL to about 20000 mL of cell culture medium, or about 20000 mL to about 25000 mL of cell culture medium. In one embodiment, expanding the number of TILs requires the use of no more than one type of cell culture medium. Any suitable cell culture medium may be used, such as AIM-V cell culture medium (L-glutamine, 50 μM streptomycin sulfate, and 10 μM gentamicin sulfate) (Invitrogen, Carlsbad, CA). In this regard, the methods of the present invention advantageously reduce the amount of medium and number of types of medium required to expand the number of TILs. In one embodiment, expanding the number of TILs may involve feeding the cells no more frequently than once every three or four days. Expanding the number of cells in a gas-permeable container simplifies the procedures required to expand the number of cells by reducing the feeding frequency required to expand the cells.

[0179]

[0224] In one embodiment, the second expansion culture is performed using a gas-permeable container. Such an embodiment is suitable for a cell population of about 5×10 5 cells / cm 2 From 10 x 10 6 ~30×10 6 cells / cm 2The gas-permeable flask allows for expansion of TILs to a depth of 10 cm. In one embodiment, this expansion occurs without feed. In one embodiment, this expansion occurs without feed, as long as the medium is at a height of about 10 cm within the gas-permeable flask. In one embodiment, this is without feed, but with the addition of one or more cytokines. In one embodiment, cytokines can be added as a bolus without the need to mix them with the medium. Such vessels, devices, and methods are known in the art and have been used for the expansion of TILs, and are described in U.S. Patent Application Publication Nos. 2014 / 0377739A1, WO 2014 / 210036A1, U.S. Patent Application Publication Nos. 2013 / 0115617A1, WO 2013 / 188427A1, U.S. Patent Application Publication No. 2011 / 0136228A1, U.S. Patent No. 8,809,050, WO 2011 / 072088A1, and the like. No. 2, U.S. Patent Application Publication No. 2016 / 0208216A1, U.S. Patent Application Publication No. 2012 / 0244133A1, WO 2012 / 129201A1, U.S. Patent Application Publication No. 2013 / 0102075A1, U.S. Patent No. 8,956,860, WO 2013 / 173835A1, and U.S. Patent Application Publication No. 2015 / 0175966A1, the disclosures of which are incorporated herein by reference. Such processes are also described in Jin, et al., J. Immunotherapy 2012, 35, 283-292, the disclosure of which is incorporated herein by reference.

[0180]

[0225] In one embodiment, the gas-permeable container is a G-Rex 10 flask (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). 2 In one embodiment, the gas permeable container comprises a gas permeable culture surface of 100-300 million TILs after two medium changes. In one embodiment, the gas permeable container comprises a cell culture medium capacity of 40 mL. In one embodiment, the gas permeable container provides 100-300 million TILs after two medium changes.

[0181]

[0226] In one embodiment, the gas-permeable container is a G-Rex 100 flask (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). 2 In one embodiment, the gas permeable container comprises a gas permeable culture surface of 450 mL. In one embodiment, the gas permeable container comprises a cell culture medium volume of 450 mL. In one embodiment, the gas permeable container provides 1 to 3 billion TILs after two medium changes.

[0182]

[0227] In one embodiment, the gas-permeable container is a G-Rex 100M flask (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). 2 In one embodiment, the gas permeable container comprises a gas permeable culture surface of 1000 mL. In one embodiment, the gas permeable container comprises a cell culture medium capacity of 1000 mL. In one embodiment, the gas permeable container provides 1 to 3 billion TILs without medium changes.

[0183]

[0228] In one embodiment, the gas-permeable container is a G-Rex 100 L flask (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). 2 In one embodiment, the gas permeable container comprises a gas permeable culture surface of 1000 mL. In one embodiment, the gas permeable container comprises a cell culture medium capacity of 2000 mL. In one embodiment, the gas permeable container provides 1 to 3 billion TILs without medium changes.

[0184]

[0229] In one embodiment, the gas-permeable container is a G-Rex 24-well plate (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In one embodiment, the gas-permeable container comprises a plate with wells, each well having a diameter of 2 cm. 2In one embodiment, the gas-permeable container comprises a plate having wells, each well containing 8 mL of cell culture medium. In one embodiment, the gas-permeable container provides 20 to 60 million cells per well after two medium changes.

[0185]

[0230] In one embodiment, the gas-permeable container is a G-Rex 6-well plate (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In one embodiment, the gas-permeable container comprises a plate with wells, each well having a diameter of 10 cm. 2 In one embodiment, the gas-permeable container comprises a plate having wells, each well containing 40 mL of cell culture medium. In one embodiment, the gas-permeable container provides 100-300 million cells per well after two medium changes.

[0186]

[0231] In one embodiment, the cell culture medium in the first and / or second gas permeable container is unfiltered. The use of unfiltered cell culture medium can simplify the procedures required to expand cell numbers. In one embodiment, the cell culture medium in the first and / or second gas permeable container lacks beta-mercaptoethanol (BME).

[0187]

[0232] 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 cell culture medium therein; obtaining TILs from the tumor tissue sample; and expanding the number of TILs in a second gas-permeable container containing cell culture medium therein for about 14 to about 42 days, e.g., about 28 days.

[0188]

[0233] 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 contains 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.

[0189]

[0234] In one embodiment, the cell culture medium comprises an OKT-3 antibody. In a preferred embodiment, the cell culture medium comprises about 30 ng / mL of the 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, or about 1 μg / mL of the OKT-3 antibody. In one embodiment, the cell culture medium contains 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL, and 50 ng / mL to 100 ng / mL of OKT-3 antibody.

[0190]

[0235] In one embodiment, TILs are expanded in a gas-permeable container. Gas-permeable containers have been used to expand TILs using PBMCs using methods, compositions, and devices known in the art, including those described in U.S. Patent Application Publication No. 2005 / 0106717A1, the disclosure of which is incorporated herein by reference. In one embodiment, TILs are expanded in a gas-permeable bag. In one embodiment, TILs are expanded using a cell expansion system that expands TILs in a gas-permeable bag, such as the Xuri Cell Expansion System W25 (GE Healthcare). In one embodiment, TILs are expanded using a cell expansion system that expands TILs in a gas-permeable bag, such as the WAVE Bioreactor System, also known as the Xuri Cell Expansion System W5 (GE Healthcare). In one embodiment, the cell expansion culture system 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 culture system comprises a gas permeable cell bag having a volume in a range 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 culture system comprises a gas permeable cell bag having a volume in a range 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 culture system comprises a gas permeable cell bag having a volume in a range selected from the group consisting of 0.5L-5L, 5L-10L, 10L-15L, 15L-20L, 20L-25L, and 25L-30L. In one embodiment, the cell expansion culture system uses a rocking time of about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, and about 28 days. In one embodiment, the cell expansion culture system uses rocking times of 30 minutes to 1 hour, 1 hour to 12 hours, 12 hours to 1 day, 1 day to 7 days, 7 days to 14 days, 14 days to 21 days, and 21 days to 28 days. In one embodiment, the cell expansion culture system uses rocking rates of about 2 rocking / min, about 5 rocking / min, about 10 rocking / min, about 20 rocking / min, about 30 rocking / min, and about 40 rocking / min. In one embodiment, the cell expansion culture system uses rocking rates of 2 rocking / min to 5 rocking / min, 5 rocking / min to 10 rocking / min, 10 rocking / min to 20 rocking / min, 20 rocking / min to 30 rocking / min, and 30 rocking / min to 40 rocking / min. In one embodiment, the cell expansion culture system uses rocking angles of about 2°, about 3°, about 4°, about 5°, about 6°, about 7°, about 8°, about 9°, about 10°, about 11°, and about 12°. In one embodiment, the cell expansion culture system uses rocking angles of 2°-3°, 3°-4°, 4°-5°, 5°-6°, 6°-7°, 7°-8°, 8°-9°, 9°-10°, 10°-11°, and 11°-12°.

[0191]

[0236] In one embodiment, the method for expanding TILs obtained from a liquid tumor further comprises selecting TILs for superior tumor responsiveness. Any selection method known in the art may 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) may be used to select TILs for superior tumor responsiveness.

[0192]

[0237] In one embodiment, the present invention provides a method for expanding a population of TILs 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 disrupted for approximately 1 minute. The mixture may then be incubated at 37°C in 5% CO2 for 30 minutes, and then mechanically disrupted again for approximately 1 minute. After 30 minutes of incubation at 37°C in 5% CO2, the tumor or a portion thereof may be mechanically disrupted a third time for approximately 1 minute. After the third mechanical disruption, if large tissue fragments are present, the sample may be subjected to one or two additional mechanical disruptions, with or without an additional 30-minute incubation at 37°C in 5% CO2. If the cell suspension contains a large number of red blood cells or dead cells at the end of the final incubation, density gradient separation using Ficoll may be performed to remove these cells. TIL cultures were initiated in 24-well plates (Costar 24-well cell culture cluster, flat bottom; Corning Incorporated, Corning, NY), with each well receiving 1 × 10 cells in 2 mL of complete medium (CM) containing IL-2 (6000 IU / mL; Chiron Corp., Emeryville, CA). 6 tumor digestive cells or approximately 1-8 mm 3 The 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. The cultures are grown in a 40 mL volume and 10 cm 2 Gas-permeable flasks with gas-permeable silicone bottoms (G-Rex 10; Wilson Wolf Manufacturing, New Brighton) were prepared. Each flask contained 10-40 × 10 cells in 10-40 mL of CM containing IL-2. 6Viable tumor digested cells or 5-30 tumor fragments can be loaded into the wells. G-Rex 10- and 24-well plates can be incubated in a humidified incubator at 37°C in 5% CO2. Five days after initiation of culture, half of the medium can be removed and replaced with fresh CM and IL-2. After day 5, half of the medium can be replaced every 2-3 days. Using TILs obtained from liquid tumors of the present disclosure, a second expansion protocol (REP) of TILs can be performed using T-175 flasks and gas-permeable bags or gas-permeable G-Rex flasks, as described elsewhere herein. For REP in T-175 flasks, 1 x 10 cells can be cultured. 6 TILs can be suspended in 150 mL of 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 IL-2 and 30 ng / mL anti-CD3 antibody (OKT-3). T-175 flasks can be incubated at 37°C in 5% CO2. Half of the medium can be replaced on day 5 with 50 / 50 medium containing 3000 IU / mL IL-2. On day 7, cells from two T-175 flasks can be combined in a 3 L 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 daily or every other day, and fresh medium can be added to keep the cell count at 0.5–2.0 × 10. 6 cells / mL. 100cm 2 For REP in a 500 mL flask with a gas permeable silicone bottom (e.g., G-Rex 100, Wilson Wolf Manufacturing, as described elsewhere herein), 5 x 10 6 or 10 x 10 6TILs can be cultured in 400 mL of 50 / 50 medium supplemented with 3000 IU / mL IL-2 and 30 ng / mL anti-CD3 antibody (OKT-3). The G-Rex 100 flasks can be incubated at 37°C under 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 resulting TIL pellet can be resuspended in 150 mL of fresh 50 / 50 medium containing 3000 IU / mL IL-2 and added back to the G-Rex 100 flask. If TILs are to be continuously expanded in G-Rex 100 flasks, on day 7, the TILs in each G-Rex 100 flask can be suspended in the 300 mL of medium present in each flask, and the cell suspension can be divided into three 100 mL aliquots that can be used to inoculate three G-Rex 100 flasks. Approximately 150 mL of AIM-V containing 5% human AB serum and 3000 UI / mL IL-2 per mL can then be added to each flask. The G-Rex 100 flasks are then incubated at 37°C in 5% CO2, and after 4 days, 150 mL of AIM-V with 3000 IU / mL IL-2 can be added to each G-Rex 100 flask. REP can then be completed by harvesting the cells on day 14 of culture.

[0193]

[0238] In one embodiment, the method for expanding or treating cancer includes obtaining TILs from a patient tumor sample. Patient tumor samples can be obtained using methods known in the art. For example, TILs can be obtained from enzymatic tumor digests and sharp dissection tumor fragments (about 1 to about 8 mm in size). 3) can be cultured from tumor cells. Such tumor digests can be produced by incubation in an enzyme medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg / mL gentamicin, 30 units / mL DNase, and 1.0 mg / mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociation agent). Tumor digests can be produced by placing the tumor in the enzyme medium, mechanically dissociating the tumor for approximately 1 minute, followed by incubation at 37°C under 5% CO2 for 30 minutes, and then repeating the cycle of mechanical dissociation and incubation under the aforementioned conditions until only minimal tissue is present. If the cell suspension contains a large number of red blood cells or dead cells at the end of this process, density gradient separation using FICOLL branched hydrophilic polysaccharide can be performed to remove these cells. Alternative methods known in the art, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1, the disclosure of which is incorporated herein by reference, can be used. Any of the aforementioned methods can be used in any of the embodiments described herein for methods of expanding TILs or methods of treating cancer.

[0194]

[0239] In one embodiment, the second / REP expansion process of TILs can be performed using T-175 flasks and gas-permeable bags as previously described (Tran, et al., J. Immunother. 2008, 31, 742-51; Dudley, et al., J. Immunother. 2003, 26, 332-42) or gas-permeable culture equipment (G-Rex flasks, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). For TIL expansion in T-175 flasks, 1 x 10 cells suspended in 150 mL of medium are cultured. 6TILs can be added to each T-175 flask. TILs can be cultured in a 1:1 mixture of CM and AIM-V medium supplemented with 3000 IU (international units) of IL-2 per mL and 30 ng per mL of anti-CD3 antibody (e.g., OKT-3). The T-175 flasks can be incubated at 37°C in 5% CO2. Half of the medium can be replaced on day 5 with 50 / 50 medium containing 3000 IU of IL-2 per mL. On day 7, 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 of IL-2 per mL are added to the 300 mL TIL suspension. The number of cells in each bag is counted daily or every other day, and fresh medium is added to keep the cell count at 0.5–2.0 × 10. 6 The cell density was maintained at 100 cells / mL.

