Combination of CAR T cell therapy and immunomodulatory compounds for the treatment of lymphoma

Abstract

Methods, compositions, uses, and articles of manufacture are provided for adoptive cell therapy, e.g., immunotherapy, such as T cell therapy, and combination therapy involving the use of (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione or an enantiomer or a mixture of enantiomers thereof, or a pharma- ceutically acceptable salt, solvate, hydrate, cocrystal, clathrate, or polymorph, and related methods, compositions, uses, and articles of manufacture, for treating subjects suffering from diseases and conditions, such as certain B cell malignancies. The cells generally express a recombinant receptor, such as a chimeric antigen receptor (CAR). In some embodiments, the disease or condition is non-Hodgkin's lymphoma (NHL), e.g., relapsed or refractory NHL or a particular NHL subtype.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 167,599, filed March 29, 2021, and U.S. Provisional Patent Application No. 63 / 277,134, filed November 8, 2021, the entire contents of each of which are incorporated herein by reference for all purposes.

[0002] Incorporation by reference to sequence listing This application is being filed with an electronic Sequence Listing, which is provided as a file entitled 735042024540SeqList.txt, created on March 27, 2022, and which is 36 kilobytes in size. The information in the electronic Sequence Listing is incorporated by reference in its entirety.

[0003] Field The present disclosure relates, in some aspects, to methods, compositions, uses, and articles of manufacture for adoptive cell therapy, e.g., immunotherapy such as T cell therapy, and combination therapy involving the use of (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or an enantiomer or a mixture of enantiomers thereof, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph, and related methods, compositions, uses, and articles of manufacture, for treating subjects suffering from diseases and conditions, such as certain B-cell malignancies. The T cell therapy includes cells expressing a recombinant receptor, such as a chimeric antigen receptor (CAR). In some embodiments, the disease or condition is non-Hodgkin's lymphoma (NHL), e.g., relapsed or refractory NHL or a particular NHL subtype. [Background technology]

[0004] For example, various strategies are available for immunotherapy, such as administering engineered T cells for adoptive therapy. For example, a strategy for engineering T cells is available, such as expressing a genetically engineered antigen receptor, such as CAR, and administering a composition containing such cells to a subject. Improved strategies are needed to improve the efficacy of cells, for example, to improve the persistence, activity and / or proliferation of cells when administered to a subject. Methods, compositions, kits and systems that meet such needs are provided. Summary of the Invention

[0005] In some embodiments, provided herein are methods of treating a CD19-expressing cancer, the method comprising administering to a subject afflicted with a CD19-expressing cancer: (i) a T cell therapy comprising a dose of engineered cells, including T cells expressing a chimeric antigen receptor (CAR) against the cancer; and (ii) a combination therapy comprising a compound that is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof.

[0006] In some of any of the provided methods, the CD19-expressing cancer is lymphoma.

[0007] Also provided herein in some embodiments is a method of treating lymphoma, the method comprising administering to a subject afflicted with a cancer that is lymphoma (i) a T cell therapy comprising a dose of engineered cells, the T cell comprising a T cell expressing a chimeric antigen receptor (CAR) that binds to Cluster of Differentiation 19 (CD19); and (ii) a combination therapy comprising a compound that is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof.

[0008] In some of any of the provided methods, administration of the compound is initiated prior to administration of the T cell therapy.

[0009] Also provided herein in some embodiments is a method of treating a CD19-expressing cancer, comprising administering to a subject afflicted with a CD19-expressing cancer a T cell therapy comprising a dose of engineered cells, including T cells expressing a chimeric antigen receptor (CAR) that targets the cancer, wherein the T cell therapy is administered in combination therapy with a compound that is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein administration of the compound is initiated prior to administration of the T cell therapy.

[0010] In some of any of the provided methods, the CD19-expressing cancer is lymphoma.

[0011] Also provided herein in some embodiments is a method of treating lymphoma, comprising administering to a subject afflicted with a cancer that is lymphoma a T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds to Cluster of Differentiation 19 (CD19), wherein the T cell therapy is administered in combination therapy with a compound that is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein administration of the compound is initiated prior to administration of the T cell therapy.

[0012] In some of any of the provided methods, administration of the compound is initiated after administration of the T cell therapy.

[0013] Also provided herein in some embodiments is a method of treating a CD19-expressing cancer, comprising administering to a subject afflicted with a CD19-expressing cancer a compound that is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein the compound is administered in combination therapy with a T cell therapy comprising a dose of engineered cells, including T cells expressing a chimeric antigen receptor (CAR) that targets the cancer, and wherein administration of the compound is initiated after administration of the T cell therapy.

[0014] In some of any of the provided methods, the CD19-expressing cancer is lymphoma.

[0015] Also provided herein in some embodiments is a method of treating lymphoma, comprising administering to a subject afflicted with cancer that is lymphoma a compound that is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein the compound is administered in combination therapy with a T cell therapy that includes a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds to cluster of differentiation 19 (CD19), and wherein administration of the compound is initiated after administration of the T cell therapy.

[0016] In some of the provided methods, the T cell therapy is administered on day 1 of the combination therapy.

[0017] In some of any of the provided methods, administration of the compound begins between days 1 and 29, inclusive, of combination therapy.

[0018] In some of any of the provided methods, the compound is administered in multiple doses, each dose being from 0.1 mg or about 0.1 mg to 0.6 mg or about 0.6 mg, inclusive.

[0019] In some of any of the provided methods, the compound is administered as multiple intermittent doses administered no more than once a week.

[0020]

[0013] In some embodiments, a method of treating lymphoma comprises administering to a subject afflicted with lymphoma (i) a T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds to cluster of differentiation 19 (CD19), wherein the T cell therapy is administered on day 1 of the combination therapy; and (ii) a compound having the following structure: [ka] Also provided herein are methods comprising administering a combination therapy comprising a compound which is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione having the formula:

[0021] In some embodiments, a method of treating lymphoma comprises administering to a subject suffering from lymphoma a compound having the following structure: [ka] or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein the compound is administered in combination therapy with a T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds to Cluster of Differentiation 19 (CD19), wherein the T cell therapy is administered on day 1 of the combination therapy, and the compound is administered in an intermittent (i.e., not daily) dosing regimen.

[0022] In some of the provided methods, the compound is administered as multiple intermittent doses. In some of the provided methods, the compound is administered no more than once a week.

[0023] In some of any of the provided methods, each dose of the compound is 0.1 mg or about 0.1 mg to 0.6 mg or about 0.6 mg, inclusive.

[0024] In some of any of the provided methods, administration of the compound begins between days 1 and 29, inclusive, of combination therapy.

[0025]

[0013] In some embodiments, a method of treating lymphoma comprises administering to a subject afflicted with lymphoma (i) a T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds to cluster of differentiation 19 (CD19), wherein the T cell therapy is administered on day 1 of the combination therapy; and (ii) a compound having the following structure: [ka] or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein the compound is administered as multiple intermittent doses administered no more than once a week, each dose being 0.1 mg or about 0.1 mg to 0.6 mg or about 0.6 mg, inclusive, and wherein administration of the compound begins between day 1 and day 29, inclusive, of the combination therapy.

[0026] In some embodiments, a method of treating lymphoma comprises administering to a subject suffering from lymphoma a compound having the following structure: [ka] or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein the compound is administered in combination with a T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds to cluster of differentiation 19 (CD19), wherein the T cell therapy is administered on day 1 of the combination therapy; and the compound is administered as multiple intermittent doses administered no more than once a week, each dose being 0.1 mg at or about 0.1 mg to 0.6 mg at or about 0.6 mg, inclusive, and wherein administration of the compound begins between days 1 and 29, inclusive, of the combination therapy.

[0027] In some of the provided methods, administration of the compound begins between days 1 and 22, inclusive. In some of the provided methods, administration of the compound begins between days 1 and 15, inclusive. In some of the provided methods, administration of the compound begins between days 8 and 15, inclusive.

[0028] In some of the provided methods, administration of the compound begins on or about day 1. In some of the provided methods, administration of the compound begins on or about day 8. In some of the provided methods, administration of the compound begins on or about day 15.

[0029] In some of any of the provided methods, each dose of the multiple intermittent doses is the same.

[0030] In some of the provided methods, the compound is administered once weekly. In some of the provided methods, the compound is administered once every seven days (Q7D). In some of the provided methods, the compound is administered once every two weeks. In some of the provided methods, the compound is administered once every 14 days (Q14D).

[0031] In some of the provided methods, the compound is administered for at least 12 weeks after administration of the T cell therapy. In some of the provided methods, the compound is administered for up to 12 weeks after administration of the T cell therapy.

[0032] In some of the provided methods, the compound is administered on days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85. In some of the provided methods, the compound is administered on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85. In some of the provided methods, the compound is administered on days 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85. In some of the provided methods, the compound is administered on days 8, 22, 36, 50, 64, and 78.

[0033] In some of the provided methods, the dose of the compound is at or about 0.3 mg to 0.6 mg or about 0.6 mg, inclusive. In some of the provided methods, the dose of the compound is at or about 0.6 mg.

[0034] In some of the provided methods, the dose of the compound is 0.2 mg or about 0.2 mg to 0.4 mg or about 0.4 mg (inclusive). In some of the provided methods, the dose of the compound is 0.4 mg or about 0.4 mg. In some of the provided methods, the dose of the compound is less than 0.4 mg. In some of the provided methods, the dose of the compound is 0.3 mg or about 0.3 mg. In some of the provided methods, the dose of the compound is 0.2 mg or about 0.2 mg.

[0035] In some embodiments, a method of treating lymphoma comprises administering to a subject suffering from lymphoma a compound having the following structure: [ka] or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein the compound is administered in combination therapy with a T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds to cluster of differentiation 19 (CD19), wherein the T cell therapy is administered on day 1 of the combination therapy; and the compound is administered as multiple intermittent doses administered once every seven days (Q7D) on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85, wherein each dose is 0.3 mg.

[0036] In some embodiments, a method of treating lymphoma comprises administering to a subject suffering from lymphoma a compound having the following structure: [ka] or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein the compound is administered in combination therapy with a T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds to cluster of differentiation 19 (CD19), wherein the T cell therapy is administered on day 1 of the combination therapy; and the compound is administered as multiple intermittent doses administered once every seven days (Q7D) on days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85, wherein each dose is 0.3 mg.

[0037] In some embodiments, a method of treating lymphoma comprises administering to a subject suffering from lymphoma a compound having the following structure: [ka] or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein the compound is administered in combination therapy with a T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds to cluster of differentiation 19 (CD19), wherein the T cell therapy is administered on day 1 of the combination therapy; and the compound is administered as multiple intermittent doses administered once every seven days (Q7D) on days 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85, wherein each dose is 0.3 mg.

[0038] In some embodiments, a method of treating lymphoma comprises administering to a subject suffering from lymphoma a compound having the following structure: [ka] or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein the compound is administered in combination therapy with a T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds to cluster of differentiation 19 (CD19), wherein the T cell therapy is administered on day 1 of the combination therapy; and the compound is administered as multiple intermittent doses administered once every seven days (Q7D) on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85, wherein each dose is 0.4 mg.

[0039] In some embodiments, a method of treating lymphoma comprises administering to a subject suffering from lymphoma a compound having the following structure: [ka] or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein the compound is administered in combination therapy with a T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds to cluster of differentiation 19 (CD19), wherein the T cell therapy is administered on day 1 of the combination therapy; and the compound is administered as multiple intermittent doses administered once every 14 days (Q14D) on days 8, 22, 36, 50, 64, and 78, wherein each dose is 0.3 mg.