[0195]

[0240] In one embodiment, 100 cm 2 For the second / REP TIL expansion culture, 5 × 10 cells were cultured in a 500 mL gas-permeable flask (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA) with a gas-permeable silicone bottom. 6 or 10 x 10 6The TILs can be cultured in 400 mL of 50 / 50 medium supplemented with 5% human AB serum, 3000 IU of IL-2 per mL, and 30 ng of anti-CD3 (OKT-3) per mL. The G-Rex 100 flask can be incubated at 37°C under 5% CO2. On day 5, 250 mL of supernatant can be removed and placed in a centrifuge bottle and centrifuged at 1500 rpm (491 x g) for 10 minutes. The TIL pellet can be resuspended in 150 mL of fresh medium containing 5% human AB serum, 3000 IU of IL-2 per mL, and added back to the original G-Rex 100 flask. If TILs are to be continuously expanded in G-Rex 100 flasks, on day 7, the TILs in each G-Rex 100 flask can be suspended in the 300 mL of medium present in each flask, and the cell suspension can be divided into three 100 mL aliquots that can be used to inoculate three G-Rex 100 flasks. Next, 150 mL of AIM-V containing 5% human AB serum and 3000 IU of IL-2 per mL can be added to each flask. The G-Rex 100 flasks can be incubated at 37°C in 5% CO2, and after four days, 150 mL of AIM-V with 3000 IU of IL-2 per mL can be added to each G-Rex 100 flask. Cells can be harvested on day 14 of culture.

[0196]

[0241] In one embodiment, TILs can be prepared as follows: 3Tumor fragments were cultured in complete medium (CM) containing AIM-V medium (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 2 mM glutamine (Mediatech, Inc., Manassas, VA), 100 U / mL penicillin (Invitrogen Life Technologies), 100 μg / mL streptomycin (Invitrogen Life Technologies), 5% heat-inactivated human AB serum (Valley Biomedical, Inc., Winchester, VA), and 600 IU / mL rhIL-2 (Chiron, Emeryville, CA). For enzymatic digestion of liquid tumors, tumor specimens were diced into RPMI-1640, washed, centrifuged at 800 rpm for 5 minutes at 15–22°C, resuspended in enzymatic digestion buffer (0.2 mg / mL collagenase and 30 units / mL DNase in RPMI-1640), and then rotated overnight at room temperature. TILs generated from the fragments can be grown in CM for 3-4 weeks and expanded fresh, or cryopreserved in heat-inactivated HAB serum containing 10% dimethyl sulfoxide (DMSO) and stored at -180°C until study. Tumor-associated lymphocytes (TALs) obtained from ascites collections were cultured in CM at 3 x 10 in 24-well plates. 6 TILs were seeded at 1000 cells / well. TIL proliferation was examined every other day using a low-magnification inverted microscope.

[0197] Exemplary Embodiments of the TIL Fabrication Process ("2A Process")

[0242] An exemplary TIL production / expansion process, known as Process 2A, is shown schematically in Figure 22. In certain embodiments, the method produces TILs that are capable of increasing replication cycles upon administration to a subject / patient, and thus may provide an additional therapeutic advantage over mature TILs (i.e., TILs that have undergone more rounds of replication before administration to a subject / patient). Characteristics of juvenile TILs have been described in the literature. For example, Donia, at al., Scandinavian Journal of Immunology, 75:157-167 (2012);Dudley et al., Clin Cancer Res, 16:6122-6131 (2010);Huang et al., J Immunother, 28(3):258-267 (2005);Besser et al., Clin Cancer Res, 19(17):OF1-OF9 (2013);Besser et al., J Immunother 32:415-423 (2009);Robbins, et al., J Immunol 2004;173:7125-7130;Shen et al., J Immunother, 30:123-129 (2007);Zhou, et al., J Immunother, 28:53-62 (2005); and Tran, et al., J Immunother, 31:742-751 (2008), all of which are incorporated herein by reference in their entireties.

[0198]

[0243] As discussed herein, the invention may include steps relating to restimulating cryopreserved TILs prior to transplantation into a patient to increase their metabolic activity, and therefore relative health, and methods for testing said metabolic health. As generally outlined herein, TILs are typically harvested from a patient sample and manipulated to expand their numbers prior to transplantation into a patient. In some embodiments, TILs may optionally be genetically engineered, as discussed below.

[0199]

[0244] In some embodiments, the TILs can be cryopreserved. After thawing, the TILs can be restimulated to enhance their metabolism before infusion into the patient.

[0200]

[0245] In some embodiments, as discussed in detail below and in the Examples and Figures, the first expansion culture (including a process called pre-REP) is shortened to 7-14 days, and the second expansion culture (including a process called REP) is shortened to 7-14 days, compared to conventional expansion culture methods.

[0201]

[0246] FIG. 23 illustrates an exemplary 2A process. As shown in FIG. 23 and described in further detail below, in some embodiments, the first expansion culture (step B) is shortened to 11 days, and the second expansion culture (step D) is shortened to 11 days. In some embodiments, as discussed in detail herein, the combined first and second expansion cultures (steps B and D) are shortened to 22 days. As will be understood, the process illustrated in FIG. 23 and described below is exemplary, and the methods described herein encompass modifications and additions to the steps described, as well as any combination. An exemplary embodiment of this process is described in U.S. Patent Application Publication No. 2018 / 0282694 A1, the entire contents of which are incorporated herein by reference.

[0202] Step A: Obtaining patient tumor samples

[0247] Generally, TILs are initially obtained from patient tumor samples ("primary TILs") and then expanded into larger populations for further manipulation as described herein, optionally cryopreserved, restimulated as outlined herein, and optionally have phenotypic and metabolic parameters determined as indicators of TIL health.

[0203]

[0248] Patient tumor samples can be obtained using methods known in the art, typically by surgical resection, needle biopsy, apheresis, or other means for obtaining a sample containing a mixture of tumor and TIL cells. Generally, tumor samples can be from any solid tumor, including primary, invasive, or metastatic tumors. Tumor samples can also be from liquid tumors, such as tumors obtained from hematological malignancies. Solid tumors can be from any cancer type, including, but not limited to, breast, pancreatic, prostate, colorectal, lung, brain, renal, gastric, and skin cancers (including, but not limited to, squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, useful TILs are obtained from melanoma tumors, which have been reported to have particularly high levels of TILs. In some embodiments, tumors are larger than about 1.5 cm and smaller than about 4 cm. In some embodiments, tumors are smaller than 4 cm.

[0204]

[0249] Once obtained, tumor samples are typically separated into sections of 1 to approximately 8 mm using sharp dissection. 3 fragmented into small pieces of about 2-3 mm 3are particularly useful. TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests can be produced by incubation in an enzymatic medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg / mL gentamicin, 30 units / mL DNase, and 1.0 mg / mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be produced by placing the tumor in the enzymatic medium, mechanically dissociating the tumor for approximately 1 minute, followed by incubation at 37°C under 5% CO2 for 30 minutes, and then repeating the cycle of mechanical dissociation and incubation under the aforementioned conditions until only small tissue fragments are present. If the cell suspension contains a large number of red blood cells or dead cells at the end of this process, density gradient separation using FICOLL branched hydrophilic polysaccharide can be performed to remove these cells. Alternative methods known in the art may be used, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1, the disclosure of which is incorporated herein by reference. Any of the foregoing methods may be used in any of the embodiments described herein for the methods of expanding TILs or treating cancer.

[0205]

[0250] The collected cell suspension is generally referred to as a "primary cell population" or "freshly harvested" cell population.

[0206]

[0251] In one embodiment, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from the patient.

[0207]

[0252] In some embodiments, the TILs are obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharp abrasion. In some embodiments, the tumor fragments are about 1 mm 3 ~10mm 3 In some embodiments, the tumor fragment is about 1 mm 3 ~8mm 3 In some embodiments, the tumor fragment is about 1 mm3 In some embodiments, the tumor fragment is about 2 mm 3 In some embodiments, the tumor fragment is about 3 mm 3 In some embodiments, the tumor fragment is about 4 mm 3 In some embodiments, the tumor fragment is about 5 mm 3 In some embodiments, the tumor fragment is about 6 mm 3 In some embodiments, the tumor fragment is about 7 mm 3 In some embodiments, the tumor fragment is about 8 mm 3 In some embodiments, the tumor fragment is about 9 mm 3 In some embodiments, the tumor fragment is about 10 mm 3 In some embodiments, the tumor fragment is about 8 to 27 mm 3 In some embodiments, the tumor fragment is about 10 to 25 mm 3 In some embodiments, the tumor fragment is about 15 to 25 mm 3 In some embodiments, the tumor fragment is about 8 to 20 mm 3 In some embodiments, the tumor fragment is about 15-20 mm 3 In some embodiments, the tumor fragment is about 8 to 15 mm 3 In some embodiments, the tumor fragment is about 8-10 mm 3 is.

[0208]

[0253] In some embodiments, the number of tumor fragments is about 40 to about 50 tumor fragments. In some embodiments, the number of tumor fragments is about 40 tumor fragments. In some embodiments, the number of tumor fragments is about 50 tumor fragments. In some embodiments, the tumor fragment size is about 8 to 27 mm. 3 and there are less than about 50 tumor fragments.

[0209]

[0254] In some embodiments, TILs are obtained from tumor digests. In some embodiments, tumor digests are generated by incubation in an enzyme medium, such as, but not limited to, RPMI 1640, 2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase, followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, the tumor may be mechanically dissociated for approximately 1 minute. The solution may then be incubated for 30 minutes at 37°C under 5% CO2, after which it may be mechanically disrupted again for approximately 1 minute. After another 30-minute incubation at 37°C under 5% CO2, the tumor may be mechanically disrupted a third time for approximately 1 minute. In some embodiments, if large tissue debris was present after the third mechanical disruption, the sample was subjected to one or two additional mechanical dissociations, with or without an additional 30-minute incubation at 37°C under 5% CO2. In some embodiments, if the cell suspension contains a large number of red blood cells or dead cells at the end of the final incubation, density gradient separation using Ficoll may be performed to remove such cells.

[0210] Step B: First expansion culture

[0255] After detachment 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 over tumor and other cells. In some embodiments, tumor digests are incubated in 2 mL wells in medium containing inactivated human AB serum with 6000 IU / mL of IL-2. This primary cell population is cultured for several days, typically 3-14 days, thereby generating a bulk TIL population, typically about 1 x 10 8 In some embodiments, this primary cell population is cultured for a period of 7-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for a period of 10-14 days, resulting in a bulk TIL population, generally about 1 x 108 In some embodiments, this primary cell population is cultured for a period of about 11 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for a period of about 11 days, resulting in a bulk TIL population, generally about 200 x 10 6 No more than 100 bulk TIL cells are obtained.

[0211]

[0256] In a preferred embodiment, expansion of TILs can be performed using an initial bulk TIL expansion step (step B shown in FIG. 23, which may include a process referred to as pre-REP), as described below and herein, followed by a second expansion (step D, which includes a process referred to as a rapid expansion protocol (REP) step), as described below under step D and herein, followed by optional cryopreservation and a subsequent second step D (which includes a process referred to as a restimulation REP step), as described below and herein. TILs obtained by this process can optionally be characterized for phenotypic characteristics and metabolic parameters, as described herein.

[0212]

[0257] In an embodiment, when TIL cultures are initiated in 24-well plates, e.g., using Costar 24-well cell culture clusters, flat bottom (Corning Incorporated, Corning, NY), each well contains 1 x 10 cells in 2 mL of complete medium (CM) containing IL-2 (6000 IU / mL; Chiron Corp., Emeryville, CA). 6 Tumor digest cells or 1 tumor fragment can be seeded. In some embodiments, tumor fragments are about 1 mm 3 ~10mm 3 is.

[0213]

[0258] In some embodiments, the CM in step B consists of RPMI 1640 with GlutaMAX supplemented with 10% human AB serum, 25 mM HEPES, and 10 mg / mL gentamicin. The culture is grown in a 40 mL volume and 10 cm 2 In embodiments initiated in gas-permeable flasks with gas-permeable silicone bottoms (e.g., G-Rex 10; Wilson Wolf Manufacturing, New Brighton, MN) (Figure 1), each flask contains 10-40 x 10 cells in 10-40 mL of IL-2-containing CM. 6 Live tumor digest cells or 5–30 tumor fragments were loaded into G-Rex 10- and 24-well plates. Both plates were incubated in a humidified incubator at 37°C under 5% CO2. After 5 days of culture, half of the medium was removed and replaced with fresh CM and IL-2. From day 5 onward, half of the medium was replaced every 2–3 days.

[0214]

[0259] In one embodiment, the cell culture medium further 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 between 1000-2000 IU / mL, 2000-3000 IU / mL, 3000-4000 IU / mL, 4000-5000 IU / mL, 5000-6000 IU / mL, 6000-7000 IU / mL, 7000-8000 IU / mL or 8000 IU / mL of IL-2.