[0040] In some embodiments, a method of treating lymphoma comprises administering to a subject suffering from lymphoma a compound having the following structure: [ka] or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein the compound is administered in combination therapy with a T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds to cluster of differentiation 19 (CD19), wherein the T cell therapy is administered on day 1 of the combination therapy; and the compound is administered as multiple intermittent doses administered once every seven days (Q7D) on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85, wherein each dose is 0.2 mg.

[0041] In some embodiments, a method of treating lymphoma comprises administering to a subject suffering from lymphoma a compound having the following structure: [ka] or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein the compound is administered in combination therapy with a T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds to cluster of differentiation 19 (CD19), wherein the T cell therapy is administered on day 1 of the combination therapy; and the compound is administered as multiple intermittent doses administered once every seven days (Q7D) on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85, wherein each dose is 0.6 mg.

[0042] In some of the provided methods, the compound is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt thereof.

[0043] In some of the provided methods, the compound is or comprises a pharmaceutically acceptable salt of (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione. In some of the provided methods, the compound is or comprises a hydrate of (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione. In some of the provided methods, the compound is or comprises a solvate of (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione. In some of the provided methods, the compound is or comprises (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione.

[0044] In some of the provided methods, at the start of administration of the compound, the subject does not exhibit severe toxicity after administration of the T cell therapy. In some of the provided methods, the severe toxicity is severe cytokine release syndrome (CRS), optionally Grade 3 or higher CRS, prolonged Grade 3 or higher CRS, Grade 4 CRS, or Grade 5 CRS; and / or the severe toxicity is severe neurotoxicity, optionally Grade 3 or higher neurotoxicity, prolonged Grade 3 or higher neurotoxicity, Grade 4 neurotoxicity, or Grade 5 neurotoxicity. In some embodiments, the severe toxicity is severe CRS. In some embodiments, the severe toxicity is severe neurotoxicity.

[0045] In some of the provided methods, if the subject exhibits toxicity, optionally hematological toxicity, after administration of the compound, administration of the compound is withheld and / or the dose of the compound is modified, optionally reduced. In some of the provided methods, if the subject exhibits toxicity after administration of the compound, administration of the compound is withheld. In some of the provided methods, if the subject exhibits toxicity after administration of the compound, the dose of the compound is reduced. In some of the provided methods, the toxicity is hematological toxicity. In some of the provided methods, the toxicity is severe thrombocytopenia, optionally Grade 4 thrombocytopenia or prolonged Grade 4 thrombocytopenia. In some of the provided methods, the toxicity is severe neutropenia, optionally Grade 4 neutropenia, prolonged Grade 4 neutropenia, or febrile neutropenia, optionally Grade 3 or higher febrile neutropenia or prolonged Grade 3 or higher febrile neutropenia.

[0046] In some of any of the provided methods, administration of the compound is resumed after the subject no longer exhibits toxicity.

[0047] In some of any of the provided methods, the lymphoma expresses CD19.

[0048] In some of the provided methods, the lymphoma is a B-cell malignancy. In some of the provided methods, the lymphoma is a relapsed / refractory lymphoma. In some of the provided methods, the lymphoma is an aggressive lymphoma. In some of the provided methods, the lymphoma is a non-Hodgkin's lymphoma (NHL), and optionally, the NHL includes aggressive NHL; diffuse large B-cell lymphoma (DLBCL); DLBCL-NOS, optionally transformed low-grade; EBV-positive DLBCL-NOS; T-cell / histiocytocyte-rich large B-cell lymphoma; primary mediastinal large B-cell lymphoma (PMBCL); follicular lymphoma (FL), optionally follicular lymphoma grade 3B (FL3B); and / or high-grade B-cell lymphoma with MYC and BCL2 and / or BCL6 rearrangements with DLBCL histology (double / triple hit).

[0049] In some of the provided methods, the CD19 is human CD19.

[0050] In some of the provided methods, the chimeric antigen receptor (CAR) comprises an extracellular antigen recognition domain that specifically binds to CD19 and an intracellular signaling domain comprising an ITAM. In some of the provided methods, the intracellular signaling domain comprises the signaling domain of the CD3-zeta (CD3ζ) chain, optionally human CD3-zeta chain. In some of the provided methods, the intracellular signaling domain comprises the signaling domain of the human CD3-zeta (CD3ζ) chain. In some of the provided methods, the chimeric antigen receptor (CAR) further comprises a costimulatory signaling region. In some of the provided methods, the costimulatory signaling region comprises the signaling domain of CD28 or 4-1BB, optionally human CD28 or human 4-1BB. In some of the provided methods, the costimulatory signaling region comprises the signaling domain of human CD28. In some of the provided methods, the costimulatory signaling region comprises the signaling domain of human 4-1BB.

[0051] In some of the provided methods, the CAR comprises an scFv specific for CD19; a transmembrane domain; optionally a cytoplasmic signaling domain derived from a costimulatory molecule that is or comprises 4-1BB, optionally human 4-1BB; optionally a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule that is or comprises a CD3 zeta signaling domain, optionally a human CD3 zeta signaling domain; and optionally the CAR further comprises a spacer between the transmembrane domain and the scFv. In some of the provided methods, the CAR comprises an scFv specific for CD19; a transmembrane domain; a cytoplasmic signaling domain derived from human 4-1BB; a cytoplasmic signaling domain that is or comprises a human CD3 zeta signaling domain; and a spacer between the transmembrane domain and the scFv.

[0052] In some of the provided methods, the CAR comprises, in order, an scFv specific for CD19; a transmembrane domain; a cytoplasmic signaling domain derived from a costimulatory molecule that is or comprises a 4-1BB signaling domain, optionally a human 4-1BB signaling domain; and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule that is a CD3 zeta signaling domain, optionally a human CD3 zeta signaling domain. In some of the provided methods, the CAR comprises, in order, an scFv specific for CD19; a transmembrane domain; a cytoplasmic signaling domain that is or comprises a human 4-1BB signaling domain; and a cytoplasmic signaling domain that is a human CD3 zeta signaling domain.

[0053] In some of the provided methods, the CAR comprises, in order, an scFv specific for CD19; a spacer; a transmembrane domain; a cytoplasmic signaling domain derived from a costimulatory molecule that is optionally a 4-1BB signaling domain; and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule that is or includes a CD3 zeta signaling domain. In some of the provided methods, the CAR comprises, in order, an scFv specific for CD19; a spacer; a transmembrane domain; a cytoplasmic signaling domain that is a 4-1BB signaling domain; and a cytoplasmic signaling domain that is or includes a CD3 zeta signaling domain.

[0054] In some of the provided methods, the spacer comprises or consists of all or part of an immunoglobulin hinge or a modified version thereof, or is a polypeptide spacer comprising about 15 amino acids or less. In some of the provided methods, the spacer comprises or consists of all or part of an immunoglobulin hinge, optionally an IgG4 hinge or a modified version thereof, and / or comprises about 15 amino acids or less. In some embodiments, the spacer comprises or consists of all or part of an immunoglobulin hinge, such as IgG4, or a modified version thereof. In some of the provided methods, the spacer comprises or consists of all or part of an IgG4 hinge or a modified version thereof. In some of the provided methods, the spacer comprises about 15 amino acids or less. In some of the provided methods, the spacer is 12 amino acids or about 12 amino acids in length. In some of the provided methods, the spacer is 12 amino acids or about 12 amino acids in length. In some embodiments, the spacer has or consists of the sequence of SEQ ID NO: 1; a sequence encoded by SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34; or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the spacer has or consists of the sequence of SEQ ID NO: 1. In some embodiments, the spacer has a sequence encoded by SEQ ID NO: 2.

[0055] In some of the provided methods, the cytoplasmic signaling domain derived from the costimulatory molecule comprises the sequence set forth in SEQ ID NO: 12, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some of the provided methods, the cytoplasmic signaling domain derived from the primary signaling ITAM-containing molecule comprises the sequence of any of SEQ ID NOs: 13-15, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0056] In some of the provided methods, the scFv comprises the CDRL1 sequence of RASQDISKYLN (SEQ ID NO:35), the CDRL2 sequence of SRLHSGV (SEQ ID NO:36), and / or the CDRL3 sequence of GNTLPYTFG (SEQ ID NO:37); and / or the CDRH1 sequence of DYGVS (SEQ ID NO:38), the CDRH2 sequence of VIWGSETTYYNSALKS (SEQ ID NO:39), and / or the CDRH3 sequence of YAMDYWG (SEQ ID NO:40). In some of the provided methods, the scFv comprises the CDRL1 sequence of RASQDISKYLN (SEQ ID NO:35), the CDRL2 sequence of SRLHSGV (SEQ ID NO:36), and the CDRH3 sequence of GNTLPYTFG (SEQ ID NO:37); and / or the CDRH1 sequence of DYGVS (SEQ ID NO:38), the CDRH2 sequence of VIWGSETTYYNSALKS (SEQ ID NO:39), and the CDRH3 sequence of YAMDYWG (SEQ ID NO:40). In some of the provided methods, the scFv comprises the CDRL1 sequence of RASQDISKYLN (SEQ ID NO:35), the CDRL2 sequence of SRLHSGV (SEQ ID NO:36), and the CDRL3 sequence of GNTLPYTFG (SEQ ID NO:37); and the CDRH1 sequence of DYGVS (SEQ ID NO:38), the CDRH2 sequence of VIWGSETTYYNSALKS (SEQ ID NO:39), and the CDRH3 sequence of YAMDYWG (SEQ ID NO:40). In some of the provided methods, the scFv comprises a variable light chain region comprising the CDRL1 sequence of FMC63, the CDRL2 sequence of FMC63, and the CDRL3 sequence of FMC63, and a variable heavy chain region comprising the CDRH1 sequence of FMC63, the CDRH2 sequence of FMC63, and the CDRH3 sequence of FMC63. In some of the provided methods, the scFv comprises the variable heavy chain region of FMC63 and the variable light chain region of FMC63. In some embodiments, the scFv comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 41, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 42. In some of any of the provided methods, the scFv comprises the CDRL1 sequence of FMC63, the CDRL2 sequence of FMC63, the CDRL3 sequence of FMC63, the CDRH1 sequence of FMC63, the CDRH2 sequence of FMC63, and the CDRH3 sequence of FMC63.In some of the provided methods, the scFv comprises a variable heavy chain region of FMC63 and a variable light chain region of FMC63. In some of the provided methods, the scFv comprises a VH comprising SEQ ID NO: 41 and a VL comprising the amino acid sequence set forth as SEQ ID NO: 42. In some of the provided methods, the scFv has the sequence of amino acids set forth as SEQ ID NO: 43.

[0057] In some of the provided methods, the dose of engineered cells is 1 x 10 5 or approximately 1 x 10 5 pieces~5×10 8 pieces or approximately 5 x 10 8 Total CAR-expressing T cells, 1 x 10 6 or approximately 1 x 10 6 pieces~2.5×10 8 or approximately 2.5 x 10 8 Total CAR-expressing T cells, 5 x 10 6 pieces or approximately 5 x 10 6 pieces~1×10 8 or approximately 1 x 10 8 Total CAR-expressing T cells, 1 x 10 7 Or about 1 x 10 7 ~2.5×10 8 or approximately 2.5 x 10 8 total CAR-expressing T cells or 5 x 10 7 pieces or approximately 5 x 10 7 pieces~1×10 8 or approximately 1 x 10 8 In some of the provided methods, the dose of engineered cells is 1 x 10 total CAR-expressing T cells (each including the upper and lower limits). 5 ~5×10 8 In some of the provided methods, the dose of engineered cells comprises 1 x 10 total CAR-expressing T cells or approximately 1 x 10 total CAR-expressing T cells, inclusive. 6 or approximately 1 x 10 6 pieces~2.5×10 8 or approximately 2.5 x 10 8In some of the provided methods, the dose of engineered cells is 5 x 10 total CAR-expressing T cells (inclusive). 6 pieces or approximately 5 x 10 6 pieces~1×10 8 or approximately 1 x 10 8 In some of the provided methods, the dose of engineered cells is 1 x 10 total CAR-expressing T cells (inclusive). 7 or approximately 1 x 10 7 pieces~2.5×10 8 or approximately 2.5 x 10 8 In some of the provided methods, the dose of engineered cells is 5 x 10 total CAR-expressing T cells (inclusive). 7 pieces or approximately 5 x 10 7 pieces~1×10 8 or approximately 1 x 10 8 total CAR-expressing T cells (upper and lower limits included).