[0215]

[0260] In some embodiments, as discussed in the Examples and Figures, the first expansion culture (including a process called pre-REP; step B) process is shortened to 3 to 14 days. In some embodiments, as discussed in the Examples and shown in Figures 4 and 5, the first expansion culture in step B is shortened to 7 to 14 days. In some embodiments, as discussed in the Examples, the first expansion culture in step B is shortened to 10 to 14 days. In some embodiments, as discussed in the Examples, the first expansion culture in step B is shortened to 11 days.

[0216]

[0261] In some embodiments, IL-2, IL-7, IL-15, and IL-21, and combinations thereof, may be included in the Step B process, as described herein.

[0217]

[0262] In some embodiments, step B is performed in a closed bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100.

[0218] Step C: Transition from the first expansion culture to the second expansion culture

[0263] 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 culture (REP), as discussed below, and then cryopreserved.

[0219]

[0264] In some embodiments, the TILs from step B are not preserved, and the TILs from step B proceed directly to step D. In some embodiments, the transfer is performed in a closed system, as further described herein.

[0220] Step D: Second expansion culture

[0265] In some embodiments, the TIL cell population is expanded in number after collection and initial bulk processing (i.e., after steps A and B). This may include an expansion process referred to herein as a second expansion culture and commonly referred to in the art as a rapid expansion process (REP). The second expansion culture may generally be accomplished using culture medium in a gas-permeable container containing several components, including feeder cells, a cytokine source, and an anti-CD3 antibody. In some embodiments, the second expansion culture may include scaling up to increase the number of TILs obtained in the second expansion culture.

[0221]

[0266] In one embodiment, the 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 OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA). TILs can be rapidly expanded by further stimulation in vitro with one or more antigens of the cancer, including an antigenic portion thereof, such as an epitope, optionally expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2)-binding peptide, e.g., 0.3 μM MART-1:26-35(27L) or gpl 00:209-217(210M), optionally in the presence of a T cell growth factor, such as 300 IU / mL IL-2 or IL-15. 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. TILs can also be rapidly expanded by restimulation with the same antigen of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, TILs can be further restimulated with, for example, irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0222]

[0267] In one embodiment, the cell culture medium further 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 contains 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.

[0223]

[0268] In one embodiment, the cell culture medium comprises an OKT3 antibody. In a preferred embodiment, the cell culture medium comprises about 30 ng / mL of the OKT3 antibody. In certain embodiments, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, or about 1 μg / mL of the OKT3 antibody. In certain embodiments, the cell culture medium contains 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL, and 50 ng / mL to 100 ng / mL of OKT3 antibody.

[0224]

[0269] In some embodiments, IL-2, IL-7, IL-15 and IL-21, and combinations thereof, may be included in the second expansion culture in the Step D process, as described herein.

[0225]

[0270] In some embodiments, the second expansion culture may be performed in a supplemented cell culture medium containing IL-2, OKT-3, and antigen-presenting feeder cells.

[0226]

[0271] In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In certain embodiments, the ratio of TILs to PBMCs and / or antigen-presenting cells in the rapid expansion culture and / or second expansion culture is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In certain embodiments, the ratio of TILs to PBMCs in the rapid expansion culture and / or second expansion culture is 1:50 to 1:300. In certain embodiments, the ratio of TILs to PBMCs in the rapid expansion culture and / or second expansion culture is 1:100 to 1:200.

[0227]

[0272] In one embodiment, REP and / or secondary expansion is performed in flasks by mixing bulk TILs with a 100-fold or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody, and 3000 IU / mL IL-2 in 150 ml of medium. Media replenishment is performed (typically two-thirds via respiration with fresh medium) until the cells are transferred to a separate growth chamber. Separate growth chambers include GRex flasks and gas-permeable vessels, as discussed more fully below.

[0228]

[0273] In some embodiments, the second expansion culture (also referred to as the REP process) is shortened to 7-14 days, as discussed in the Examples and Figures, hi some embodiments, the second expansion culture is shortened to 11 days.

[0229]

[0274] In certain embodiments, the REP and / or second expansion may be performed using T-175 flasks and gas-permeable bags or gas-permeable cultureware (G-Rex flasks) as previously described (Tran, et al., J. Immunother. 2008, 31, 742-51; Dudley, et al., J. Immunother. 2003, 26, 332-42). The TIL rapid expansion and / or second expansion in T-175 flasks involves 1 x 10 TILs suspended in 150 mL of medium. 6 TILs can be added to each T-175 flask. TILs can be cultured in a 1:1 mixture of CM and AIM-V medium supplemented with 3000 IU per mL of IL-2 and 30 ng per mL of anti-CD3. T-175 flasks can be incubated at 37°C in 5% CO2. Half of the medium can be replaced on day 5 with 50 / 50 medium containing 3000 IU of IL-2 per mL. On day 7, 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 of IL-2 per mL are added to the 300 mL TIL suspension. The number of cells in each bag is counted daily or every other day, and fresh medium is added to keep the cell count at 0.5–2.0 × 10. 6 cells / mL.

[0230]

[0275] In one embodiment, the REP and / or second expansion culture may be performed in a 500 mL gas-permeable flask with a 100 cm gas-permeable silicone bottom (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA), containing 5×10 6 or 10 x 10 6TILs can be cultured with PBMCs in 400 mL of 50 / 50 medium supplemented with 5% human AB serum, 3000 IU of IL-2 per mL, and 30 ng / mL of anti-CD3 (OKT-3). The G-Rex 100 flask can be incubated at 37°C under 5% CO2. On day 5, 250 mL of supernatant can be removed and placed in a centrifuge bottle and centrifuged at 1500 rpm (491 x g) for 10 minutes. The TIL pellet can be resuspended in 150 mL of fresh medium containing 5% human AB serum, 3000 IU of IL-2 per mL, and added back to the original G-Rex 100 flask. When serially expanding TILs in G-Rex 100 flasks, on day 7, TILs from each G-Rex 100 flask can be suspended in the 300 mL of medium present in each flask, and the cell suspension can be divided into three 100 mL aliquots, which can be used to seed three G-Rex 100 flasks. Next, 150 mL of AIM-V containing 5% human AB serum and 3000 IU of IL-2 per mL can be added to each flask. The G-Rex 100 flasks are incubated at 37°C in 5% CO2, and after 4 days, 150 mL of AIM-V with 3000 IU of IL-2 per mL can be added to each G-Rex 100 flask. Cells can be harvested on day 14 of culture.

[0231]

[0276] In one embodiment, REP and / or secondary expansion is performed in flasks by mixing bulk TILs with a 100-fold or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody, and 3000 IU / mL IL-2 in 150 ml of medium. Media replenishment is performed (typically two-thirds via respiration with fresh medium) until the cells are transferred to a separate growth chamber. Separate growth chambers include GRex flasks and gas-permeable vessels, as discussed more fully below.

[0232]

[0277] In one embodiment, REP and / or a second expansion culture is performed, which further includes selecting TILs for superior tumor response. Any selection method known in the art can be used. For example, the method described in U.S. Patent Application Publication No. 2016 / 0010058A1 (the disclosure of which is incorporated herein by reference) can be used to select TILs for superior tumor response.

[0233]

[0278] REP and / or secondary expansion of TILs can be performed using T-175 flasks and gas-permeable bags as previously described (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 is performed using gas-permeable G-Rex flasks. For TIL REP and / or secondary expansion in T-175 flasks, approximately 1 x 10 6 TILs are suspended in approximately 150 mL of medium and added to each T-175 flask. The TILs are cultured at a 1:100 ratio with irradiated (50 Gy) allogeneic PBMCs as "feeder" cells, and the cells are cultured in a 1:1 mixture of CM and AIM-V medium (50 / 50 medium) supplemented with 3000 IU / mL IL-2 and 30 ng / mL anti-CD3. The T-175 flasks are incubated at 37°C under 5% CO. In some embodiments, half of the medium is replaced on day 5 using 50 / 50 medium containing 3000 IU / mL IL-2. In some embodiments, on day 7, cells from two T-175 flasks are combined into a 3 L bag, and 300 mL of the TIL suspension is added to 300 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2. The number of cells in each bag can be counted daily or every two days, and fresh medium can be added to increase the cell count to about 0.5 to about 2.0 × 10 6cells / mL.

[0234]

[0279] 100cm 2 For TIL REP and / or secondary expansion in 500 mL volumetric flasks with gas-permeable silicone bottoms (G-Rex 100, Wilson Wolf), approximately 5 × 10 cells were cultured in 400 mL of 50 / 50 medium supplemented with 3000 IU / mL IL-2 and 30 ng / mL anti-CD3. 6 or 10 x 10 6 TILs are cultured with irradiated allogeneic PBMCs at a 1:100 ratio. G-Rex 100 flasks are incubated at 37°C under 5% CO2. In some embodiments, on day 5, 250 mL of supernatant is removed, placed in a centrifuge bottle, and centrifuged at 1500 rpm (491 g) for 10 minutes. The TIL pellet can then be resuspended in 150 mL of fresh 50 / 50 medium containing 3000 IU / mL of IL-2 and added back to the original G-Rex 100 flask. In embodiments where TILs are continuously expanded in G-Rex 100 flasks, on day 7, the TILs in each G-Rex 100 are suspended in the 300 mL of medium present in each flask, and the cell suspension is divided into three 100 mL aliquots, which are used to seed three G-Rex 100 flasks. Next, 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2 was added to each flask. The G-Rex 100 flasks were incubated at 37°C under 5% CO2. After 4 days, 150 mL of AIM-V containing 3000 IU / mL IL-2 was added to each G-Rex 100 flask. Cells were harvested on day 14 of culture.

[0235] Feeder cells and antigen-presenting cells

[0280] In certain embodiments, the second expansion procedure described herein (including step D, REP) requires an excess of feeder cells during the REP TIL expansion and / or during the second expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard whole blood unit from a healthy donor. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation.

[0236]

[0281] Generally, allogeneic PBMCs are inactivated by either 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 the replicative capacity of irradiated allogeneic PBMCs.

[0237]

[0282] In some embodiments, if the total number of viable cells on day 14 is less than the initial number of viable cells entered into culture on day 0 of REP and / or day 0 of the second expansion culture (i.e., the start day of the second expansion culture), the PBMCs are considered non-replicating and are approved for use in the TIL expansion culture procedures described herein.

[0238]

[0283] In some embodiments, PBMCs are considered non-replicating and approved for use in the TIL expansion procedures described herein if the total number of viable cells cultured in the presence of OKT3 and IL-2 has not increased on days 7 and 14 from the initial number of viable cells entered into culture on REP day 0 and / or second expansion day 0 (i.e., the start of the second expansion). In some embodiments, PBMCs are cultured in the presence of 30 ng / ml OKT3 antibody and 3000 IU / ml IL-2.

[0239]

[0284] 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 from the initial number of viable cells introduced into culture on day 0 of REP and / or day 0 of the second expansion culture (i.e., the start of the second expansion culture), the PBMCs are considered replication-incompetent and are approved for use in the TIL expansion procedures described herein. In some embodiments, the PBMCs are cultured in the presence of 5-60 ng / ml of OKT3 antibody and 1000-6000 IU / ml of IL-2. In some embodiments, the PBMCs are cultured in the presence of 10-50 ng / ml of OKT3 antibody and 2000-5000 IU / ml of IL-2. In some embodiments, the PBMCs are cultured in the presence of 20-40 ng / ml of OKT3 antibody and 2000-4000 IU / ml of IL-2. In some embodiments, PBMCs are cultured in the presence of 25-35 ng / ml of OKT3 antibody and 2500-3500 IU / ml of IL-2.

[0240]

[0285] In one embodiment, artificial antigen-presenting cells are used in the REP stage, either as an alternative to or in combination with PBMCs.

[0241] cytokines

[0286] The expansion methods described herein generally use culture media containing high doses of cytokines, particularly IL-2, as is known in the art.

[0242]

[0287] Alternatively, it is further possible to use combinations of cytokines in the rapid and / or secondary expansion of TILs, such as combinations of two or more of IL-2, IL-15, and IL-21, as generally outlined in U.S. Patent Application Publication No. 2017 / 0107490A1, WO 2015 / 189356, U.S. Patent Application Publication No. 2017 / 0107490A1, and WO 2015 / 189357 (each of which is expressly incorporated by reference in its entirety herein). Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15, IL-21, and IL-2, and IL-15 and IL-21, the latter of which finds particular use in many embodiments. As described therein, the use of combinations of cytokines is particularly advantageous for the generation of lymphocytes, particularly T cells.

[0243] Anti-CD3 antibody

[0288] In some embodiments, the culture medium (including REP) used in the expansion methods described herein also contains an anti-CD3 antibody. Anti-CD3 antibodies, when used in combination with IL-2, induce T cell activation and cell division in 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; see, e.g., Tsoukas et al., J. Immunol. 1985, 135, 1719 (hereby incorporated by reference in its entirety).

[0244]

[0289] As one of skill in the art will appreciate, there are a number of suitable anti-human CD3 antibodies that find use in the present invention, including anti-human CD3 polyclonal and monoclonal antibodies from a variety of mammals, including, but not limited to, murine, human, primate, rat, and canine antibodies. In a particular embodiment, the OKT3 anti-CD3 antibody is used (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA).

[0245] Step E: Harvesting TILs

[0290] After the second expansion step, the cells can be harvested. In some embodiments, the TILs are harvested after one, two, three, four or more second expansion steps.