[0058] In some of the provided methods, the dose of engineered cells is at least 1 x 10 5 or at least about 1×10 5 CAR-expressing cells, at least 2.5 x 10 5 or at least about 2.5 × 10 5 CAR-expressing cells, at least 5 x 10 5 or at least about 5×10 5 CAR-expressing cells, at least 1 x 10 6 or at least about 1×10 6 CAR-expressing cells, at least 2.5 x 10 6 or at least about 2.5 × 10 6 CAR-expressing cells, at least 5 x 10 6 or at least about 5×10 6 CAR-expressing cells, at least 1 x 10 7 or at least about 1×10 7 CAR-expressing cells, at least 2.5 x 10 7 or at least about 2.5 × 10 7CAR-expressing cells, at least 5 x 10 7 or at least about 5×10 7 CAR-expressing cells, at least 1 x 10 8 or at least about 1×10 8 CAR-expressing cells, at least 2.5 x 10 8 or at least about 2.5 × 10 8 CAR-expressing cells, or at least 5 x 10 8 or at least about 5×10 8 Contains CAR-expressing cells.

[0059] In some of the provided methods, the dose of engineered cells is at least 1 x 10 5 or at least about 1 x 10 5 In some of the provided methods, the dose of engineered cells comprises at least 2.5 x 10 CAR-expressing cells. 5 or at least about 2.5 x 10 5 In some of the provided methods, the dose of engineered cells comprises at least 5 x 10 CAR-expressing cells. 5 or at least about 5 x 10 5 In some of the provided methods, the dose of engineered cells comprises at least 1 x 10 CAR-expressing cells. 6 or at least about 1 x 10 6 In some of the provided methods, the dose of engineered cells comprises at least 2.5 x 10 CAR-expressing cells. 6 or at least about 2.5 x 10 6 In some of the provided methods, the dose of engineered cells comprises at least 5 x 10 CAR-expressing cells. 6 or at least about 5 x 10 6 In some of the provided methods, the dose of engineered cells comprises at least 1 x 10 CAR-expressing cells. 7 or at least about 1 x 10 7 In some of the provided methods, the dose of engineered cells comprises at least 2.5 x 10 CAR-expressing cells.7 or at least about 2.5 x 10 7 In some of the provided methods, the dose of engineered cells comprises at least 5 x 10 CAR-expressing cells. 7 or at least about 5 x 10 7 In some of the provided methods, the dose of engineered cells comprises at least 1 x 10 CAR-expressing cells. 8 or at least about 1 x 10 8 In some of the provided methods, the dose of engineered cells comprises at least 2.5 x 10 CAR-expressing cells. 8 or at least about 2.5 x 10 8 In some of the provided methods, the dose of engineered cells comprises at least 5 x 10 CAR-expressing cells. 8 or at least about 5 x 10 8 Contains CAR-expressing cells.

[0060] In some of the provided methods, the dose of engineered cells is 1 x 10 8 pieces or approximately 1 x 10 8 Contains CAR-expressing T cells.

[0061] In some of the provided methods, the dose of engineered cells is administered parenterally, optionally intravenously. In some of the provided methods, the dose of engineered cells is administered intravenously.

[0062] In some of the provided methods, the T cells are primary T cells obtained from the subject. In some of the provided methods, the T cells are autologous to the subject. In some of the provided methods, the T cells are allogeneic to the subject.

[0063] In some of any of the provided methods, the dose of engineered cells comprises CD4+ T cells expressing a CAR and CD8+ T cells expressing a CAR, and administering the dose comprises administering a plurality of separate compositions, said plurality of separate compositions comprising a first composition comprising one of the CD4+ T cells and CD8+ T cells, and a second composition comprising the other of the CD4+ T cells and CD8+ T cells.

[0064] In some of the provided methods, the first composition and the second composition are administered 0 to 12 hours, 0 to 6 hours, or 0 to 2 hours apart, or the administration of the first composition and the administration of the second composition are administered on the same day and are administered about 0 to 12 hours, 0 to 6 hours, or 0 to 2 hours apart; and / or the start of administration of the first composition and the start of administration of the second composition are administered about 1 minute to 1 hour, or about 5 minutes to 30 minutes apart. In some of the provided methods, the first composition and the second composition are administered 2 hours or less, 1 hour or less, 30 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less apart.

[0065] In some of the provided methods, the first composition comprises CD4+ T cells. In some of the provided methods, the first composition comprises CD8+ T cells.

[0066] In some of any of the provided methods, the first composition is administered before the second composition.

[0067] In some of the provided methods, prior to administration of the T cell therapy, the subject is preconditioned with lymphodepleting therapy comprising administration of fludarabine and / or cyclophosphamide. In some of the provided methods, the method further comprises administering to the subject lymphodepleting therapy comprising administration of fludarabine and / or cyclophosphamide immediately prior to administration of the T cell therapy.

[0068] In some of the provided methods, the lymphodepletion therapy is about 200-400 mg / m daily for 2-4 days, optionally 3 days. 2 (including upper and lower limits), 300 mg / m 2 or approximately 300 mg / m 2 of cyclophosphamide and / or approximately 20-40 mg / m 2 , as appropriate, 30mg / m 2 or lymphodepleting therapy includes administration of fludarabine at a dose of approximately 500 mg / m 2 In some of the provided methods, the lymphodepletion therapy includes administration of cyclophosphamide at a dose of 300 mg / m daily for 3 days. 2 or approximately 300 mg / m 2 of cyclophosphamide and approximately 30 mg / m 2 and / or lymphodepletion therapy includes administration of fludarabine at 500 mg / m daily for 3 days. 2 or approximately 500 mg / m 2 of cyclophosphamide and approximately 30 mg / m 2 This includes administration of fludarabine.

[0069] In some of the methods provided, the subject is a human.

[0070] In some of the provided methods, at least 35%, at least 40%, or at least 50% of subjects treated according to the method achieve a complete response (CR) that is sustained for 6 months or more or for 9 months or more, or a CR that is sustained in at least 60, 70, 80, 90, or 95% of subjects who achieve a CR; and / or at least 60, 70, 80, 90, or 95% of subjects who achieve a CR by 6 months remain in response, remain in CR for 3 months or more and / or for 6 months or more and / or for 9 months or more. and / or survive or survive without progression; and / or at least 50%, at least 60%, or at least 70% of subjects treated according to the method achieve an objective response (OR) for 6 months or more or for 9 months or more, where appropriate, the OR is sustained or is sustained in at least 60, 70, 80, 90, or 95% of subjects who achieve an OR; and / or at least 60, 70, 80, 90, or 95% of subjects who achieve an OR by 6 months remain in response or survive for 3 months or more and / or for 6 months or more.

[0071] In some of the provided methods, at or immediately prior to administration of the dose of engineered cells, the subject has relapsed or become refractory following remission after treatment with one or more previous therapies for NHL, optionally one, two, or three previous therapies other than another dose of engineered cells expressing a CAR. In some of the provided methods, at or immediately prior to administration of the dose of engineered cells, the subject has relapsed or become refractory following remission after treatment with one previous therapy for NHL. In some of the provided methods, at or immediately prior to administration of the dose of engineered cells, the subject has relapsed or become refractory following remission after treatment with two previous therapies for NHL. In some of the provided methods, at or immediately prior to administration of the dose of engineered cells, the subject has relapsed or become refractory following remission after treatment with three previous therapies for NHL. In some of the provided methods, the one or more previous therapies do not include another dose of engineered cells expressing a CAR.

[0072] In some of the provided methods, at or before the administration of the dose of engineered cells, the subject has or has been identified as having double / triple hit lymphoma; the subject has or has been identified as having chemotherapy-resistant lymphoma, optionally chemotherapy-resistant DLBCL; and / or the subject has not achieved a complete remission (CR) in response to a previous therapy. In some of the provided embodiments, the subject has not achieved a complete remission (CR) in response to a previous therapy.

[0073] In some of any of the provided methods, administration of the compound reverses an exhaustion phenotype in CAR-expressing T cells in the subject; prevents, inhibits, or delays the development of an exhaustion phenotype in CAR-expressing T cells in the subject; reduces the level or degree of an exhaustion phenotype in CAR-expressing T cells in the subject; or reduces the percentage or total number of CAR-expressing T cells in the subject that have an exhaustion phenotype.

[0074] In some of any of the provided methods, initiation of administration of the compound occurs after administration of T cell therapy, and after administration or initiation of the compound, the subject exhibits restoration or rescue of antigen- or tumor-specific activity or function of CAR-expressing T cells in said subject, and optionally, said restoration, rescue, and / or initiation of administration of the compound occurs at a time after the subject or CAR-expressing T cells in the subject's blood exhibit an exhausted phenotype.

[0075] In some of any of the provided methods, administering a compound comprises administering in an amount, frequency, and / or duration effective to (a) result in an increase in antigen-specific or antigen receptor-driven activity of naive or non-exhausted T cells in the subject, which may comprise T cells expressing the CAR, following exposure of the T cells to a CD19 antigen or antigen receptor-specific agent, compared to the absence of said administering of the compound; or (b) prevent, inhibit, or delay the development of an exhausted phenotype in naive or non-exhausted T cells in the subject, which may comprise T cells expressing the CAR, following exposure of the T cells to a CD19 antigen or antigen receptor-specific agent, compared to the absence of said administering of the compound; or (c) reverse an exhausted phenotype in exhausted T cells, which may comprise T cells expressing the CAR, in the subject, compared to the absence of said administering of the compound.

[0076] In some of any of the provided methods, administering the compound includes administration in an amount, frequency and / or duration effective to (i) result in said increase in activity, and (ii) prevent, inhibit or delay said onset of said exhaustion phenotype and / or reverse said exhaustion phenotype.

[0077] In some of any of the provided methods, the T cells in the subject comprise T cells that express the CAR, and / or the antigen is CD19, e.g., the exposure is exposure to a CD19 antigen.

[0078] In some of any of the provided methods, the exhaustion phenotype comprises, in relation to a T cell or population of T cells, an increase in the level or degree of surface expression on T cells, or the percentage of said population of T cells exhibiting surface expression, of one or more exhaustion markers, optionally 2, 3, 4, 5 or 6 exhaustion markers, compared to a reference T cell population under the same conditions; or a decrease in the level or degree of activity exhibited by said T cell or population of T cells following exposure to a CD19 antigen or antigen receptor-specific agent, compared to a reference T cell population under the same conditions.

[0079] In some of the provided methods, the level, magnitude, or percentage increase is greater than or equal to 1.2-fold, 1.5-fold, or about 1.5-fold, 2.0-fold, or about 2.0-fold, 3-fold, or about 3-fold, 4-fold, or about 4-fold, 5-fold, or about 5-fold, 6-fold, or about 6-fold, 7-fold, or about 7-fold, 8-fold, or about 8-fold, 9-fold, or about 9-fold, 10-fold, or about 10-fold or more. In some of the provided methods, the level, magnitude, or percentage decrease is less than or equal to 1.2-fold, 1.5-fold, or about 1.5-fold, 2.0-fold, or about 2.0-fold, 1 / 3, 1 / 4, or about 1 / 4, 1 / 5, or about 1 / 5, 1 / 6, or about 1 / 6, 1 / 7, or about 1 / 7, 1 / 8, or about 1 / 8, 1 / 9, or about 1 / 9, 1 / 10, or about 1 / 10 or less.

[0080] In some of the provided methods, the reference T cell population is optionally a population of T cells known to have a non-exhausted phenotype from the same subject or species as the subject from which the T cells with an exhausted phenotype are derived, a population of naive T cells, a population of central memory T cells, or a population of stem central memory T cells. In some of the provided methods, the reference T cell population is a subject-matched population comprising (a) bulk T cells isolated from the blood of the subject from which the T cells with an exhausted phenotype are derived, optionally the bulk T cells do not express a CAR and / or (b) obtained from the subject from which the T cells with an exhausted phenotype are derived prior to receiving a dose of T cells that express a CAR. In some of the provided methods, the reference T cell population is a T cell therapy sample, e.g., a composition comprising a sample of CAR-expressing T cells or a pharmaceutical composition comprising CAR-expressing T cells, prior to its administration to the subject, optionally the composition is a cryopreserved sample.

[0081] In some of the provided methods, the one or more exhaustion markers are inhibitory receptors. In some of the provided methods, the one or more exhaustion markers are selected from among PD-1, CTLA-4, TIM-3, LAG-3, BTLA, 2B4, CD160, CD39, VISTA, and TIGIT.

[0082] In some of any of the provided methods, the activity is one or more of proliferative, cytotoxic, or production of one or a combination of inflammatory cytokines, optionally wherein the one or combination of cytokines is selected from the group consisting of IL-2, IFN-gamma, and TNF-alpha.

[0083] In some of the provided methods, the exposure to the CD19 antigen or antigen receptor-specific agent comprises incubation with a CD19 antigen or antigen receptor-specific agent, optionally an agent that binds to the antigen-binding domain of the CAR. In some of the provided methods, the exposure to the CD19 antigen or antigen receptor-specific agent comprises exposing the T cells to CD19 antigen-expressing target cells, optionally cells of a B-cell malignancy.

[0084] Also provided is a combination therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that targets a CD19-expressing cancer, and a compound that is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, for use in a method of treating cancer according to any of the provided methods.

[0085] Also provided is a compound which is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, for use in a method of treating a CD19-expressing cancer according to any of the provided methods.

[0086] Also provided is a T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that targets a CD19-expressing cancer, for use in a method of treating cancer according to any of the provided methods.

[0087] Also provided is the use of a T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that targets a CD19-expressing cancer, and a combination therapy comprising a compound that is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, to treat cancer according to any of the provided methods.

[0088] Also provided is the use of a compound that is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, to treat a CD19-expressing cancer according to any of the provided methods.

[0089] Also provided is the use of T cell therapy comprising a dose of engineered cells, including T cells expressing a chimeric antigen receptor (CAR) that targets a CD19-expressing cancer, to treat cancer according to any of the provided methods.

[0090] Also provided is the use of a combination therapy comprising a dose of engineered cells, including T cells expressing a chimeric antigen receptor (CAR) that targets a CD19-expressing cancer, and a compound that is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, for the manufacture of a medicament for treating cancer according to any of the provided methods.

[0091] Also provided is the use of a compound that is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, for the manufacture of a medicament for treating a CD19-expressing cancer, according to any of the provided methods.

[0092] Also provided is the use of a T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that targets a CD19-expressing cancer for the manufacture of a medicament for treating cancer according to any of the provided methods.

[0093] In some of any of the provided embodiments, the CAR binds to Cluster of Differentiation 19 (CD19). In some embodiments, the CD19-expressing cancer is a lymphoma.

[0094] In some of any of the provided embodiments, the compound is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt thereof. [Brief explanation of the drawings]

[0095] [Figure 1] Figure 1 shows the expression levels of Aiolos and Ikaros transcription factors in anti-CD19 CAR T cells after stimulation with a CAR-specific anti-idiotypic antibody in the presence of various immunomodulatory compounds. [Figure 2A] Figure 2A shows the percentage of viable anti-CD19 CAR T cells after stimulation with a CAR-specific anti-idiotypic antibody in the presence of various immunomodulatory compounds. [Figure 2B]Figure 2B shows the number of cell doublings of anti-CD19 CAR T cells after stimulation with a CAR-specific anti-idiotypic antibody in the presence of various immunomodulatory compounds. [Figure 2C] Figure 2C shows the number of cell doublings of anti-CD19 CAR T cells after stimulation with a CAR-specific anti-idiotypic antibody in the presence of various immunomodulatory compounds. [Figure 2D] Figure 2D shows the percentage of anti-CD19 CAR T cells in the G1 phase of the cell cycle after stimulation with a CAR-specific anti-idiotypic antibody in the presence of various immunomodulatory compounds. [Figure 2E] Figure 2E shows the percentage of anti-CD19 CAR T cells in the G1 phase of the cell cycle after stimulation with a CAR-specific anti-idiotypic antibody in the presence of various immunomodulatory compounds. [Figure 2F] Figure 2F shows cytokine levels of anti-CD19 CAR T cells after stimulation with CAR-specific anti-idiotypic antibodies in the presence of various immunomodulatory compounds. Log2 fold change values ​​above zero are indicated with a "+". [Figure 2G] Figure 2G shows cell counts of RL tumor cells treated with anti-CD19 CAR T cells and compound C. [Figure 2H] Figure 2H shows cell counts of RL tumor cells treated with anti-CD19 CAR T cells and compound 2. [Figure 3A] Figure 3A shows cytokine production of anti-CD19 CAR T cells after chronic stimulation. [Figure 3B] Figure 3B shows the cytolytic function of anti-CD19 CAR T cells after chronic stimulation. [Figure 3C] Figure 3C shows a representative image of CD19-expressing tumor spheroids on day 9 of co-culture with anti-CD19 CAR T cells stimulated in the presence of compound C (concomitant treatment). [Figure 3D] Figure 3D shows the volume of CD19-expressing tumor spheroids co-cultured with anti-CD19 CAR T cells stimulated in the presence of compound C after chronic stimulation (concomitant treatment). [Figure 3E] Figure 3E shows the volume of CD19-expressing tumor spheroids co-cultured with anti-CD19 CAR T cells stimulated in the presence of compound C after chronic stimulation (concomitant treatment). [Figure 3F] FIG. 3F shows the IFNγ concentration in the supernatants of co-cultured cells. [Figure 3G] Figure 3G shows Ikaros expression in anti-CD19 CAR T cells after 6 days of chronic stimulation in the presence of Compound C or Compound 2. [Figure 3H] Figure 3H depicts a volcano plot showing differentially expressed genes in chronically stimulated anti-CD19 CAR T cells concomitantly treated with compound C. [Figure 3I] Figure 3I shows a comparison of the effects on gene expression profiles (log2 fold change) induced by Compound C concomitant treatment in chronically stimulated anti-CD19 CAR T cells. [Figure 3J] Figure 3J shows KEGG pathway enrichment analysis of differentially expressed genes in chronically stimulated anti-CD19 CAR T cells after concomitant treatment with compound C. [Figure 4A] Figure 4A shows a representative image of CD19-expressing tumor spheroids at day 9 of co-culture with anti-CD19 CAR T cells in the presence of compound C (rescue treatment). [Figure 4B] Figure 4B shows the volume of CD19-expressing tumor spheroids co-cultured with anti-CD19 CAR T cells in the presence of compound C (rescue treatment). [Figure 4C] Figure 4C shows the volume of CD19-expressing tumor spheroids co-cultured with anti-CD19 CAR T cells in the presence of compound C (rescue treatment). [Figure 4D] FIG. 4D shows the IFNγ concentration in the supernatants of the co-cultured cells. [Figure 4E] Figure 4E shows a volcano plot showing differentially expressed genes in chronically stimulated anti-CD19 CAR T cells after rescue treatment with compound C. [Figure 4F] Figure 4F shows a comparison of the effects on gene expression profiles (log2 fold change) induced by Compound C rescue treatment in chronically stimulated anti-CD19 CAR T cells. [Figure 4G] Figure 4G shows KEGG pathway enrichment analysis of differentially expressed genes in chronically stimulated anti-CD19 CAR T cells after rescue treatment with compound C. [Figure 5A] Figure 5A shows tumor cell counts upon reloading of chronically stimulated CAR T cells in the presence of Compound C (concurrent treatment). [Figure 5B] Figure 5B shows tumor cell counts upon CAR T cell reloading in the presence of Compound C (rescue therapy). [Figure 6A] FIG. 6A shows the expected plasma concentration levels of Compound C based on a once every seven days (Q7D) dosing regimen. [Figure 6B] Figures 6B and 6C show Ikaros levels in anti-CD19 CAR T cells after 24 hours (Figure 6B) or 6 days (Figure 6C) exposure to Compound C. [Figure 6C] Figures 6B and 6C show Ikaros levels in anti-CD19 CAR T cells after 24 hours (Figure 6B) or 6 days (Figure 6C) exposure to Compound C. [Figure 6D] FIG. 6D shows predicted absolute neutrophil count (ANC) profiles based on a Q7D dosing regimen of Compound C generated using four different pharmacodynamic models. [Figure 7A] Figure 7A shows tumor cell counts upon re-challenge of CAR T cells chronically stimulated in the presence of Compound C, including for CAR T cells chronically stimulated in the presence of Compound C for 1 day with a high concentration of Compound C, followed by 5 days with a low concentration of Compound C. [Figure 7B]Figure 7B shows the cytolytic activity and CD27 / CCR7 expression, respectively, of CAR T cells chronically stimulated in the presence of Compound C, including for CAR T cells chronically stimulated in the presence of Compound C for 1 day at a high concentration of Compound C, followed by 6 days at a low concentration of Compound C. [Figure 7C] Figure 7C shows the cytolytic activity and CD27 / CCR7 expression, respectively, of CAR T cells chronically stimulated in the presence of Compound C, including for CAR T cells chronically stimulated in the presence of Compound C for 1 day at a high concentration of Compound C, followed by 6 days at a low concentration of Compound C. DETAILED DESCRIPTION OF THE INVENTION

[0096] 1. A cell therapy, such as a cell therapy comprising engineered T cells (e.g., CAR-T cells), for the treatment of a subject suffering from cancer or a proliferative disease, and a compound having the structure: [ka] (Formula I) or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, or racemic mixture thereof (Compound C), and compositions thereof. In some embodiments, the cell therapy is T cell therapy. In some embodiments, the cell therapy is adoptive T cell therapy, which comprises T cells that specifically recognize and / or target an antigen associated with a cancer or proliferative disease, such as an antigen associated with a B cell malignancy, e.g., a lymphoma, e.g., non-Hodgkin's lymphoma (NHL) or a subtype thereof. In some embodiments, the cell therapy comprises T cells engineered with a chimeric antigen receptor (CAR) that comprises an antigen-binding domain that binds, e.g., specifically binds, the antigen. In some cases, the antigen targeted by the cell therapy is CD19. In some embodiments, the cancer, e.g., lymphoma, expresses CD19. Also provided herein are compositions comprising the cell therapy and / or compositions comprising Compound C, and combinations and articles of manufacture, e.g., kits, containing such compositions and combinations for treating or preventing diseases, conditions, and disorders, including cancers, such as lymphoma.

[0097] (S)-2-(2,6-Dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound C) is an oral cereblon-modulating agent (CELMoD). Cereblon functions as a substrate receptor for the CRL4 ubiquitin E3 ligase, and binding of cereblon-modulating compounds induces the recruitment, ubiquitination, and destruction of key target substrates, such as Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3, also known as Ikaros and Aiolos, respectively), to mediate cellular effects. As demonstrated herein, administration of Compound C results in significant degradation of Ikaros and Aiolos, even more so than with other CELMoDs in its class (e.g., lenalidomide, avadomide, and iberdomide). However, a potential side effect of Ikaros and Aiolos degradation is the development of neutropenia secondary to neutrophil maturation arrest. Neutropenia can develop as a result of depletion of Ikaros, a critical transcription factor regulating hematopoietic development, resulting in late maturation arrest of neutrophil precursors.