[0246]

[0291] The TILs can be harvested by any suitable and sterile method, including, for example, by centrifugation. TIL harvesting methods are well known in the art, and any such known method can be used with the present process. In some embodiments, the TILs are harvested using an automated system. In some embodiments, the TILs are harvested using a semi-automated system. In some embodiments, the TILs are harvested using a semi-automated system. In some embodiments, the TILs from the second expansion culture are harvested using a semi-automated device. In some embodiments, a LOVO system is used (e.g., commercially available from Benchmark Electronics). In some embodiments, the harvesting step includes washing the TILs, blending the TILs, and / or sorting the TILs. In some embodiments, the cells are optionally frozen after harvesting or as part of the harvesting process.

[0247] Step F: Final formulation / transfer to infusion bag

[0292] After steps A through E are completed, the cells are transferred to a container for use in administration to a patient.

[0248]

[0293] In some embodiments, TILs expanded using the APCs of the present disclosure are administered to a patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded using PBMCs of the present disclosure can be administered by any suitable route as known in the art. In some embodiments, T cells are administered as a single intra-arterial or intravenous infusion, preferably lasting approximately 30-60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic.

[0249] Additional expansion steps

[0294] It will be appreciated that any of the above steps A-F may be repeated any number of times and may even be performed in a different order than that shown above.

[0250]

[0295] In some embodiments, one or more expansion steps may be repeated before the final formulation step F. Such additional expansion steps may include elements of the first and / or second expansion steps described above (e.g., including components described in the cell culture medium). The additional expansion steps may further include additional elements, including additional components in the cell culture medium that are supplemented to the cell culture medium before and / or during the additional expansion steps.

[0251]

[0296] In further embodiments, any of the expansion steps described in Figure 23 and in the paragraphs above may be preceded or followed by a cryopreservation step in which the cells produced during the expansion step are stored using methods known in the art for storage until needed for the remaining steps of the manufacturing / expansion process.

[0252] Pharmaceutical Compositions, Doses and Administration Regimen of TILs, MILs and PBLs

[0297] In one embodiment, the invention provides a therapeutic population of TILs prepared by any of the methods of expanding TILs described herein, and optionally modified to express a chimeric antigen receptor (CAR) and / or to express a modified T cell receptor and / or to suppress or reduce expression of one or more immune checkpoint genes in a transient or stable manner as described herein.

[0253]

[0298] In another embodiment, the invention provides a therapeutic population of MILs prepared by any of the methods of expanding MILs described herein, and optionally modified to express a chimeric antigen receptor (CAR), and / or to express a modified T cell receptor, and / or to suppress or reduce expression of one or more immune checkpoint genes as described herein.

[0254]

[0299] In another embodiment, the invention provides therapeutic populations of PBLs prepared by any of the methods of expanding PBLs described herein, and optionally modified to express a chimeric antigen receptor (CAR) and / or to express a modified T cell receptor and / or to suppress or reduce expression of one or more immune checkpoint genes as described herein.

[0255]

[0300] In another embodiment, the invention provides a pharmaceutical composition comprising a therapeutic population of TILs prepared by any of the methods of expanding TILs described herein, and optionally modified to express a chimeric antigen receptor (CAR), and / or express a modified T cell receptor, and / or to suppress or reduce expression of one or more immune checkpoint genes as described herein, and a pharmaceutically acceptable carrier.

[0256]

[0301] In another embodiment, the invention provides a pharmaceutical composition comprising a therapeutic population of MILs prepared by any of the methods of expanding MILs described herein, and optionally modified to express a chimeric antigen receptor (CAR), and / or to express a modified T cell receptor, and / or to suppress or reduce expression of one or more immune checkpoint genes as described herein, and a pharmaceutically acceptable carrier.

[0257]

[0302] In another embodiment, the invention provides a pharmaceutical composition comprising a therapeutic population of PBLs prepared by any of the methods of expanding PBLs described herein, and optionally modified to express a chimeric antigen receptor (CAR) and / or express a modified T cell receptor and / or to suppress or reduce expression of one or more immune checkpoint genes as described herein, and a pharmaceutically acceptable carrier.

[0258]

[0303] In one embodiment, TILs expanded using the methods of the present disclosure are administered to a patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded using the methods of the present disclosure can be administered by any suitable route known in the art. Preferably, TILs are administered as a single intra-arterial or intravenous infusion, which preferably lasts approximately 30-60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

[0259]

[0304] Any suitable dose of TILs can be administered, preferably an average of about 7.8 x 10, particularly when the cancer is a hematological malignancy. 10 TIL is approximately 2.3 x 10 10 ~Approx. 13.7×10 10 In one embodiment, about 1.2 x 10 TILs are administered. 10 ~Approx. 4.3×10 10 of TILs will be administered.

[0260]

[0305] In some embodiments, the number of TILs provided in the pharmaceutical composition of the present invention is about 1 x 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 , 8×108 , 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 x 10 13 In one embodiment, the number of TILs provided in the pharmaceutical composition of the present invention is 1 x 10 6 ~5×10 6 , 5×10 6 ~1×10 7 , 1×10 7 ~5×10 7 , 5×10 7~1×10 8 , 1×10 8 ~5×10 8 , 5×10 8 ~1×10 9 , 1×10 9 ~5×10 9 , 5×10 9 ~1×10 10 , 1×10 10 ~5×10 10 , 5×10 10 ~1×10 11 , 5×10 11 ~1×10 12 , 1×10 12 ~5×10 12 and 5 x 10 12 ~1×10 13 In one embodiment of the present invention, the number of TILs provided in the pharmaceutical composition of the present invention is in the range of about 4 x 10 8 ~Approx. 2.5×10 9 In another embodiment, the number of TILs provided in the pharmaceutical composition of the present invention is in the range of 9.5 x 10 8 In another embodiment, the number of TILs provided in the pharmaceutical composition of the present invention is 4.1 x 10 8 In another embodiment, the number of TILs provided in the pharmaceutical composition of the present invention is 2.2 x 10 9 is.

[0261]

[0306] In one embodiment of the present invention, the number of TILs provided in the pharmaceutical composition of the present invention is about 0.1 x 10 9 ~Approx. 15×10 9 TIL, approximately 0.1 x 10 9 ~Approx. 15×10 9 TIL, approximately 0.12 x 10 9 ~Approx. 12×10 9 TIL, approximately 0.15 x 10 9 ~Approx. 11×10 9 TIL, approximately 0.2 x 10 9 ~About 10×10 9 TIL, approximately 0.3 x 10 9 ~Approx. 9×10 9 TIL, approximately 0.4 x 10 9 ~Approx. 8×109 TIL, approximately 0.5 x 10 9 ~Approx. 7×10 9 TIL, approximately 0.6 x 10 9 ~about 6×10 9 TIL, approximately 0.7 x 10 9 ~Approx. 5×10 9 TIL, approximately 0.8 x 10 9 ~Approx. 4×10 9 TIL, approximately 0.9 x 10 9 ~Approx. 3×10 9 TILs or approximately 1 x 10 9 ~about 2×10 9 This is a range of TILs.

[0262]

[0307] In some embodiments, the concentration of TILs provided in the pharmaceutical compositions of the present invention is, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.09%, 0.10 ... less than 7%, 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.

[0263]

[0308] In some embodiments, the concentration of TILs provided in the pharmaceutical composition of the present invention is 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%, 15.5%, 16.5% of the pharmaceutical composition. 0%, 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. 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 higher than 0.0001% w / w, w / v or v / v.

[0264]

[0309] In some embodiments, the concentration of TILs provided in the pharmaceutical composition of the present invention is from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0.02% to about 29%, from about 0.03% to about 28%, from about 0.04% to about 27%, from about 0.05% to about 26%, from about 0.06% to about 25%, from about 0.07% to about 24% of the pharmaceutical composition. , 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.

[0265]

[0310] In some embodiments, the concentration of TILs provided in the pharmaceutical compositions of the present invention is within the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, or about 0.1% to about 0.9% w / w, w / v, or v / v of the pharmaceutical composition.

[0266]

[0311] In some embodiments, the amount of TILs provided in the pharmaceutical composition of the present invention is 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g , 0.2g, 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, 0.005g, 0.004g, 0.003g, 0.002g, 0.001g, 0.0009g, 0.0008g, 0.0007g, 0.0006g, 0.0005g, 0.0004g, 0.0003g, 0.0002g or 0.0001g.

[0267]

[0312] In some embodiments, the amount of TILs provided in the pharmaceutical composition of the present invention is 0.0001g, 0.0002g, 0.0003g, 0.0004g, 0.0005g, 0.0006g, 0.0007g, 0.0008g, 0.0009g, 0.001g, 0.0015g, 0.002g, 0.002 5g, 0.003g, 0.0035g, 0.004g, 0.0045g, 0.005g, 0.0055g, 0.006g, 0.0065g, 0.00 7g, 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. 08g, 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, 2.5, 3g, 3.5, 4g, 4.5g, 5g, 5.5g, 6g, 6.5g, 7g, 7.5g, 8g, 8.5g, 9g, 9.5g or more than 10g.

[0268]

[0313] The TILs provided in the pharmaceutical compositions of the present invention are effective over a wide dosage range. The exact dosage will depend on the route of administration, the form in which the compound is administered, the gender and age of the subject to be treated, the weight of the subject to be treated, and the preferences and experience of the attending physician. Clinically established dosages of TILs can also be used where appropriate. The amount of the pharmaceutical composition administered using the methods herein, such as the dosage of TILs, will depend on the human or mammal being treated, the severity of the disease or condition, the rate of administration, the nature of the active pharmaceutical ingredient, and the discretion of the prescribing physician.

[0269]

[0314] In some embodiments, TILs can be administered in a single dose. Such administration can be by injection, for example, intravenous injection. In some embodiments, TILs can be administered in multiple doses. Administration can be once, twice, three times, four times, five times, six times, or more than six times per year. Administration can be once a month, once every two weeks, once a week, or once every other day. Administration of TILs can be continued as long as necessary.

[0270]

[0315] In some embodiments, an effective dose of TILs is about 1×10 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2 × 10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2 × 10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10, 7×10 10 , 8×10 10 , 9×10 10 , 1×10 11 , 2 × 10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 , 1×10 12 , 2 × 10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 1×10 13 , 2 × 10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 and 9 x 10 13 In some embodiments, an effective dose of TILs is 1 x 10 6 ~5×10 6 , 5×10 6 ~1×10 7 , 1×10 7 ~5×10 7 , 5×10 7 ~1×10 8 , 1×10 8 ~5×10 8 , 5×10 8 ~1×10 9 , 1×10 9 ~5×10 9 , 5×10 9 ~1×10 10 , 1×10 10 ~5×10 10 , 5×10 10 ~1×10 11 , 5×10 11 ~1×10 12 , 1×10 12 ~5×10 12and 5 x 10 12 ~1×10 13 It is a range of individuals.

[0271]

[0316] In one embodiment of the present invention, the clinical dose of MIL useful for patients with acute myeloid leukemia (AML) is about 4×10 8 ~Approx. 2.5×10 9 In another embodiment, the number of MILs provided in the pharmaceutical composition of the present invention is within the range of 9.5 x 10 8 In another embodiment, the number of MILs provided in the pharmaceutical composition of the present invention is 4.1 x 10 8 In another embodiment, the number of MILs provided in the pharmaceutical composition of the present invention is 2.2 x 10 9 is.

[0272]

[0317] In some embodiments, an effective dose of TILs is 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 0.7 mg / kg to about 1.5 mg / kg, about 0.5 mg / kg to about 1.6 mg / kg, about 0.65 mg / kg to about 1.7 mg / kg, about 0.75 mg / kg to about 1.5 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 1.5 ...75 mg / kg to about 1.5 mg / kg, about 0.75 mg / kg to about 1.5 mg / kg, about 0.75 mg The range is about 0.75 mg / kg, about 0.7 mg / kg to about 2.15 mg / kg, about 0.85 mg / kg to about 2 mg / kg, about 1 mg / kg to about 1.85 mg / kg, about 1.15 mg / kg to about 1.7 mg / kg, about 1.3 mg / kg to about 1.6 mg / kg, about 1.35 mg / kg to about 1.5 mg / kg, about 2.15 mg / kg to about 3.6 mg / kg, about 2.3 mg / kg to about 3.4 mg / kg, about 2.4 mg / kg to about 3.3 mg / kg, about 2.6 mg / kg to about 3.15 mg / kg, about 2.7 mg / kg to about 3 mg / kg, about 2.8 mg / kg to about 3 mg / kg, or about 2.85 mg / kg to about 2.95 mg / kg.

[0273]

[0318] In some embodiments, the effective dose of TILs is about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg, about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg to about 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, The range is about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, or about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 to about 207 mg.

[0274]

[0319] An effective amount of TILs can be administered in either a single dose or multiple doses by any of the accepted modes of administration for agents with similar utilities, including intranasal and transdermal routes, intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, topical, implanted or injected directly into the tumor, or by inhalation.

[0275] Optional Genetic Engineering of TILs, PBLs and / or MILs

[0320] In some embodiments, the expanded TILs, PBLs, and / or MILs of the present invention are further manipulated before, during, or after the expansion step, including during a closed, sterile manufacturing process, each of which is provided herein, to transiently alter protein expression. In some embodiments, the transiently altered protein expression is by transient gene editing. In some embodiments, the expanded TILs, PBLs, and / or MILs of the present invention are treated with transcription factors (TFs) and / or other molecules that can transiently alter protein expression in the TILs, PBLs, and / or MILs. In some embodiments, the TFs and / or other molecules that can transiently alter protein expression provide for altered expression of tumor antigens and / or altered numbers of tumor antigen-specific T cells in the population of TILs, PBLs, and / or MILs.