[0098] Cellular therapies, e.g., T cell-based therapies, e.g., adoptive T cell therapies (including therapies involving the administration of cells expressing chimeric receptors specific for a disease or disorder of interest, such as chimeric antigen receptors (CARs) and / or other recombinant antigen receptors, as well as other adoptive immune cell and adoptive T cell therapies), can be effective in treating diseases and disorders, such as B cell malignancies. The engineered expression of recombinant receptors, such as chimeric antigen receptors (CARs), on the surface of T cells allows for the redirection of T cell specificity. In clinical studies, CAR-T cells, such as anti-CD19 CAR-T cells, have produced durable complete responses in both leukemia and lymphoma patients (Porter et al. (2015) Sci Transl Med., 7:303ra139; Kochenderfer (2015) J. Clin. Oncol., 33: 540-9; Lee et al. (2015) Lancet, 385:517-28; Maude et al. (2014) N Engl J Med, 371:1507-17).

[0099] In certain situations, available approaches to adoptive cell therapy may not always be completely satisfactory. For example, CAR T cell persistence can be detected in many subjects with lymphoma, but fewer complete responses (CRs) have been observed in subjects with NHL compared with subjects with ALL. More specifically, higher overall response rates of up to 80% (CR rates of 47%-60%) have been reported after CAR T cell infusion, but some responses were transient, and subjects were shown to relapse in the presence of persistent CAR T cells (Neelapu, 58th Annual Meeting of the American Society of Hematology (ASH): 2016; San Diego, CA, USA. Abstract No. LBA-6.2016; Abramson, Blood. 2016 Dec 01;128(22):4192). Another study reported a long-term CR rate of 40% (Schuster, Ann Hematol. 2016 Oct;95(11):1805-10).

[0100] In some embodiments, the explanation for this is immunological exhaustion of circulating CAR-expressing T cells and / or changes in T lymphocyte populations, as in some situations optimal efficacy may depend on the ability of the administered cells to become activated, expand, exert various effector functions including cytotoxic killing and secretion of various factors such as cytokines, persist, including long-term, differentiate into, transition to, or be associated with reprogramming into certain phenotypic states (such as long-term memory, less differentiated, and effector states), avoid or reduce immunosuppressive states in the local microenvironment of the disease, provide an effective and robust recall response after clearance and re-exposure to the target ligand or antigen, and avoid or reduce exhaustion, anergy, peripheral tolerance, terminal differentiation, and / or differentiation into suppressive states.

[0101] In some embodiments, the exposure, persistence, and function of the engineered cells are reduced or diminished after administration to a subject. In some embodiments, after long-term stimulation or exposure to an antigen and / or exposure under conditions in the tumor microenvironment, T cells can become hypofunctional and / or exhibit characteristics associated with an exhausted state over time. In some embodiments, this reduces the persistence and effectiveness of the T cells against the antigen and limits their ability to be effective. Despite this, observations indicate that in some cases, administered cells expressing recombinant receptors can be re-expanded and / or reactivated in vivo (e.g., exhibiting increased numbers of cells or duration over time), improving efficacy and therapeutic outcomes in adoptive cell therapy. There is a need for methods to improve the efficacy and function of CAR T cells, particularly to minimize, reduce, prevent, or reverse hypofunctional or exhausted states.

[0102] The provided methods are based on the observation that treatment with Compound C can improve T cell function, including functions related to the ability of T cells to produce one or more cytokines, cytotoxicity, expansion, proliferation, and persistence. In some embodiments, the provided methods enhance or modulate the proliferation and / or activity of T cells (e.g., CAR-expressing T cells) associated with the administration of cell therapy. It is found that such methods and uses provide or achieve improved or better T cell functionality and thereby improved anti-tumor efficacy.

[0103] It is also found herein that in addition to enhancing T cell function, treatment with Compound C can reverse, delay, or prevent T cell exhaustion, including by increasing T cell signaling and / or altering one or more genes that are differentially regulated after chronic stimulation. While in some cases, agents that increase or enhance T cell activity can drive cells into an exhausted state, it is found herein that Compound C's activity in exerting an enhancing effect on T cell activity is uncoupled from T cell exhaustion. Furthermore, observations herein indicate that Compound C exhibits activity in rescuing T cells from T cell exhaustion, such as by restoring or partially restoring one or more T cell activities after the cells exhibit characteristics of exhaustion. Notably, results herein indicate that T cells that have been chronically stimulated and exhibit characteristics of exhausted T cells can regain activity, or do so by partially restoring that activity, after exposure to Compound C. The observations herein support the idea that the provided methods can also achieve improved or more durable responses compared to certain alternative methods, such as in certain groups of treated subjects.

[0104] It is also shown herein that low-dose Compound C treatment (e.g., 1 nM) can significantly degrade both Ikaros and Aiolos expression in chronically stimulated CAR T cells. However, it is also shown herein that higher doses of Compound C (e.g., 10 and 100 nM) can cause sustained targeted degradation of Ikaros and Aiolos, resulting in reduced cytolytic function. Such results suggest that excessive sustained targeted degradation of Ikaros and Aiolos can have adverse effects, leading to CAR T cell dysfunction. Based on these results, and because Compound C can significantly degrade Ikaros and Aiolos more than other CELMoD compounds that target Ikaros and Aiolos, it is hypothesized herein that a single weekly dose is sufficient to cause profound but transient degradation of Ikaros and Aiolos, potentially allowing for restoration of Ikaros and / or Aiolos expression between doses. As shown herein, pharmacokinetic modeling of Compound C at a weekly dose of 0.3 mg (Q7D) indicates a C on day 7 of approximately 1 nM. min The rapid C max (approximately 20 nM) and demonstrated biphasic elimination. Modeling also indicated that a weekly dose of 0.3 mg would not substantially affect neutrophil counts. Also, as shown herein, in preclinical models, anti-CD19 CAR T cell lytic activity against CD19+ target cells was most pronounced after transient (1-day) exposure to high concentrations of Compound C (20-100 nM) followed by washout and chronic low concentrations of Compound C (1 nM for 5 days). This high-to-low exposure range was selected to model the pharmacokinetic profile of Compound C at 0.3 mg once every 7 days (Q7D). Conversely, higher chronic concentrations of Compound C had a detrimental effect on cytolytic function.

[0105] Thus, based on the results presented herein, cell therapy, e.g., CAR T cell therapy, in combination with intermittent (e.g., once every seven days) dosing of Compound C may provide a useful therapeutic approach to prevent premature exhaustion of CAR T cells, enhance and prolong T cell functionality, and produce more pronounced and durable responses in cancers such as lymphoma, while also reducing the risk of patients developing neutropenia secondary to neutrophil maturation arrest.

[0106] In some embodiments, T cell function (including functions related to T cell expansion, proliferation, and persistence) of engineered T cell therapy administered according to the provided methods is improved by Compound C. In some embodiments, the methods are beneficial based on the administration of T cell therapy, e.g., a composition comprising cells for adoptive cell therapy, such as T cell therapy (e.g., CAR-expressing T cells), in combination with Compound C. In some aspects, the provided methods and uses provide or achieve improved or more durable responses or efficacy compared to certain alternative methods. In some aspects, the provided methods enhance or modulate T cell proliferation and / or activity associated with the administration of T cell therapy (e.g., CAR-expressing T cells). In certain embodiments, combination therapy with Compound C can provide a useful therapeutic approach for enhancing and prolonging the activity of CAR T cells across B cell malignancies by modulating the tumor microenvironment, thereby improving the sustained anti-tumor function of CAR T cells.

[0107] In some embodiments, Compound C is administered to the subject a sufficient time after receiving lymphodepleting therapy such that the myelosuppressive effects of Compound C and the lymphodepleting therapy are minimized.

[0108] In some embodiments, provided methods are used at a time when T cell therapy (e.g., CAR T cells) can or may exhibit characteristics of exhaustion. In some embodiments, the exhaustion phenotype is evident after T cells have reached peak expansion and begin to decline in number in the subject's blood. In some embodiments, the method of exposing or contacting T cells of a T cell therapy (CAR T cells) with Compound C is performed at a time when the T cells exhibit increased reduced function or exhaustion compared to a time point immediately prior to exposure of the T cells to an antigen (baseline), or compared to a time point when the cells have been exposed to an antigen but continue to proliferate and have not yet reached peak expansion. In some embodiments, the increased reduced function or exhaustion can be determined by increased expression of exhaustion markers compared to a previous, earlier time point. In some embodiments, the increased reduced function or exhaustion, such as increased expression of exhaustion markers, occurs at a time point after administration of T cell therapy (e.g., CAR T cells) to a subject suffering from a disease or condition associated with the antigen targeted by the T cell therapy. T cells, such as T cells in the peripheral blood after administration to a subject, can be monitored for markers of T cell activation or exhaustion, such as PD-1, TIM-3, and LAG-3.

[0109] In some embodiments, provided methods involve administration of T cell therapy, e.g., CAR T cells, and initiation of administration of Compound C at a time before the CAR T cells exhibit or are likely to exhibit an exhausted phenotype. In some embodiments, administration of Compound C is initiated at a time when the CAR T cells are still expanding or still capable of expansion. In some embodiments, administration of Compound C is initiated before or at a suspected time before the presence of peak CAR T cell numbers in the subject's blood. In some aspects, initiation of administration of Compound C at this time enhances CAR T cell function. In some aspects, initiation of administration of Compound C at this time also delays or prevents CAR T cell exhaustion.

[0110] In some embodiments, administration of Compound C is initiated before or about the time when peak CAR-T cells are present in the subject's blood, e.g., within 21 days after initiation of administration of T cells, or at a time suspected or likely to be present. In some cases, peak CAR-T cells occur within 11-15 days after administration of CAR T cells. In some embodiments, administration of Compound C is initiated 1-15 days, e.g., 1 day, 8 days, or 15 days, or any number thereof, after initiation of administration of cell therapy. In some embodiments, Compound C is administered at a time when the subject is not experiencing severe toxicity following administration of cell therapy.

[0111] In some embodiments, the provided methods do not result in a high rate or likelihood of toxicity or a toxic outcome, or reduce the rate or likelihood of toxicity or a toxic outcome, e.g., neurotoxicity (NT), cytokine release syndrome (CRS), or hematologic toxicity, e.g., neutropenia, as compared to certain other cell therapy or immunomodulatory regimens, etc.

[0112] In some embodiments, the method does not result in or increase the risk of certain hematological toxicities, such as neutropenia or thrombocytopenia. In some embodiments, 50% or less of the subjects exhibit neutropenia greater than Grade 3, e.g., prolonged Grade 3 neutropenia or Grade 4 neutropenia, and / or thrombocytopenia greater than Grade 3, e.g., Grade 3 or Grade 4 thrombocytopenia. In some embodiments, at least 50% of the subjects treated according to the method (e.g., at least 60%, at least 70%, at least 80%, at least 90% or more of the treated subjects) do not exhibit Grade 3 or severe neutropenia greater than Grade 3 or severe thrombocytopenia.