[0276]

[0321] In certain embodiments, the method includes gene editing a population of TILs, PBLs, and / or MILs. In certain embodiments, the method includes gene editing a first population of TILs, PBLs, and / or MILs, a second population of TILs, PBLs, and / or MILs, and / or a third population of TILs, PBLs, and / or MILs.

[0277]

[0322] In some embodiments, the invention involves gene editing via nucleotide insertion into a population of TILs, PBLs and / or MILs, such as ribonucleic acid (RNA) insertion, including insertion of messenger RNA (mRNA) or small (or short) interfering RNA (siRNA), to promote expression of one or more proteins or inhibit expression of one or more proteins, and a combination of both promoting one set of proteins and inhibiting another set of proteins simultaneously.

[0278]

[0323] In some embodiments, the expanded TILs, PBLs, and / or MILs of the present invention undergo a transient change in protein expression. In some embodiments, the transient change in protein expression occurs at any time before, during, or after the expansion process. In some embodiments, the transient change in protein expression occurs at any step during the expansion process. In some embodiments, the transient change in protein expression occurs in the bulk TIL, PBL, and / or MIL population before the first expansion. In some embodiments, the transient change in protein expression occurs during the first expansion. In some embodiments, the transient change in protein expression occurs after the first expansion, including, for example, in a transitional TIL, PBL and / or MIL population between the first and second expansion (e.g., a second population of TILs, PBLs and / or MILs described herein). In some embodiments, the transient change in protein expression occurs in a bulk TIL, PBL and / or MIL population before the second expansion. In some embodiments, the transient change in protein expression occurs during the second expansion, including, for example, in a TIL, PBL and / or MIL population being expanded (e.g., a third population of TILs, PBLs and / or MILs). In some embodiments, the transient change in protein expression occurs after the second expansion.

[0279]

[0324] In one embodiment, the method for transiently altering protein expression in a population of TILs, PBLs, and / or MILs comprises the step of electroporation. Electroporation methods are known in the art and are described, for example, in Tsong, Biophys. J. 1991, 60, 297-306 and U.S. Patent Application Publication No. 2014 / 0227237A1, the disclosures of each of which are incorporated herein by reference. In one embodiment, the method for transiently altering protein expression in a population of TILs, PBLs, and / or MILs comprises the step of calcium phosphate transfection. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and are described in Graham and van der Eb, Virology 1973, 52, 456-467; Wigler, et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; and Chen and Okarea, Mol. Cell. Biol. 1987, 7, 2745-2752; and U.S. Patent No. 5,593,875, the disclosures of each of which are incorporated herein by reference. In one embodiment, the method for transiently altering protein expression in a population of TILs, PBLs, and / or MILs includes a liposomal transfection step.Liposome transfection methods, such as those using a 1:1 (w / w) liposome formulation of the cationic lipids N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoylphosphotidylethanolamine (DOPE) in filtered water, are known in the art and are described in Rose, et al., Biotechniques 1991, 10, 520-525 and Felgner, et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417 and U.S. Patent Nos. 5,279,833; 5,908,635; 6,056,938; 6,110,490; 6,534,484; and 7,687,070, the disclosures of each of which are incorporated herein by reference. In one embodiment, the method of transiently altering protein expression in a population of TILs, PBLs, and / or MILs comprises a transfection step using the methods described in U.S. Patent Nos. 5,766,902; 6,025,337; 6,410,517; 6,475,994; and 7,189,705, the disclosures of each of which are incorporated herein by reference.

[0280]

[0325] In some embodiments, transient alterations in protein expression result in an increase in stem cell-like memory T cells (TSCM). TSCM are early precursors of antigen-experienced central memory T cells. TSCM generally exhibit the long-term survival, self-renewal, and multipotency that define stem cells, making them generally desirable for the generation of effective TIL products. TSCM have demonstrated enhanced anti-tumor activity compared to other T cell subsets in mouse models of adoptive cell transfer (Gattinoni et al. Nat Med 2009, 2011; Gattinoni, Nature Rev. Cancer, 2012; Cieri et al. Blood 2013). In some embodiments, transient alterations in protein expression result in a TIL population with a composition containing a high proportion of TSCM. In some embodiments, the transient alteration in protein expression results in an increase in the proportion of TSCMs of at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, the transient alteration in protein expression results in an increase in TSCMs of at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold in the TIL population. In some embodiments, the transient alteration in protein expression results in a TIL population with at least at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% TSCM.In some embodiments, the transient alteration of protein expression results in a therapeutic TIL population with at least at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% TSCM.

[0281]

[0326] In some embodiments, the transient change in protein expression results in the transformation of antigen-experienced T cells, which in some embodiments includes, for example, increased proliferation, increased T cell activation, and / or increased antigen recognition.

[0282]

[0327] In some embodiments, the transient alteration of protein expression alters expression in a majority of T cells to preserve the tumor-derived TCR repertoire. In some embodiments, the transient alteration of protein expression does not alter the tumor-derived TCR repertoire. In some embodiments, the transient alteration of protein expression maintains the tumor-derived TCR repertoire.

[0283]

[0328] In some embodiments, the transient alteration of a protein results in altered expression of a particular gene. In some embodiments, the transient alteration of protein expression targets genes including, but not limited to, PD-1 (also referred to as PDCD1 or CC279), TGFBR2, CCR4 / 5, CBLB (CBL-B), CISH, CCR (chimeric costimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1 / 2 ICD, TIM3, LAG3, TIGIT, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1 / CXCL8, CCL22, CCL17, CXCL1 / CXCL8, VHL, CD44, PIK3CD, SOCS1, and / or cAMP-protein kinase A (PKA). In some embodiments, the transient alteration of protein expression targets a gene selected from the group consisting of PD-1, TGFBR2, CCR4 / 5, CBLB (CBL-B), CISH, CCR (chimeric costimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1 / 2 ICD, TIM3, LAG3, TIGIT, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1 / CXCL8, CCL22, CCL17, CXCL1 / CXCL8, VHL, CD44, PIK3CD, SOCS1, and / or cAMP protein kinase A (PKA). In some embodiments, the transient alteration of protein expression targets PD-1. In some embodiments, the transient alteration of protein expression targets TGFBR2. In some embodiments, the transient alteration of protein expression targets CCR4 / 5. In some embodiments, the transient alteration of protein expression targets CBLB. In some embodiments, the transient alteration of protein expression targets CISH. In some embodiments, the transient alteration of protein expression targets CCR (chimeric costimulatory receptor).In some embodiments, the transient change in protein expression targets IL-2. In some embodiments, the transient change in protein expression targets IL-12. In some embodiments, the transient change in protein expression targets IL-15. In some embodiments, the transient change in protein expression targets IL-21. In some embodiments, the transient change in protein expression targets NOTCH 1 / 2 ICD. In some embodiments, the transient change in protein expression targets TIM3. In some embodiments, the transient change in protein expression targets LAG3. In some embodiments, the transient change in protein expression targets TIGIT. In some embodiments, the transient change in protein expression targets TGFβ. In some embodiments, the transient change in protein expression targets CCR1. In some embodiments, the transient change in protein expression targets CCR2. In some embodiments, the transient change in protein expression targets CCR4. In some embodiments, the transient change in protein expression targets CCR5. In some embodiments, the transient change in protein expression targets CXCR1. In some embodiments, the transient change in protein expression targets CXCR2. In some embodiments, the transient change in protein expression targets CSCR3. In some embodiments, the transient change in protein expression targets CCL2 (MCP-1). In some embodiments, the transient change in protein expression targets CCL3 (MIP-1α). In some embodiments, the transient change in protein expression targets CCL4 (MIP1-β). In some embodiments, the transient change in protein expression targets CCL5 (RANTES). In some embodiments, the transient change in protein expression targets CXCL1. In some embodiments, the transient change in protein expression targets CXCL8. In some embodiments, the transient change in protein expression targets CCL22. In some embodiments, the transient change in protein expression targets CCL17.In some embodiments, the transient alteration of protein expression targets VHL. In some embodiments, the transient alteration of protein expression targets CD44. In some embodiments, the transient alteration of protein expression targets PIK3CD. In some embodiments, the transient alteration of protein expression targets SOCS1. In some embodiments, the transient alteration of protein expression targets cAMP protein kinase A (PKA).

[0284]

[0329] In some embodiments, the transient alteration of protein expression results in an increase and / or overexpression of a chemokine receptor. In some embodiments, the chemokine receptor that is overexpressed by transient protein expression comprises a receptor with a ligand, including, but not limited to, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1, CXCL8, CCL22, and / or CCL17.

[0285]

[0330] In some embodiments, the transient alteration in protein expression results in a decrease and / or down-regulation of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, TGFβR2, and / or TGFβ (e.g., including resulting in blockage of the TGFβ pathway). In some embodiments, the transient alteration in protein expression results in a decrease and / or down-regulation of CBLB (CBL-B). In some embodiments, the transient alteration in protein expression results in a decrease and / or down-regulation of CISH.

[0286]

[0331] In some embodiments, the transient alteration of protein expression results in an increase and / or overexpression of a chemokine receptor, e.g., to improve trafficking or migration of TILs to tumor sites. In some embodiments, the transient alteration of protein expression results in an increase and / or overexpression of a CCR (chimeric costimulatory receptor). In some embodiments, the transient alteration of protein expression results in an increase and / or overexpression of a chemokine receptor selected from the group consisting of CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR2, and / or CSCR3.

[0287]

[0332] In some embodiments, the transient alteration in protein expression results in an increase and / or overexpression of an interleukin, hi some embodiments, the transient alteration in protein expression results in an increase and / or overexpression of an interleukin selected from the group consisting of IL-2, IL-12, IL-15 and / or IL-21.

[0288]

[0333] In some embodiments, the transient alteration in protein expression results in an increase and / or overexpression of NOTCH 1 / 2 ICD. In some embodiments, the transient alteration in protein expression results in an increase and / or overexpression of VHL. In some embodiments, the transient alteration in protein expression results in an increase and / or overexpression of CD44. In some embodiments, the transient alteration in protein expression results in an increase and / or overexpression of PIK3CD. In some embodiments, the transient alteration in protein expression results in an increase and / or overexpression of SOCS1.

[0289]

[0334] In some embodiments, the transient change in protein expression results in a decrease and / or decreased expression of cAMP protein kinase A (PKA).

[0290]

[0335] In some embodiments, the transient alteration in protein expression results in a decrease and / or down-regulation of one molecule selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF(BR3), and combinations thereof. In some embodiments, the transient alteration in protein expression results in a decrease and / or down-regulation of two molecules selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF(BR3), and combinations thereof. In some embodiments, the transient alteration in protein expression results in a decrease and / or down-regulation of PD-1 and one molecule selected from the group consisting of LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF(BR3), and combinations thereof. In some embodiments, the transient alteration in protein expression results in a decrease and / or down-regulation of PD-1, LAG-3, CISH, CBLB, TIM3, and combinations thereof. In some embodiments, the transient alteration in protein expression results in a decrease and / or down-regulation of PD-1 and one of LAG-3, CISH, CBLB, TIM3, and combinations thereof. In some embodiments, the transient alteration in protein expression results in a decrease and / or down-regulation of PD-1 and LAG3. In some embodiments, the transient alteration in protein expression results in a decrease and / or down-regulation of PD-1 and CISH. In some embodiments, the transient alteration in protein expression results in a decrease and / or down-regulation of PD-1 and CBLB. In some embodiments, the transient alteration in protein expression results in a decrease and / or down-regulation of LAG3 and CISH. In some embodiments, the transient alteration in protein expression results in a decrease and / or down-regulation of LAG3 and CBLB. In some embodiments, the transient alteration in protein expression results in a decrease and / or down-regulation of CISH and CBLB. In some embodiments, the transient changes in protein expression result in a decrease and / or down-regulation of TIM3 and PD-1.In some embodiments, the transient alteration of protein expression results in a decrease and / or under-expression of TIM3 and LAG3. In some embodiments, the transient alteration of protein expression results in a decrease and / or under-expression of TIM3 and CISH. In some embodiments, the transient alteration of protein expression results in a decrease and / or under-expression of TIM3 and CBLB.

[0291]

[0336] In some embodiments, an adhesion molecule selected from the group consisting of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof is inserted (e.g., expression of the adhesion molecule is increased) into the first population of TILs, PBLs, and / or MILs, the second population of TILs, PBLs, and / or MILs, or the population of harvested TILs, PBLs, and / or MILs by gammaretroviral or lentiviral methods.

[0292]

[0337] In some embodiments, the transient alteration in protein expression results in a decrease and / or decreased expression of one molecule selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3), and combinations thereof, and an increase and / or enhanced expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof. In some embodiments, the transient alteration in protein expression results in a decrease and / or decreased expression of one molecule selected from the group consisting of PD-1, LAG3, TIM3, CISH, CBLB, and combinations thereof, and an increase and / or enhanced expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof.

[0293]

[0338] In some embodiments, there is about a 5%, 10%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% reduction in expression. In some embodiments, there is at least about a 65%, 70%, 75%, 80%, 85%, 90%, or 95% reduction in expression. In some embodiments, there is at least about a 75%, 80%, 85%, 90%, or 95% reduction in expression. In some embodiments, there is at least about a 80%, 85%, 90%, or 95% reduction in expression. In some embodiments, there is at least about a 85%, 90%, or 95% reduction in expression. In some embodiments, there is at least about a 80% reduction in expression. In some embodiments, there is at least about an 85% reduction in expression. In some embodiments, there is at least about a 90% reduction in expression. In some embodiments, there is at least about a 95% reduction in expression. In some embodiments, there is at least about a 99% reduction in expression.

[0294]

[0339] In some embodiments, there is an increase in expression of about 5%, about 10%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is an increase in expression of at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is an increase in expression of at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is an increase in expression of at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is an increase in expression of at least about 80%. In some embodiments, there is an increase in expression of at least about 85%. In some embodiments, there is an increase in expression of at least about 90%. In some embodiments, there is an increase in expression of at least about 95%. In some embodiments, there is an increase in expression of at least about 99%.

[0295]

[0340] In some embodiments, transient changes in protein expression are induced by treatment of TILs, PBLs, and / or MILs with transcription factors (TFs) and / or other molecules capable of transiently altering protein expression in TILs, PBLs, and / or MILs. In some embodiments, the SQZ vector-free microfluidic platform is utilized for intracellular delivery of transcription factors (TFs) and / or other molecules capable of transiently altering protein expression. Such methods have been described (Sharei et al. PNAS 2013 and Sharei et al. PLOS ONE 2015 and Greisbeck et al. J. Immunology vol. 195, 2015) that demonstrate the ability to deliver proteins, including transcription factors, to a variety of primary human cells, including T cells. See, e.g., WO 2013 / 059343A1, WO 2017 / 008063A1, and WO 2017 / 123663A1, all of which are incorporated by reference in their entirety. Such methods described in WO 2013 / 059343A1, WO 2017 / 008063A1, and WO 2017 / 123663A1 can be utilized with the present invention to expose a population of TILs, PBLs, and / or MILs to transcription factors (TFs) and / or other molecules capable of inducing transient protein expression, wherein the TFs and / or other molecules capable of inducing transient protein expression provide increased expression of tumor antigens and / or increased numbers of tumor antigen-specific T cells in the population of TILs, PBLs, and / or MILs, thus resulting in reprogramming of the TIL population and increased therapeutic efficacy of the reprogrammed TIL population compared to a non-reprogrammed TIL population. In some embodiments, reprogramming results in an increase in a subpopulation of effector T cells and / or central memory T cells compared to a starting population or a pre-population (i.e., pre-programming) population of TILs, PBLs and / or MILs as described herein.

[0296]

[0341] In some embodiments, the transcription factor (TF) includes, but is not limited to, TCF-1, NOTCH 1 / 2 ICD, and / or MYB. In some embodiments, the transcription factor (TF) is TCF-1. In some embodiments, the transcription factor (TF) is NOTCH 1 / 2 ICD. In some embodiments, the transcription factor (TF) is MYB. In some embodiments, the transcription factor (TF) is administered together with induced pluripotent stem cell culture (iPSC), such as commercially available KNOCKOUT Serum Replacement (Gibco / ThermoFisher), to induce additional TIL reprogramming. In some embodiments, the transcription factor (TF) is administered together with an iPSC cocktail to induce additional TIL reprogramming. In some embodiments, the transcription factor (TF) is administered without an iPSC cocktail. In some embodiments, the reprogramming results in an increase in the proportion of TSCM. In some embodiments, the initialization results in an increase in the proportion of TSCM of about 5%, about 10%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.

[0297]

[0342] In some embodiments, the method of transiently altering protein expression, as described above, can be combined with a method of genetically modifying a population of TILs, PBLs, and / or MILs, comprising the step of stable integration of genes for producing one or more proteins. In certain embodiments, the method comprises genetically modifying a population of TILs, PBLs, and / or MILs. In certain embodiments, the method comprises genetically modifying a first population of TILs, PBLs, and / or MILs, a second population of TILs, PBLs, and / or MILs, and / or a third population of TILs, PBLs, and / or MILs. In one embodiment, the method of genetically modifying a population of TILs, PBLs, and / or MILs comprises the step of retroviral transduction. In one embodiment, the method of genetically modifying a population of TILs, PBLs, and / or MILs comprises the step of lentiviral transduction. Lentiviral transduction systems are known in the art and are described, for example, in Levine, et al., Proc. Nat'l Acad. Sci. 2006, 103, 17372-77; Zufferey, et al., Nat. Biotechnol. 1997, 15, 871-75; Dull, et al., J. Virology 1998, 72, 8463-71 and U.S. Patent No. 6,627,442, the disclosures of each of which are incorporated herein by reference. In one embodiment, the method for genetically modifying a population of TILs, PBLs and / or MILs comprises a step of gammaretroviral transduction. Gammaretroviral transduction systems are known in the art and are described, for example, in Cepko and Pear, Cur.Prot.Mol.Biol.1996, 9.9.1-9.9.16, the disclosure of which is incorporated herein by reference. In one embodiment, the method for genetically modifying a population of TILs, PBLs and / or MILs comprises the step of transposon-mediated gene transfer.Transposon-mediated gene transfer systems are known in the art and include systems in which the transposase is provided as a DNA expression vector or as an expressible RNA or protein, such as mRNA (e.g., mRNA containing a cap and polyA tail), so that long-term expression of the transposase does not occur in transgenic cells. Suitable transposon-mediated gene transfer systems, including salmonid-type Tel-like transposases (SB or Sleeping Beauty transposase), such as SB10, SB11, and SB100x, as well as engineered enzymes with increased enzymatic activity, are described, for example, in Hackett, et al., Mol. Therapy 2010, 18, 674-83 and U.S. Patent No. 6,489,458, the disclosures of each of which are incorporated herein by reference.

[0298]

[0343] In some embodiments, the transient alteration of protein expression is a reduction in expression induced by self-delivering RNA interference (sdRNA), which is a chemically synthesized asymmetric siRNA duplex with a high 2'-OH substitution rate (typically fluorine or -OCH3), comprising a 20-nucleotide antisense (guide) strand and a 13-15-nucleotide sense (passenger) strand linked to cholesterol at its 3' end using a tetraethylene glycol (TEG) linker. In some embodiments, the method comprises transient alteration of protein expression in a population of TILs, PBLs, and / or MILs, involving the use of self-delivering RNA interference (sdRNA), which is a chemically synthesized asymmetric siRNA duplex with a high 2'-OH substitution rate (typically fluorine or -OCH3), comprising a 20-nucleotide antisense (guide) strand and a 13-15-nucleotide sense (passenger) strand linked to cholesterol at its 3' end using a tetraethylene glycol (TEG) linker. Methods for using sdRNA are described in Khvorova and Watts, Nat. Biotechnol. 2017, 35, 238-248; Byrne, et al., J. Ocul. Pharmacol. Ther. 2013, 29, 855-864; and Ligtenberg, et al., Mol. Therapy, 2018, 26, 1482-1493, the disclosures of which are incorporated herein by reference. In one embodiment, delivery of sdRNA to a TIL population is achieved without the use of electroporation, SQZ, or other methods, but instead using a 1-3 day period in which the TIL population is exposed to sdRNA at a concentration of 1 μM / 10,000 TILs, PBLs, and / or MILs in culture medium. In certain embodiments, the method comprises delivering sdRNA to a population of TILs, PBLs and / or MILs, which comprises exposing the population of TILs, PBLs and / or MILs to the sdRNA at a concentration of 1 μM / 10,000 TILs, PBLs and / or MILs in culture medium for a period of 1 to 3 days.In one embodiment, delivery of sdRNA to a TIL population is achieved using a 1-3 day period in which the TIL population is exposed to sdRNA at a concentration of 10 μM / 10,000 TILs, PBLs, and / or MILs in medium. In one embodiment, delivery of sdRNA to a TIL population is achieved using a 1-3 day period in which the TIL population is exposed to sdRNA at a concentration of 50 μM / 10,000 TILs, PBLs, and / or MILs in medium. In one embodiment, delivery of sdRNA to a TIL population is achieved using a 1-3 day period in which the TIL population is exposed to sdRNA at a concentration of 0.1 μM / 10,000 TILs, PBLs, and / or MILs to 50 μM / 10,000 TILs, PBLs, and / or MILs in medium. In one embodiment, delivery of sdRNA to a TIL population is achieved using a 1-3 day period in which the TIL population is exposed to sdRNA at a concentration of 0.1 μM / 10,000 TILs, PBLs, and / or MILs to 50 μM / 10,000 TILs, PBLs, and / or MILs in the culture medium, where the exposure to sdRNA is repeated 2, 3, 4, or 5 times by adding fresh sdRNA to the culture medium. Other suitable processes are described, for example, in U.S. Patent Application Publication Nos. 2011 / 0039914 A1, 2013 / 0131141 A1, and 2013 / 0131142 A1, and U.S. Patent No. 9,080,171, the disclosures of which are incorporated herein by reference.

[0299]

[0344] In some embodiments, sdRNA is inserted into a population of TILs, PBLs, and / or MILs during production. In some embodiments, the sdRNA encodes an RNA that interferes with NOTCH 1 / 2 ICD, PD-1, CTLA-4 TIM-3, LAG-3, TIGIT, TGFβ, TGFBR2, cAMP protein kinase A (PKA), BAFF BR3, CISH, and / or CBLB. In some embodiments, the reduction in expression is determined based on the percentage of gene silencing, as assessed, for example, by flow cytometry and / or qPCR. In some embodiments, there is a reduction in expression of about 5%, about 10%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is a reduction in expression of at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is a reduction in expression of at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is a reduction in expression of at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is a reduction in expression of at least about 85%, about 90%, or about 95%. In some embodiments, there is a reduction in expression of at least about 80%. In some embodiments, there is a reduction in expression of at least about 85%. In some embodiments, there is a reduction in expression of at least about 90%. In some embodiments, there is a reduction in expression of at least about 95%. In some embodiments, there is a reduction in expression of at least about 99%.

[0300]

[0345] As described herein, self-deliverable RNAi technology based on chemical modification of siRNA can be used in conjunction with the methods of the present invention to successfully deliver sdRNA to TILs, PBLs, and / or MILs. The combination of backbone modifications with asymmetric siRNA structures and hydrophobic ligands (e.g., Ligtenberg, et al., Mot. Therapy, 2018 and U.S. Patent Application Publication No. 20160304873) exploits the nuclease stability of sdRNA, allowing it to penetrate cultured mammalian cells without additional formulations or methods by simply adding it to the culture medium. This stability allows for the support of a constant level of RNAi-mediated reduction in target gene activity simply by maintaining an active concentration of sdRNA in the culture medium. Without being bound by theory, sdRNA backbone stabilization provides a significant reduction in gene expression effects that can last for several months in non-dividing cells.

[0301]

[0346] In some embodiments, greater than 95% transfection efficiency of TILs, PBLs, and / or MILs and reduction of target expression by various specific sdRNAs occur. In some embodiments, sdRNAs containing several unmodified ribose residues are replaced with fully modified sequences to increase the potency and / or longevity of the RNAi effect. In some embodiments, the reduction in expression effect is maintained for 12 hours, 24 hours, 36 hours, 48 ​​hours, 5 days, 6 days, 7 days, or 8 days or more. In some embodiments, the reduction in expression effect decreases 10 days or more after sdRNA treatment of TILs, PBLs, and / or MILs. In some embodiments, greater than 70% reduction in expression of target expression is maintained. In some embodiments, greater than 70% reduction in expression of target expression is maintained in TILs, PBLs, and / or MILs. In some embodiments, reduced expression of the PD-1 / PD-L1 pathway allows TILs, PBLs, and / or MILs to exhibit more potent in vivo effects, which in some embodiments is due to circumvention of the inhibitory effects of the PD-1 / PD-L1 pathway. In some embodiments, reducing the expression of PD-1 by sdRNA results in increased TIL proliferation.

[0302]

[0347] Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a double-stranded RNA molecule typically 19-25 base pairs in length. siRNA is used in RNA interference (RNAi) to interfere with the expression of specific genes that have complementary nucleotide sequences.

[0303]

[0348] Double-stranded DNA (dsRNA) can be generally used to define any molecule that comprises a pair of complementary strands of RNA, generally being sense (passenger) and antisense (guiding) strands, and this may comprise a single-stranded overhang region.In contrast to siRNA, the term dsRNA generally refers to the precursor molecule that comprises the sequence of siRNA molecule that is released from larger dsRNA molecule by the action of the cleavage enzyme system that comprises Dicer.

[0304]

[0349] Self-deliverable RNA (sdRNA) is a new class of covalently modified RNAi compounds that do not require a delivery vehicle to enter cells and have improved pharmacology compared to conventional siRNA. "Self-deliverable RNA" or "sdRNA" is a hydrophobically modified RNA interference antisense hybrid that has been demonstrated to be highly effective in primary cells in vitro and in vivo when administered locally. It has demonstrated potent uptake and / or silencing without toxicity. sdRNA is generally a chemically modified, asymmetric nucleic acid molecule with minimal double-stranded regions. sdRNA molecules typically contain single-stranded and double-stranded regions and may contain various chemical modifications in both the single-stranded and double-stranded regions of the molecule. Furthermore, sdRNA molecules can be attached to hydrophobic linkages, such as conventional and cutting-edge sterol-type molecules, as described herein.

[0003] sdRNAs and related methods for making such sdRNAs are also described extensively in, for example, U.S. Patent Application Publication No. 20160304873, WO 2010033246, WO 2017070151, WO 2009102427, WO 2011119887, WO 2010033247A2, WO 2009045457, and WO 2011119852, all of which are incorporated by reference in their entirety for all purposes. Proprietary algorithms have been developed to optimize sdRNA structure, chemistry, target location, sequence preferences, etc., and are utilized to predict sdRNA potency (see, e.g., U.S. Patent Application Publication No. 20160304873). Based on these analyses, functional sdRNA sequences are generally defined as those that have a greater than 40% probability of having a greater than 70% reduction in expression at a concentration of 1 μM.