[0113] In some embodiments, the methods do not result in or increase the risk of severe NT (sNT), severe CRS (sCRS), macrophage activation syndrome, tumor lysis syndrome, fever of at least about 38 degrees Celsius for three or more days, and plasma levels of CRP of at least about 20 mg / dL. In some embodiments, more than or about 30%, 35%, 40%, 50%, 55%, 60% or more of subjects treated according to the provided methods do not exhibit any grade of CRS or any grade of neurotoxicity. In some embodiments, 50% or less of treated subjects (e.g., at least 60%, at least 70%, at least 80%, at least 90% or more of treated subjects) exhibit cytokine release syndrome (CRS) greater than Grade 2 and / or neurotoxicity greater than Grade 2. In some embodiments, at least 50% of subjects treated according to the method (e.g., at least 60%, at least 70%, at least 80%, at least 90% or more of the treated subjects) do not experience a severe toxic outcome (e.g., severe CRS or severe neurotoxicity), e.g., do not experience grade 3 or higher neurotoxicity and / or do not experience severe CRS, or do not do so within a certain period of time following treatment, such as within 1 week, 2 weeks, or 1 month of administration of the cells.

[0114] In some embodiments, the dose, timing of doses, or number of doses is not expected to cause severe toxicity, such as grade 4 neutropenia or grade 4 thrombocytopenia. In some embodiments, the methods provided minimize or avoid toxicity following administration of T cell therapy and / or Compound C to a subject, e.g., throughout intermittent dosing of Compound C. In some embodiments, the methods provided herein involve administering a dose that is substantially lower and given at a reduced frequency than would be used for Compound C in a monotherapy approach. In some embodiments, the methods provided herein involve administering a lower dose than would be used for Compound C in a monotherapy approach. In some embodiments, the methods provided herein involve administering intermittent doses of Compound C with a longer period between doses of Compound C than would be used for Compound C in a monotherapy approach.

[0115] In some cases, Compound C is administered at a time when it can efficiently / effectively enhance or prime the cells. In some embodiments, administration of Compound C is initiated at or before the peak or maximum level of cell therapy cells is detectable in the subject's blood. In some embodiments, the provided methods can enhance T cell therapy, e.g., CAR-T cell therapy, which, in some aspects, can improve outcomes for treatment. In some embodiments, the methods are particularly advantageous in subjects in which T cell therapy cells show weak expansion, become exhausted, show reduced or decreased persistence in the subject, and / or in subjects with cancer that is resistant or refractory to other therapies and / or is an aggressive or high-risk cancer.

[0116] In some embodiments, a subject receiving a T cell therapy, e.g., CAR-T cells, is monitored for the presence, absence, or level of therapeutic T cells in the subject in a biological sample from the subject, e.g., in the subject's blood. In some embodiments, the provided methods result in engineered cells with increased persistence and / or better efficacy in the subject to which they are administered. In some embodiments, the persistence of engineered cells, such as CAR-expressing T cells, in the subject is greater than the persistence that would be achieved by alternative methods, such as methods involving administration of T cell therapy but in the absence of administration of Compound C. In some embodiments, the persistence is increased by at least or about at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more.

[0117] In some embodiments, the degree or extent of persistence of administered cells can be detected or quantified after administration to a subject. For example, in some aspects, quantitative PCR (qPCR) is used to assess the content of cells expressing the recombinant receptor (e.g., CAR-expressing cells) in the subject's blood or serum or organs or tissues (e.g., disease sites). In some aspects, persistence is quantified as copies of the receptor, e.g., CAR-encoding DNA or plasmid, per microgram of DNA, or as the number of receptor-expressing, e.g., CAR-expressing cells, per microliter of sample, e.g., blood or serum, or per microliter of sample, per total number of peripheral blood mononuclear cells (PBMCs) or white blood cells or T cells. In some embodiments, flow cytometry assays can be performed to detect receptor-expressing cells, typically using receptor-specific antibodies. Cell-based assays can also be used to detect the number or percentage of functional cells, such as cells that can bind to and / or neutralize and / or respond to, e.g., induce a cytotoxic response, disease or condition cells or cells expressing an antigen recognized by the receptor. In any of such embodiments, the degree or level of expression of another marker associated with the recombinant receptor (e.g., CAR-expressing cells) can be used to distinguish the administered cells from endogenous cells in the subject.

[0118] In some embodiments, Compound C is administered over a period of time to enhance, increase, or optimize the durability of the response. In some aspects, the provided methods are based on the observation that subjects who achieve or are in complete remission (CR) at 3 months are more likely to sustain the response for a longer period of time, such as surviving or surviving without progression for more than or about 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after initially achieving complete response (CR) after stopping treatment or after administration of a combination therapy. In some aspects, the methods are performed to administer Compound C, such as in a particular intermittent dosing regimen described, for a period of at least 3 months or about 3 months after initiation of administration of T cell therapy.

[0119] In some aspects, the provided methods and uses provide or achieve improved or more durable responses or efficacy, such as in certain groups of treated subjects, compared to certain alternative methods, e.g., methods that do not include administration of T cell therapy or Compound C as monotherapy, or administration together as a combination therapy as described herein. In some embodiments, the methods are advantageously based on administration of T cell therapy, e.g., a composition comprising cells for adoptive cell therapy, such as T cell therapy (e.g., CAR-expressing T cells), and Compound C. In some embodiments, such responses are observed in high-risk patients with poor prognosis, such as patients with high-risk disease, e.g., high-risk NHL. In some aspects, the methods treat subjects with aggressive and / or poor-prognosis forms of B-cell non-Hodgkin's lymphoma (NHL), such as NHL that has relapsed or is refractory (R / R) to standard therapy or has a poor prognosis. In some embodiments, subjects treated according to the provided methods have diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma.

[0120] In some embodiments, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% or more of subjects treated according to the provided methods and / or with the provided articles of manufacture, kits, or compositions achieve a complete response (CR). In some embodiments, the subjects are in CR and exhibit minimal residual disease (MRD). In some embodiments, the subjects are in CR and are MRD-. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of subjects treated according to the provided methods and / or with the provided articles of manufacture, kits, or compositions achieve an objective response of partial response (PR). In some embodiments, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more of subjects treated according to the provided methods and / or with the provided articles of manufacture, kits, or compositions achieve a CR or PR at 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year after initiation of administration of the cell therapy.

[0121] In some embodiments, by 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more after initiation of cell therapy administration, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more of subjects treated according to the provided methods and / or using the provided articles of manufacture, kits, or compositions remain responsive, such as remaining in CR or objective response (OR). In some embodiments, such a response, such as a CR or OR, is durable for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, such as in at least or about at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more of subjects treated according to the provided methods, or in such subjects achieving a CR by 3, 4, 5, or 6 months. In some embodiments, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more of subjects treated according to the provided methods and / or with the provided articles of manufacture, kits, or compositions, or those subjects who achieve a CR by 3, 4, 5, or 6 months, survive or survive progression-free for more than or about 6, 7, 8, 9, 10, 11, 12, or more months.

[0122] All publications, including patent documents, scientific articles, and databases, referenced in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated herein by reference. To the extent that a definition set forth herein contradicts or is otherwise inconsistent with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein takes precedence over the definition incorporated herein by reference.

[0123] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0124] I. Combination Therapy and (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a compound represented by Formula I, for the treatment of a subject suffering from cancer. [ka] (Formula I) or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, or racemic mixture thereof (Compound C), including compositions thereof. In some embodiments, the method is for treating a subject suffering from lymphoma. In some embodiments, the lymphoma is a B-cell malignancy. In some aspects, the lymphoma is non-Hodgkin's lymphoma (NHL). In some embodiments, the cancer, e.g., lymphoma, expresses CD19. In some aspects, the method and use provide or achieve an improved response and / or a more durable response or efficacy, e.g., in certain groups of treated subjects, compared to certain alternative methods.

[0125] Methods for preparing Compound C are described in US2019 / 0322647, which is incorporated herein by reference in its entirety.

[0126] In some embodiments, the methods and uses include: 1) administering to a subject a cell therapy comprising T cells expressing an engineered cell surface receptor (e.g., a recombinant antigen receptor), e.g., a chimeric receptor, e.g., a chimeric antigen receptor (CAR), that recognizes an antigen expressed by, associated with, and / or specific to lymphoma (e.g., a B-cell malignancy such as NHL) and / or a cell type from which the lymphoma is derived; and 2) administering Compound C to the subject. In some embodiments, administration of Compound C is initiated after (following) administration of the cell therapy or after (following) initiation of administration of the cell therapy. In some cases, Compound C is administered to a subject who has received cell therapy. The method can involve administering to a subject one or more doses of engineered cells and two or more doses of Compound C.

[0127] Combination therapies comprising engineered cells expressing a recombinant receptor, such as a chimeric antigen receptor (CAR), and Compound C, or compositions comprising the engineered cells and / or Compound C described herein, are useful in a variety of therapeutic, diagnostic, and prophylactic indications. For example, combinations are useful in treating various diseases and disorders in a subject. Such methods and uses include therapeutic methods and uses involving administration of engineered cells, Compound C, and / or a composition containing one or both to a subject suffering from a disease, condition, or disorder, such as a tumor or cancer. In some embodiments, the engineered cells, Compound C, and / or a composition containing one or both are administered in an amount effective to provide treatment for the disease or disorder. Uses include the use of engineered cells, Compound C, and / or a composition containing one or both in such methods and treatments, and in the preparation of medicaments for carrying out such therapeutic methods. In some embodiments, the methods are carried out by administering a composition containing engineered cells, Compound C, and / or one or both to a subject suffering from or suspected of having a disease or condition. In some embodiments, the methods thereby treat the disease, condition, or disorder in the subject. In some embodiments, the engineered cells are any of those described in Section II.

[0128] In some embodiments, the combination therapy is administered to a subject suffering from lymphoma. In some embodiments, the combination therapy is administered to a subject suffering from a particular B cell malignancy. The B cell malignancy being treated can be one in which expression of an antigen is associated with and / or involved in the pathogenesis of the B cell malignancy, e.g., causes, exacerbates, or otherwise contributes to the B cell malignancy. Exemplary B cell malignancies can include diseases or conditions associated with cellular malignancy or transformation (e.g., cancer). Exemplary antigens, including antigens associated with various B cell malignancies that can be treated, are described herein. In certain embodiments, a chimeric antigen receptor specifically binds to an antigen associated with a disease or condition. In some embodiments, the antigen targeted by the receptor includes an antigen associated with a B cell malignancy, such as any of a number of known B cell markers. In some embodiments, the antigen is expressed by or present on the surface of B cells, including human B cells. In some embodiments, the antigen targeted by the receptor is CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig kappa, Ig lambda, CD79a, CD79b, or CD30. In some embodiments, the antigen is CD19, and the chimeric antigen receptor specifically binds to CD19. In some embodiments, the CD19 antigen is human CD19. In some embodiments, the lymphoma comprises B cells that express CD19. It is understood that any description of the methods provided herein in which the CAR-expressing T cells are specific for CD19 can also be performed by targeting another B cell antigen or an antigen associated with or expressed on the cell surface of a T cell malignancy, such as any described above.

[0129] In some embodiments, B-cell malignancies to be treated include leukemias and lymphomas, such as acute myeloid (or myeloid) leukemia (AML), chronic myeloid (or myeloid) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), anaplastic large cell lymphoma (ALCL), follicular lymphoma, refractory follicular lymphoma, and diffuse large B-cell lymphoma (DLBCL). In some embodiments, the disease or condition is a B-cell malignancy selected from among acute lymphocytic leukemia (ALL), adult ALL, chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), and diffuse large B-cell lymphoma (DLBCL). In some embodiments, the disease or condition is NHL, wherein the NHL is selected from the group consisting of aggressive NHL, diffuse large B-cell lymphoma (DLBCL), NOS (de novo and low-grade transformed), primary mediastinal large B-cell lymphoma (PMBCL), T-cell / histocyte-rich large B-cell lymphoma (TCHRBCL), Burkitt's lymphoma, mantle cell lymphoma (MCL), and / or follicular lymphoma (FL), optionally follicular lymphoma grade 3B (FL3B).