[0305]

[0350] In some embodiments, the sdRNA sequences used in the present invention exhibit a 70% reduction in target gene expression. In some embodiments, the sdRNA sequences used in the present invention exhibit a 75% reduction in target gene expression. In some embodiments, the sdRNA sequences used in the present invention exhibit a 80% reduction in target gene expression. In some embodiments, the sdRNA sequences used in the present invention exhibit a 85% reduction in target gene expression. In some embodiments, the sdRNA sequences used in the present invention exhibit a 90% reduction in target gene expression. In some embodiments, the sdRNA sequences used in the present invention exhibit a 95% reduction in target gene expression. In some embodiments, the sdRNA sequences used in the present invention exhibit a 99% reduction in target gene expression. In some embodiments, the sdRNA sequences used in the present invention exhibit a reduction in target gene expression when delivered at a concentration of about 0.25 μM to about 4 μM. In some embodiments, the sdRNA sequences used in the present invention exhibit a reduction in target gene expression when delivered at a concentration of about 0.25 μM. In some embodiments, the sdRNA sequences used in the present invention exhibit a reduction in target gene expression when delivered at a concentration of about 0.5 μM. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced target gene expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced target gene expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced target gene expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced target gene expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced target gene expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced target gene expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced target gene expression when delivered at a concentration of about 2.25 μM.In some embodiments, the sdRNA sequences used in the present invention exhibit reduced target gene expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced target gene expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced target gene expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced target gene expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced target gene expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced target gene expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequences used in the present invention exhibit reduced target gene expression when delivered at a concentration of about 4.0 μM.

[0306]

[0351] In some embodiments, the oligonucleotide agent contains one or more modifications to increase the stability and / or efficacy of the therapeutic agent and to achieve efficient delivery of the oligonucleotide to the treated cell or tissue. Such modifications may include 2'-O-methyl modifications, 2'-O-fluoro modifications, diphosphorothioate modifications, 2'F-modified nucleotides, 2'-O-methyl modifications, and / or 2'-deoxy nucleotides. In some embodiments, the oligonucleotide is modified to contain one or more hydrophobic modifications, including, for example, sterol, cholesterol, vitamin D, naphthyl, isobutyl, benzyl, indole, tryptophan, and / or phenyl. In further specific embodiments, the chemically modified nucleotide is a combination of phosphorothioate, 2'-O-methyl, 2'-deoxy, hydrophobic modifications, and phosphorothioate. In some embodiments, sugars can be modified, and modified sugars can include, but are not limited to, D-ribose, 2'-O-alkyl (including 2'-O-methyl and 2'-O-ethyl), i.e., 2'-alkoxy, 2'-amino, 2'-S-alkyl, 2'-halo (including 2'-fluoro), T-methoxyethoxy, 2'-allyloxy (-OCHCH=CH), 2'-propargyl, 2'-propyl, ethynyl, ethenyl, propenyl, and cyano. In one embodiment, the sugar moiety can be a hexose and can be incorporated into an oligonucleotide as described (Augustyns, K., et al., Nucl. Acids. Res. 18:4711 (1992)).

[0307]

[0352] In some embodiments, the double-stranded oligonucleotide of the present invention is double-stranded throughout its entire length, i.e., there is no single-stranded sequence protruding from either end of the molecule, i.e., it is blunt-ended.In some embodiments, individual nucleic acid molecules can be of different lengths.In other words, the double-stranded oligonucleotide of the present invention is not double-stranded throughout its entire length.For example, when two separate nucleic acid molecules are used, one of the molecules, for example, the first molecule that comprises antisense sequence, can be longer than the second molecule that hybridizes to it (a part of the molecule remains single-stranded).In some embodiments, when a single nucleic acid molecule is used, a part of the molecule at either end can remain single-stranded.

[0308]

[0353] In some embodiments, double-stranded oligonucleotides of the present invention contain mismatches and / or loops or bulges, but are double-stranded over at least about 70% of the length of the oligonucleotide. In some embodiments, double-stranded oligonucleotides of the present invention are double-stranded over at least about 80% of the length of the oligonucleotide. In other embodiments, double-stranded oligonucleotides of the present invention are double-stranded over at least about 90%-95% of the length of the oligonucleotide. In some embodiments, double-stranded oligonucleotides of the present invention are double-stranded over at least about 96%-98% of the length of the oligonucleotide. In some embodiments, double-stranded oligonucleotides of the present invention contain at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mismatches.

[0309]

[0354] In some embodiments, oligonucleotides can be substantially protected from nucleases, for example, by modifying the 3' or 5' linkage (e.g., U.S. Pat. No. 5,849,902 and WO 98 / 13526). For example, oligonucleotides can be made resistant by including a "blocking group." As used herein, the term "blocking group" refers to a substituent (e.g., other than an OH group) that can be attached to an oligonucleotide or nucleomonomer, either as a protecting group for synthesis or as a coupling group (e.g., FITC, propyl (CH2-CH2-CH3), glycol (-O-CH2-CH2-O-) phosphate (PO3 2’’ ), hydrogen phosphonate or phosphoramidite). "Blocking groups" can also include "terminal blocking groups" or "exonuclease blocking groups" that protect the 5' and 3' ends of oligonucleotides, including modified nucleotides and non-nucleotide exonuclease-resistant structures.

[0310]

[0355] In some embodiments, at least some of the adjacent polynucleotides within the sdRNA are joined by substitute linkages, such as phosphorothioate linkages.

[0311]

[0356] In some embodiments, the chemical modification leads to an enhancement of cellular uptake of at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500. In some embodiments, at least one of the C or U residues comprises a hydrophobic modification. In some embodiments, a plurality of C's and U's contain a hydrophobic modification. In some embodiments, at least 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or at least 95% of C's and U's may contain a hydrophobic modification. In some embodiments, all of C's and U's contain a hydrophobic modification.

[0312]

[0357] In some embodiments, sdRNA or sd-rxRNA exhibit enhanced endosomal release of sd-rxRNA molecules due to the incorporation of a protonatable amine. In some embodiments, the protonatable amine is incorporated into the sense strand (in the portion of the molecule that is truncated after RISC loading). In some embodiments, sdRNA compounds of the present invention comprise asymmetric compounds containing a double-stranded region (10-15 bases long, required for efficient RISC entry) and a single-stranded region 4-12 nucleotides long, with a 13-nucleotide double strand. In some embodiments, a single-stranded region of 6 nucleotides is utilized. In some embodiments, the single-stranded region of the sdRNA contains 2-12 phosphorothioate internucleotide linkages (referred to as phosphorothioate modifications). In some embodiments, 6-8 phosphorothioate internucleotide linkages are utilized. In some embodiments, sdRNA compounds of the present invention also contain unique chemical modification patterns that provide stability and are compatible with RISC entry.

[0313]

[0358] For example, the guide strand can be modified with any chemical modification that ensures stability without interfering with RISC entry. In some embodiments, the chemical modification pattern of the guide strand includes a majority of C and U nucleotides being 2'F modified and the 5' end being phosphorylated.

[0314]

[0359] In some embodiments, at least 30% of the nucleotides of the sdRNA or sd-rxRNA are modified. In some embodiments, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% modified. In some embodiments, 100% of the nucleotides of the sdRNA or sd-rxRNA are modified.

[0315]

[0360] In some embodiments, the sdRNA molecule has a minimal double-stranded region. In some embodiments, the region of the molecule that is double-stranded ranges from 8 to 15 nucleotides in length. In some embodiments, the region of the molecule that is double-stranded is 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In some embodiments, the double-stranded region is 13 nucleotides in length. The guide strand and passenger strand can be 100% complementary, or there can be one or more mismatches between the guide strand and the passenger strand. In some embodiments, at the end of the double-stranded molecule, the molecule is blunt-ended or has a single-nucleotide overhang. The single-stranded region of the molecule is, in some embodiments, 4 to 12 nucleotides in length. In some embodiments, the single-stranded region can be 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides in length. In some embodiments, the single-stranded region can be less than 4 nucleotides in length or more than 12 nucleotides in length. In certain embodiments, the single-stranded region is 6 or 7 nucleotides in length.

[0316]

[0361] In some embodiments, the sdRNA molecule has increased stability. In some instances, the chemically modified sdRNA or sd-rxRNA molecule has a half-life in culture medium of greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or more, including any intermediate value. In some embodiments, the sd-rxRNA has a half-life in culture medium of greater than 12 hours.

[0317]

[0362] In some embodiments, sdRNA is optimized for increased potency and / or reduced toxicity. In some embodiments, the nucleotide length of the guide strand and / or passenger strand and / or the number of phosphorothioate modifications in the guide strand and / or passenger strand can affect the potency of the RNA molecule in some aspects, and the replacement of 2'-fluoro (2'F) modifications with 2'-O-methyl (2'OMe) modifications can affect the toxicity of the molecule in some aspects. In some embodiments, reducing the 2'F content of a molecule is predicted to reduce the toxicity of the molecule. In some embodiments, the number of phosphorothioate modifications in an RNA molecule can affect the efficiency of cellular uptake of the molecule, for example, passive uptake of the molecule into cells. In some embodiments, sdRNA does not have 2'F modifications but is characterized by comparable potency in cellular uptake and tissue penetration.

[0318]

[0363] In some embodiments, the guide strand is approximately 18-19 nucleotides in length and has approximately 2-14 phosphate modifications. For example, the guide strand can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 phosphate-modified nucleotides. The guide strand can contain one or more modifications that confer increased stability without interfering with RISC entry. Phosphate-modified nucleotides, such as phosphorothioate-modified nucleotides, can be at the 3'-end, 5'-end, or spread throughout the guide strand. In some embodiments, the 3'-terminal 10 nucleotides of the guide strand contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphorothioate-modified nucleotides. The guide strand can contain 2'F and / or 2'OMe modifications, which can also be located throughout the molecule. In some embodiments, the first nucleotide of the guide strand (the 5'-most nucleotide of the guide strand) is 2'OMe-modified and / or phosphorylated. C and U nucleotides in the guide strand can be 2'F-modified. For example, C and U nucleotides at positions 2-10 of a 19-nt guide strand (or corresponding positions in guide strands of different lengths) can be 2'F-modified. C and U nucleotides in the guide strand can also be 2'OMe-modified. For example, C and U nucleotides at positions 11-18 of a 19-nt guide strand (or corresponding positions in guide strands of different lengths) can be 2'OMe-modified. In some embodiments, the 3'-most nucleotide of the guide strand is unmodified. In certain embodiments, the majority of C and U nucleotides in the guide strand are 2'F-modified, and the 5'-end of the guide strand is phosphorylated. In other embodiments, C or U nucleotides at positions 1 and 11-18 are 2'OMe-modified, and the 5'-end of the guide strand is phosphorylated. In other embodiments, C or U nucleotides at positions 1 and 11-18 are 2'OMe-modified, the 5'-end of the guide strand is phosphorylated, and C or U nucleotides at positions 2-10 are 2'F-modified.

[0319]

[0364] Self-deliverable RNAi technology provides a method for directly transfecting cells with RNAi agents without the need for additional formulations or techniques. The ability to transfect difficult-to-transfect cell lines, high in vivo activity, and ease of use are characteristics of compositions and methods that offer significant functional advantages over traditional siRNA-based technologies, and therefore sdRNA methods are utilized in some embodiments of the methods of the present invention for reducing target gene expression in TILs, PBLs, and / or MILs. The sdRNAi method allows for the direct delivery of chemically synthesized compounds to a wide range of primary cells and tissues, both ex vivo and in vivo. The sdRNAs described in some embodiments of the present invention are commercially available from Advirna LLC, Worcester, MA, USA.

[0320]

[0365] sdRNAs are formed as hydrophobically modified siRNA-antisense oligonucleotide hybrid structures, as disclosed, for example, in Byrne et al., December 2013, J. Ocular Pharmacology and Therapeutics, 29(10):855-864, which is incorporated herein by reference in its entirety.

[0321]

[0366] In some embodiments, sdRNA oligonucleotides can be delivered to the TILs, PBLs, and / or MILs described herein using sterile electroporation. In certain embodiments, the methods involve sterile electroporation of a population of TILs, PBLs, and / or MILs to deliver the sdRNA oligonucleotides.

[0322]

[0367] In some embodiments, the oligonucleotide can be delivered to cells in combination with a transmembrane delivery system. In some embodiments, the transmembrane delivery system includes a lipid, a viral vector, or the like. In some embodiments, the oligonucleotide agent is a self-delivering RNAi agent that does not require any delivery agent. In certain embodiments, the method includes using a transmembrane delivery system to deliver sdRNA oligonucleotides to a population of TILs, PBLs, and / or MILs.

[0323]

[0368] Oligonucleotides and oligonucleotide compositions can be contacted (e.g., contacted, also referred to herein as administered or delivered) with and taken up by TILs, PBLs, and / or MILs described herein, including through passive uptake by TILs, PBLs, and / or MILs. sdRNA can be added to TILs, PBLs, and / or MILs as described herein during the first expansion, e.g., step B; after the first expansion, e.g., during step C; before or during the second expansion, e.g., before or during step D; after step D and before recovery in step E; during or after recovery in step F; before or during transfer to the final formulation and / or infusion bag in step F; and before the optional cryopreservation step in step F. Additionally, sdRNA can be added after thawing from the optional cryopreservation step in step F. In one embodiment, one or more sdRNA targeting genes described herein, including PD-1, LAG-3, TIM-3, CISH, and CBLB, may be added to cell culture medium containing TILs, PBLs, and / or MILs and other agents at a concentration selected from the group consisting of 100 nM to 20 mM, 200 nM to 10 mM, 500 nM to 1 mM, 1 μM to 100 μM, and 1 μM to 100 μM.In one embodiment, one or more sdRNA targeting genes described herein, including PD-1, LAG-3, TIM-3, CISH, and CBLB, are administered to TILs, PBLs, and / or MILs and other agents at concentrations of 0.1 μM sdRNA / 10,000 TILs, PBLs, and / or MILs / 100 μL medium, 0.5 μM sdRNA / 10,000 TILs, PBLs, and / or MILs / 100 μL medium, 0.75 μM sdRNA / 10,000 TILs, PBLs, and / or MILs / 100 μL medium, 1 μM sdRNA / 10,000 TILs, PBLs, and / or MILs / 100 μL medium, 1.25 μM sdRNA / 10,000 TILs, PBLs, and / or MILs / 100 μL medium, 1.5 μM The sdRNA may be added to cell culture medium containing an amount selected from the group consisting of sdRNA / 10,000 TILs, PBLs, and / or MILs / 100 μL medium, 2 μM sdRNA / 10,000 TILs, PBLs, and / or MILs / 100 μL medium, 5 μM sdRNA / 10,000 TILs, PBLs, and / or MILs / 100 μL medium, or 10 μM sdRNA / 10,000 TILs, PBLs, and / or MILs / 100 μL medium. In one embodiment, one or more sdRNA-targeted genes described herein, including PD-1, LAG-3, TIM-3, CISH, and CBLB, may be added to TIL cultures twice a day, once a day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, or every 7 days during the pre-REP or REP stages.

[0324]

[0369] Oligonucleotide compositions of the invention comprising sdRNA can be contacted with TILs, PBLs, and / or MILs described herein during the expansion process, for example, by dissolving the sdRNA at a high concentration in cell culture medium and allowing sufficient time for passive uptake to occur. In certain embodiments, methods of the invention involve contacting a population of TILs, PBLs, and / or MILs with an oligonucleotide composition described herein. In certain embodiments, methods involve dissolving oligonucleotides, such as sdRNA, in cell culture medium and contacting the cell culture medium with a population of TILs, PBLs, and / or MILs. The TILs, PBLs, and / or MILs can be a first population, a second population, and / or a third population, as described herein.

[0325]

[0370] In some embodiments, delivery of oligonucleotides to cells may be enhanced by suitable art-recognized methods, including calcium phosphate, DMSO, glycerol, or dextran, electroporation, or by transfection, e.g., using cationic, anionic, or neutral lipid compositions or liposomes, using methods known in the art (see, e.g., WO 90 / 14074; WO 91 / 16024; WO 91 / 17424; U.S. Pat. No. 4,897,355; Bergan et al. 1993. Nucleic Acids Research. 21:3567).

[0326]

[0371] In some embodiments, two or more sdRNAs are used to reduce the expression of target genes. In some embodiments, one or more of PD-1, TIM-3, CBLB, LAG3, and / or CISH-targeting sdRNAs are used together. In some embodiments, PD-1 sdRNA is used with one or more of TIM-3, CBLB, LAG3, and / or CISH to reduce the expression of two or more gene targets. In some embodiments, LAG3 sdRNA is used in combination with CISH-targeting sdRNA to reduce the gene expression of both targets. In some embodiments, the sdRNAs targeting one or more of PD-1, TIM-3, CBLB, LAG3, and / or CISH herein are commercially available from Advirna LLC, Worcester, MA, USA.

[0327]

[0372] In some embodiments, the sdRNA targets a gene selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF(BR3), and combinations thereof. In some embodiments, the sdRNA targets a gene selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF(BR3), and combinations thereof. In some embodiments, one sdRNA targets PD-1 and another sdRNA targets a gene selected from the group consisting of LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF(BR3), and combinations thereof. In some embodiments, the sdRNA targets a gene selected from PD-1, LAG-3, CISH, CBLB, TIM3, and combinations thereof. In some embodiments, the sdRNA targets a gene selected from PD-1 and one of LAG3, CISH, CBLB, TIM3, and combinations thereof. In some embodiments, one sdRNA targets PD-1 and one sdRNA targets LAG3. In some embodiments, one sdRNA targets PD-1 and one sdRNA targets CISH. In some embodiments, one sdRNA targets PD-1 and one sdRNA targets CBLB. In some embodiments, one sdRNA targets LAG3 and one sdRNA targets CISH. In some embodiments, one sdRNA targets LAG3 and one sdRNA targets CBLB. In some embodiments, one sdRNA targets CISH and one sdRNA targets CBLB. In some embodiments, one sdRNA targets TIM3 and one sdRNA targets PD-1. In some embodiments, one sdRNA targets TIM3 and one sdRNA targets LAG3. In some embodiments, one sdRNA targets TIM3 and one sdRNA targets CISH.In some embodiments, one sdRNA targets TIM3 and one sdRNA targets CBLB.

[0328]

[0373] As described above, embodiments of the present invention provide TILs, PBLs, and / or MILs that have been genetically modified via gene editing to enhance their therapeutic effects. Embodiments of the present invention encompass gene editing by inserting nucleotides (RNA or DNA) into a population of TILs, PBLs, and / or MILs to both promote expression of one or more proteins and inhibit expression of one or more proteins, as well as combinations thereof. Embodiments of the present invention also provide methods for expanding TILs, PBLs, and / or MILs into a therapeutic population, the methods comprising genetically editing the TILs, PBLs, and / or MILs. There are several gene editing techniques that can be used to genetically modify a population of TILs, PBLs, and / or MILs suitable for use in accordance with the present invention.

[0329]

[0374] In some embodiments, the method comprises a method for genetically modifying a population of TILs, PBLs, and / or MILs, comprising the step of stable integration of a gene for producing one or more proteins. In one embodiment, the method for genetically modifying a population of TILs, PBLs, and / or MILs comprises the step of retroviral transduction. In one embodiment, the method for genetically modifying a population of TILs, PBLs, and / or MILs comprises the step of lentiviral transduction. Lentiviral transduction systems are known in the art and are described, for example, in Levine, et al., Proc. Nat'l Acad. Sci. 2006, 103, 17372-77; Zufferey, et al., Nat. Biotechnol. 1997, 15, 871-75; Dull, et al., J. Virology 1998, 72, 8463-71 and U.S. Pat. No. 6,627,442, the disclosures of each of which are incorporated herein by reference. In one embodiment, the method for genetically modifying a population of TILs, PBLs, and / or MILs comprises a step of gammaretroviral transduction. Gammaretroviral transduction systems are known in the art and are described, for example, in Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16, the disclosure of which is incorporated herein by reference. In one embodiment, the method for genetically modifying a population of TILs, PBLs, and / or MILs comprises a step of transposon-mediated gene transfer. Transposon-mediated gene transfer systems are known in the art and include systems in which the transposase is provided as a DNA expression vector or as an expressible RNA or protein, such as mRNA (e.g., mRNA comprising a cap and polyA tail), such that long-term expression of the transposase does not occur in the transgenic cells.Suitable transposon-mediated gene transfer systems including salmonid-type Tel-like transposases (SB or Sleeping Beauty transposase), such as SB10, SB11, and SB100x, as well as engineered enzymes with increased enzymatic activity, are described, for example, in Hackett, et al., Mol. Therapy 2010, 18, 674-83 and U.S. Pat. No. 6,489,458, the disclosures of each of which are incorporated herein by reference.

[0330]

[0375] In one embodiment, the method includes genetically modifying a population of TILs, PBLs, and / or MILs, such as the first, second, and / or third populations described herein. In one embodiment, the method includes stable integration of genes for producing or inhibiting (e.g., silencing) one or more proteins. In one embodiment, the method includes electroporation. Electroporation methods are known in the art and are described, for example, in Tsong, Biophys. J. 1991, 60, 297-306 and U.S. Patent Application Publication No. 2014 / 0227237A1, the disclosures of each of which are incorporated herein by reference. Other electroporation methods known in the art can be used, such as those described in U.S. Patent Nos. 5,019,034; 5,128,257; 5,137,817; 5,173,158; 5,232,856; 5,273,525; 5,304,120; 5,318,514; 6,010,613 and 6,078,490 (these disclosures are incorporated herein by reference).In one embodiment, the electroporation method is sterile electroporation.In one embo...

Claims

1. A method for expanding peripheral blood lymphocytes (PBLs) from peripheral blood, comprising: Steps below: a. culturing a sample of peripheral blood mononuclear cells (PBMCs) obtained from the peripheral blood of a patient who has been pre-treated with ibrutinib or another IL-2-inducible T-cell kinase (ITK) inhibitor and who is refractory to treatment with ibrutinib or another ITK inhibitor, in a culture comprising a first cell culture medium, IL-2, and an anti-CD3 / anti-CD28 antibody for a period of time selected from the group consisting of 9 days, 10 days, 11 days, 12 days, 13 days, and 14 days, thereby resulting in the expansion of peripheral blood lymphocytes (PBLs) from said PBMCs; b. recovering the PBLs from the culture in step a; and c. Transducing the PBLs with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single-chain variable fragment antibody fused to at least one endodomain of a T cell signaling molecule, or transducing the PBLs with an expression vector comprising a genetically engineered T cell receptor. A method comprising:

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

3. 2. The method of claim 1, 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 is replaced in the culture.

4. 4. The method of claim 3, wherein the first cell culture medium is exchanged with the second cell culture medium in the culture.

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

6. 3. The method of claim 2, wherein the magnetic beads in step a are DynaBeads®.

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

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

9. 10. The method of claim 1, wherein the first cell culture medium comprises 3000 IU / ml of IL-2.

10. Cultures were incubated at 37°C and 5% CO ２ The method of claim 1, wherein the mixture is incubated with

11. The method described in claim 1, wherein step a is carried out for 9 days.

12. The method described in claim 1, wherein step a is carried out for 11 days.

13. 10. The method of claim 1, wherein the patient has been pre-treated with ibrutinib and is refractory to treatment with ibrutinib.

14. 10. The method of claim 1, wherein the patient has not received treatment with ibrutinib or another ITK inhibitor for at least one month prior to being pretreated with ibrutinib or another ITK inhibitor.

15. 10. The method of claim 1, wherein the patient has been pretreated with ibrutinib or another ITK inhibitor for at least 3 months.

16. 10. The method of claim 1, wherein the patient has been pretreated with ibrutinib for at least 3 months.

17. A pharmaceutical composition for treating hematological malignancies, comprising peripheral blood lymphocytes (PBLs), The PBL is subjected to the following steps: a. culturing a sample of peripheral blood mononuclear cells (PBMCs) obtained from the peripheral blood of a patient with a hematological malignancy who has been pre-treated with ibrutinib or another IL-2-inducible T-cell kinase (ITK) inhibitor and who is refractory to treatment with ibrutinib or another ITK inhibitor, in a culture comprising a first cell culture medium, IL-2, and an anti-CD3 / anti-CD28 antibody for a period of time selected from the group consisting of 9 days, 10 days, 11 days, 12 days, 13 days, and 14 days, thereby resulting in the expansion of peripheral blood lymphocytes (PBLs) from said PBMCs; b. recovering the PBLs from the culture in step a; and c. Transducing the PBLs with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single-chain variable fragment antibody fused to at least one endodomain of a T cell signaling molecule, or transducing the PBLs with an expression vector comprising a genetically engineered T cell receptor. A pharmaceutical composition obtained by a method comprising:

18. 18. The pharmaceutical composition of claim 17, wherein in step a, the anti-CD3 / anti-CD28 antibodies are bound to magnetic beads, and the ratio of beads to cells is 3:1 in the culture.

19. 18. The pharmaceutical composition of claim 17, 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 is replaced in the culture.

20. 20. The pharmaceutical composition of claim 19, wherein the first cell culture medium is exchanged with the second cell culture medium in culture.

21. 21. The pharmaceutical composition of claim 20, wherein the first cell culture medium is different from the second cell culture medium.

22. 19. The pharmaceutical composition of claim 18, wherein the magnetic beads in step a are DynaBeads®.

23. 18. The pharmaceutical composition of claim 17, wherein the patient has been pre-treated with ibrutinib and is refractory to treatment with ibrutinib.

24. 24. The pharmaceutical composition of claim 23, wherein the patient is pre-treated with at least three rounds of the ibrutinib regimen.

25. 24. The pharmaceutical composition of claim 23, wherein the patient is pretreated with ibrutinib for at least 3 months.

26. 18. The pharmaceutical composition of claim 17, wherein the patient has not received treatment with ibrutinib or another ITK inhibitor for at least one month prior to being pre-treated with ibrutinib or another ITK inhibitor.

27. The hematological malignancies include 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 (BCL), 18. The pharmaceutical composition of claim 17, wherein the tumor is selected from the group consisting of lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt's lymphoma, Waldenstrom's macroglobulinemia (WM), multiple myeloma, myelodysplastic syndrome, myelofibrosis, chronic myeloid leukemia, follicular central lymphoma, indolent NHL, human immunodeficiency virus (HIV)-associated B-cell lymphoma, and Epstein-Barr virus (EBV)-associated B-cell lymphoma.

28. 18. The pharmaceutical composition of claim 17, wherein the hematological malignancy is a liquid tumor.

29. 29. The pharmaceutical composition of claim 28, wherein the liquid tumor is chronic lymphocytic leukemia or small lymphocytic lymphoma.

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