[0130] In some embodiments, the methods involve treating a subject suffering from a lymphoma, such as non-Hodgkin's lymphoma (NHL), by administering an antigen receptor-expressing cell (e.g., a CAR-expressing cell) and Compound C. In some embodiments, Compound C is administered after or following administration of the recombinant receptor-expressing cell (e.g., a CAR-expressing cell), such as after or following the initiation of administration of the recombinant receptor-expressing cell (e.g., a CAR-expressing cell).

[0131] In some embodiments, NHL can be staged based on the Lugano classification (see, e.g., Cheson et al., (2014) JCO 32(27):3059-3067; Cheson, BD (2015) Chin Clin Oncol 4(1):5). In some cases, stages are described by Roman numerals I-IV (1-4), with limited stage (I or II) lymphoma affecting organs other than the lymphatic system (extranodal organs) designated by E. Stage I represents involvement of one node or adjacent nodal groups, or a single extranodal lesion without nodal involvement (IE). Stage 2 represents involvement of two or more nodal groups on the same side of the diaphragm, or stage I or II by nodal degree with limited contiguous extranodal involvement (IIE). Stage III represents involvement of nodes on both sides of the diaphragm, or nodes above the diaphragm with splenic involvement. Stage IV describes the involvement of additional non-contiguous extranodal involvement. In addition, "bulky disease" can be used to describe large tumors in the chest, especially for stage II. The extent of disease is determined by positron emission tomography (PET)-computed tomography (CT) for lymphomas with accretion and by CT for non-accreting histology.

[0132] In some embodiments, the Eastern Cooperative Oncology Group (ECOG) performance status index can be used to evaluate or select subjects for treatment, for example, subjects who have had poor performance from previous therapy (see, e.g., Oken et al. (1982) Am J Clin Oncol. 5:649-655). In some embodiments, the subject has an ECOG status of 1 or less. The ECOG scale of performance status represents a patient's level of function in terms of their ability to care for themselves, daily activities, and physical abilities (e.g., walking, working, etc.). In some embodiments, an ECOG performance status of 0 indicates that the subject is able to carry out normal activities. In some aspects, a subject with an ECOG performance status of 1 shows some limitation in physical activity, but the subject is fully ambulatory. In some aspects, a patient with an ECOG performance status of 2 is more than 50% ambulatory. In some cases, subjects with an ECOG performance status of 2 may be capable of self-care; see, e.g., Sorensen et al., (1993) Br J Cancer 67(4) 773-775. Criteria reflecting ECOG performance status are set forth in Table 1 below: [Table 1]

[0133] In some embodiments, the subject is afflicted with or identified as afflicted with double / triple-hit lymphoma or lymphoma of a double / triple-hit molecular subtype. In some embodiments, the lymphoma is a double-hit lymphoma characterized by the presence of MYC (myelocytomatosis oncogene), BCL2 (B-cell lymphoma 2), and / or BCL6 (B-cell lymphoma 6) gene rearrangements (e.g., translocations). In some embodiments, the lymphoma is a triple-hit lymphoma characterized by the presence of MYC, BCL2, and BCL6 gene rearrangements; see, e.g., Aukema et al., (2011) Blood 117:2319-2331. In some aspects of such embodiments, the subject has an ECOG score of 0-1. In aspects, the therapy is indicated for such subjects, and / or the instructions for use indicate administration to subjects within such populations. In some embodiments, based on the 2016 WHO criteria (Swerdlow et al., (2016) Blood 127(20):2375-2390), double / triple hit lymphomas can be considered high-grade B-cell lymphomas with MYC and BCL2 and / or BCL6 rearrangements (double / triple hit) with DLBCL histology.

[0134] In some embodiments, the combination therapy is administered to a subject who is, is likely to, or is predicted to be an inadequate responder to treatment with cell therapy (e.g., CAR+ T cells) and / or who will not respond, will not respond, will not respond, or will not respond within a certain time and / or to a certain extent. In some embodiments, the combination therapy is administered to a subject who will not, is unlikely to, or is not predicted to have a complete or overall response, such as within one month, two months, or three months after initiation of cell therapy administration. In some embodiments, the combination therapy is administered to a subject who will exhibit, is likely to, or is predicted to exhibit progressive disease (PD), such as within one month, two months, or three months after administration of cell therapy. In some embodiments, the subject is likely or predicted to not respond or have a certain response based on multiple similarly affected subjects who have been so treated or previously treated with cell therapy.

[0135] In some embodiments, provided methods involve treating a specific group or subset of subjects, e.g., subjects identified as having high-risk disease, e.g., high-risk NHL. In some aspects, the methods treat subjects with an aggressive and / or poor-prognosis form of B-cell non-Hodgkin's lymphoma (NHL), such as NHL that has relapsed or is refractory (R / R) or has a poor prognosis to standard therapy. In some cases, the overall response rate (ORR) to available therapies, standard therapies, or reference therapies for the disease and / or patient population for which the therapy is indicated is less than 40%, and / or the complete response (CR) is less than 20%. In some embodiments, in chemotherapy-resistant DLBCL, the ORR with reference or available treatment or standard of care therapy is about 26%, and the CR is about 8% (Crump et al. Outomes in refractory aggressive diffuse large B-cell lymphoma (DLBCL): Results from the international SCHOLAR study. ASCO 2016 [Abstract 7516]). In some aspects, the provided methods, compositions, uses, and articles of manufacture achieve improved and superior response to available therapy.

[0136] In some embodiments, the methods and uses for treating a subject described herein involve selecting or identifying a specific group or subset of subjects based on, for example, a particular type of disease, diagnostic criteria, previous treatment, and / or response to previous treatment. In some embodiments, the methods involve treating subjects who have relapsed or become refractory to one or more previous therapies after remission; or subjects who have relapsed or become refractory to one or more previous therapies, such as first-line or higher-line standard therapies, including the therapies described herein (R / R).

[0137] In some embodiments, the subject has been exposed to 1, 2, 3, 4, 5, or more than 6 previous therapies. In some embodiments, the subject has been exposed to 1 previous therapy. In some embodiments, the subject has been exposed to about 2-4 previous therapies. In some embodiments, the subject has been exposed to about 5-6 previous therapies. In some embodiments, the subject has been exposed to more than 6 previous therapies.

[0138] In some embodiments, the subject has previously been treated with a therapy or therapeutic agent targeting lymphoma, e.g., NHL, prior to administration of the cells expressing the recombinant receptor. In some embodiments, the subject has previously been treated with cell therapy (e.g., CAR+ T cells). In some embodiments, the subject has previously been treated with hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT or autologous HSCT. In some embodiments, the subject has a poor prognosis after treatment with standard therapy and / or has failed a previous first-line or higher-line therapy. In some embodiments, the subject has been treated with or previously received at least or about at least or about 1, 2, 3, 4, 5, 6, or 7 other therapies for treating NHL other than lymphodepleting therapy. In some embodiments, the subject has been previously treated with chemotherapy or radiation therapy. In some aspects, the subject is refractory or non-responsive to other therapies or therapeutic agents. In some embodiments, the subject has persistent or recurrent disease after treatment with another therapy or intervention, including, for example, chemotherapy or radiation.

[0139] In some embodiments, the combination therapy is administered to a subject who has progressed on a previous therapy. In some embodiments, the combination therapy is administered to a subject who has ceased to respond to a previous therapy. In some embodiments, the combination therapy is administered to a subject who has relapsed following remission after a previous therapy. In some embodiments, the combination therapy is administered to a subject who is refractory to a previous therapy. In some embodiments, the combination therapy is administered to a subject who has experienced a less than optimal response (e.g., complete response, partial response, or stable disease) to a previous therapy.

[0140] In some embodiments, the subject is refractory to the last previous therapy. In some embodiments, the subject has relapsed to the last previous therapy. The condition is refractory if the subject has achieved less than a partial response to the last previous therapy. In some embodiments, the subject has had previous chemotherapy. In some embodiments, the subject is chemoresistant to the previous chemotherapy. In some embodiments, the subject is chemosensitive to the previous therapy. The condition is chemoresistant, and the subject has achieved stable disease (SD) or progressive disease (PD) on the last chemotherapy-containing regimen, or has relapsed less than 12 months after autologous stem cell transplantation. Otherwise, the condition is chemosensitive.

[0141] In some embodiments, the previous treatment or therapy includes a CD20 targeting agent. In some embodiments, the previous treatment or therapy includes an anthracycline. In some embodiments, the previous treatment or therapy includes cell therapy (e.g., T cell therapy, e.g., CAR T cell therapy).

[0142] In some embodiments, the methods, uses, and articles of manufacture involve or are used in treating subjects by selecting or identifying a particular group or subset of subjects based on a particular type of disease, diagnostic criteria, previous treatment, and / or response to previous treatment, such as, for example, any of the groups of subjects described. In some embodiments, the methods involve treating subjects who have relapsed or become refractory following remission after treatment with one or more previous therapies; or subjects who have relapsed or become refractory (R / R) to one or more previous therapies, e.g., first-line or higher-line standard therapies, e.g., cell therapy (e.g., CAR+ T cells). In some embodiments, the methods involve treating subjects with diffuse large B-cell lymphoma (DLBCL), not otherwise specified (NOS; de novo and low-grade transformed), primary mediastinal (thymic) large B-cell lymphoma (PMBCL), or follicular lymphoma grade 3B (FL3B), EBV-positive DLBCL, or EBV-positive NOS. In some embodiments, the methods involve treating a subject with an Eastern Cooperative Oncology Group (ECOG) performance status of less than 1, such as 0-1. In some embodiments, the method treats a poor prognosis population of DLBCL patients or subjects who generally respond poorly to therapy or a particular reference therapy, such as those with one or more, e.g., two or three, chromosomal translocations (such as so-called "double-hit" or "triple-hit" lymphomas, which are aggressive B-cell lymphomas with MYC and BCL2 and / or BCL6 rearrangements with DLBCL histology; which have a translocated MYC / 8q24 locus, usually in combination with the t(14;18)(q32;q21) bcl-2 gene or / and BCL6 / 3q27 chromosomal translocation; see, e.g., Xu et al. (2013) Int J Clin Exp Pathol. 6(4): 788-794), and / or those who have relapsed within 12 months, appropriately relapsed, and / or those suspected to be chemoresistant.

[0143] In some embodiments, the subject has a DLBCL that is a germinal center-like (GCB) DLBCL. In some embodiments, the subject has a non-germinal center-like (non-GCB) DLBCL. In some embodiments, the subject has double-hit lymphoma (DHL). In some embodiments, the subject has triple-hit lymphoma (THL). In some embodiments, the subject has positive expression of a gene indicative of responsiveness to treatment with Compound C. In some embodiments, the subject has negative expression of the gene. See Blood 2017 130:4118.

[0144] In some embodiments, the antigen receptor (e.g., CAR) specifically binds to a target antigen associated with a disease or condition, such as associated with NHL. In some embodiments, the antigen associated with a disease or disorder is selected from CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig kappa, Ig lambda, CD79a, CD79b, or CD30. In some embodiments, the antigen is CD19. In some embodiments, the CD19 antigen is human CD19.

[0145] In some embodiments, the methods include administering cell therapy and Compound C to a subject at risk for or suspected of having a B-cell malignancy.

[0146] In some embodiments, the method involves administering cells to a subject selected or identified as having a certain prognosis or risk of NHL. Non-Hodgkin's lymphoma (NHL) can be a variable disease. Some subjects with NHL can survive without treatment, while others may require immediate intervention. In some cases, subjects with NHL can be classified into groups that can inform disease prognosis and / or recommended treatment strategies. In some cases, such groups can be "low risk," "moderate risk," "high risk," and / or "very high risk," and patients can be classified as such depending on a number of factors, including, but not limited to, genetic abnormalities and / or morphological or physical characteristics. In some embodiments, subjects treated with the method and / or article of manufacture or composition are classified or identified based on their risk of NHL. In some embodiments, the subject is a subject with high-risk NHL.

[0147] In some embodiments, subjects to be treated include those suffering from aggressive NHL, particularly diffuse large B-cell lymphoma (DLBCL), not otherwise specified (NOS; de novo and transformed from low-grade), T-cell / histiocytic-rich large B-cell lymphoma, primary mediastinal (thymic) large B-cell lymphoma (PMBCL), follicular lymphoma grade 3B (FL3B), EBV-positive DLBCL, EBV-positive NOS, or high-grade B-cell lymphoma with DLBCL histology and MYC and BCL2 and / or BCL6 rearrangements ("double-hit" or "triple-hit" lymphoma). In some embodiments, the subject's disease has relapsed or is refractory to at least two prior lines of therapy. In some embodiments, the prior therapy includes a CD20-targeting agent and / or an anthracycline. In some embodiments, the subject has an ECOG score of 0-1 at screening. In some embodiments, the subject has positron emission tomography (PET)-positive disease according to the Lugano classification (Cheson, 2014). In some embodiments, the subject may have been previously treated with allogeneic stem cell transplantation (SCT).

[0148] In some embodiments, the subject is an adult. In some embodiments, the subject is male. In some embodiments, the subject is female. In some embodiments, the subject is at least 40 years old at the time the combination therapy is administered (e.g., at the time the cell therapy is administered). In some embodiments, the subject is under 40 years old at the time the combination therapy is administered (e.g., at the time the cell therapy is administered). In some embodiments, the subject is between about 40 and 65 years old at the time the combination therapy is administered (e.g., at the time the cell therapy is administered). In some embodiments, the subject is at least 65 years old at the time the combination therapy is administered (e.g., at the time the cell therapy is administered).

[0149] A. Cell Therapy Administration The method for administering cells for adoptive cell therapy is known, and can be used in connection with the provided methods, compositions, and products and kits.For example, the method of adoptive T cell therapy is described, for example, in U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al.; U.S. Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85).For example, see Themeli et al. (2013) Nat Biotechnol. 31(10):928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1):84-9; Davila et al. (2013) PLoS ONE 8(4):e61338.

[0150] In some embodiments, the cells used in or administered in connection with the provided methods contain or are engineered to contain engineered receptors, such as engineered antigen receptors, for example, chimeric antigen receptors (CARs) or T cell receptors (TCRs).Particularly mentioned among compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy.Also provided are methods of treatment for administering cells and compositions to subjects, for example, patients, in accordance with the provided methods and / or using the provided products or compositions.

[0151] The cells generally express recombinant receptors, such as antigen receptors, including functional non-TCR antigen receptors, e.g., chimeric antigen receptors (CARs), and other antigen-binding receptors, such as transgenic T cell receptors (TCRs). Other chimeric receptors are also specifically mentioned among receptors. Exemplary engineered cells for administration as cell therapy in the provided methods are described in Section II.

[0152] In some embodiments, cell therapy, e.g., adoptive T cell therapy, is performed by autologous transfer, whereby cells are isolated and / or otherwise prepared from a subject to receive cell therapy or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., a patient, in need of treatment, and the cells, after isolation and processing, are administered to the same subject.

[0153] In some embodiments, cell therapy, e.g., adoptive T cell therapy, is performed by allogeneic transfer, whereby cells are isolated and / or otherwise prepared from a subject other than the subject who is to receive or will ultimately receive cell therapy, e.g., a first subject. In such embodiments, the cells are then administered to a different subject of the same species, e.g., a second subject. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0154] The cells of the T cell therapy can be administered in a composition formulated for administration, or alternatively, in two or more compositions (e.g., two compositions) formulated for separate administration. A dose of cells can include a specific or relative number of cells or engineered cells, and / or two or more subtypes in a defined ratio or composition within the composition, such as CD4 vs. CD8 T cells.

[0155] The cells can be administered by any suitable means, for example, by bolus injection, injection, e.g., intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjunctival injection, sub-Tenon injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, the cells are administered parenterally, intrapulmonary, intranasally, and, if desired for localized treatment, intralesionally. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus of cells. In some embodiments, a given dose is administered by multiple boluses of cells over a period of, for example, three days or less, or by continuous infusion of cells. In some embodiments, administration of the cell dose or any additional therapy, eg, lymphodepleting therapy, interventional therapy, and / or combination therapy, is by exogenous delivery.

[0156] For treatment of disease, the appropriate dosage will depend on the type of disease being treated, the type of cells or recombinant receptor, the severity and course of the disease, previous therapy, the subject's clinical history and response to the cells, and the discretion of the treating physician. The compositions and cells, in some embodiments, are suitably administered to the subject at one time or over a series of treatments.

[0157] Preconditioning a subject with immunodepleting (eg, lymphodepleting) therapy can, in some aspects, improve the efficacy of adoptive cellular therapy (ACT).

[0158] Thus, in some embodiments, the method includes administering a preconditioning agent, such as a lymphodepleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof, to the subject prior to the initiation of cell therapy. For example, the subject can be administered the preconditioning agent at least two days, such as at least three, four, five, six, or seven days, prior to the initiation of cell therapy. In some embodiments, the subject is administered the preconditioning agent no more than seven days, such as no more than six, five, four, three, or two days, prior to the initiation of cell therapy.

[0159] In some embodiments, the subject is preconditioned with cyclophosphamide at a dose of between or about 20 mg / kg and 100 mg / kg, such as between or about 40 mg / kg and 80 mg / kg. In some aspects, the subject is preconditioned with cyclophosphamide at or about 60 mg / kg. In some embodiments, the cyclophosphamide can be administered in a single dose, or in multiple doses, such as daily, every other day, or every third day. In some embodiments, the cyclophosphamide is administered once daily for one or two days. In some embodiments, when the lymphodepleting agent includes cyclophosphamide, the subject is preconditioned with cyclophosphamide at a dose of 200 mg / m 2 ~400mg / m 2 or 250 mg / m 2 ~350mg / m 2 Between or around 100 mg / m 2 ~500mg / m 2 In some instances, the subject receives a dose of cyclophosphamide of about 300 mg / m 2 In some instances, the subject receives about 500 mg / m 2In some embodiments, the cyclophosphamide can be administered in a single dose, or in multiple doses, such as given daily, every other day, or every third day. In some embodiments, the cyclophosphamide is administered daily for 1-5 days, such as for 3-5 days. In some instances, the subject receives about 300 mg / m daily for 3 days prior to the initiation of cell therapy. 2 In some instances, the subject receives about 500 mg / m daily for three days prior to the start of cell therapy. 2 The patient is given cyclophosphamide.

[0160] In some embodiments, when the lymphodepleting agent comprises fludarabine, the subject receives 10 mg / m 2 ~75mg / m 2 , 15 mg / m 2 ~50mg / m 2 , 20 mg / m 2 ~40mg / m 2 or 24 mg / m 2 ~35mg / m 2 Between or around 1 mg / m 2 ~100mg / m 2 In some instances, the subject receives a dose of fludarabine of about 30 mg / m 2 In some embodiments, the subject is administered about 30 mg / m fludarabine daily for 3 days prior to the initiation of cell therapy. In some embodiments, the fludarabine can be administered in a single dose, or in multiple doses, such as given daily, every other day, or every third day. In some embodiments, the fludarabine is administered daily for 1-5 days, such as for 3-5 days. In some instances, the subject is administered about 30 mg / m fludarabine daily for 3 days prior to the initiation of cell therapy. 2 The patient was given fludarabine.

[0161] In some embodiments, the lymphodepleting agent comprises a combination of drugs, such as a combination of cyclophosphamide and fludarabine. Thus, the combination of drugs can include cyclophosphamide at any dose or administration schedule, such as those described above, and fludarabine at any dose or administration schedule, such as those described above. For example, in some aspects, the subject receives 60 mg / kg (approximately 2 g / m) of cyclophosphamide prior to the first or subsequent dose. 2 ) of cyclophosphamide and 3 to 5 doses of 25 mg / m 2 In some embodiments, the subject receives fludarabine at 300 mg / m daily for three days each prior to the initiation of cell therapy. 2 of cyclophosphamide and 30 mg / m 2 In some embodiments, the subject receives 500 mg / m fludarabine daily for three days each prior to the initiation of cell therapy. 2 of cyclophosphamide and 30 mg / m 2 The patient was given fludarabine.

[0162] After administration of the cells, the biological activity of the engineered cell population is measured, in some embodiments, by any of a number of known methods. Parameters for evaluation include specific binding of engineered or natural T cells or other immune cells to antigens in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable known method, such as the cytotoxicity assays described in Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009) and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells is measured by assaying the expression and / or secretion of one or more cytokines, such as CD107a, IFNγ, IL-2, and TNF. In some aspects, biological activity is measured by assessing clinical outcomes, such as reduction in tumor burden or burden.

[0163] 1. Compositions and Formulations In some embodiments, a dose of cells for cell therapy, such as T cell therapy, comprising cells engineered with a recombinant antigen receptor, e.g., a CAR or TCR, is provided as a composition or formulation, such as a pharmaceutical composition or formulation. Such compositions can be used in conjunction with the provided methods and / or using the provided articles of manufacture or compositions, such as in the treatment of B cell malignancies.

[0164] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0165] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0166] In some embodiments, cell therapies, such as engineered T cells (e.g., CAR T cells), are formulated with a pharmaceutically acceptable carrier. In some aspects, the choice of carrier is determined, in part, by the particular cell or agent and / or by the method of administration. Accordingly, a variety of suitable formulations exist. For example, the pharmaceutical composition can contain a preservative. Suitable preservatives can include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and may include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polymers; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0167] In some embodiments, a buffering agent is included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffering agents is used. The buffering agent or mixture thereof is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0168] The formulation may comprise an aqueous solution. The formulation or composition may contain two or more active ingredients useful for the particular indication, disease, or condition being treated by the cells or agent, where the activities of each do not adversely affect each other. Such active ingredients are suitably present in combination in amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises another pharmaceutically active agent or drug, such as a chemotherapeutic agent, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.

[0169] In some embodiments, the pharmaceutical composition contains cells in an amount effective for treating a disease or condition, such as a therapeutically or prophylactically effective amount. In some embodiments, the therapeutic effectiveness is monitored by periodic evaluation of the treated subject. For repeated administration over several days or longer, depending on the condition, treatment is repeated until the desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administration of the composition, or by continuous infusion administration of the composition.

[0170] Cells can be administered using standard administration techniques, formulations, and / or devices. Formulations and devices, such as syringes and vials, are provided for storing and administering the compositions. With respect to cells, administration can be autologous or xenogeneic. For example, immunoresponsive cells or precursors can be obtained from a subject and administered to the same subject or a different compatible subject. Peripheral blood-derived immunoresponsive cells or their progeny (e.g., derived in vivo, ex vivo, or in vitro) can be administered by localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition containing genetically modified immunoresponsive cells), it will generally be formulated in a unit-dosage injectable form (solution, suspension, emulsion).

[0171] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the agent or cell population is administered parenterally. The term "parenteral," as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the agent or cell population is administered to a subject using peripheral systemic delivery via intravenous, intraperitoneal, or subcutaneous injection.

[0172] In some embodiments, the composition is provided as a sterile liquid preparation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which may be buffered to a selected pH in some aspects. Liquid preparations are usually easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions are somewhat easier to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within an appropriate viscosity range to provide a longer contact period with specific tissues. Liquid or viscous compositions can include, for example, a solvent or dispersion medium carrier, including water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0173] Sterile injectable solutions can be prepared by incorporating the cells in a solvent such as sterile water, saline, glucose, dextrose, or the like, mixed with a suitable carrier, diluent, or excipient.

[0174] Formulations to be used for in vivo administration are generally sterile. Sterility is readily accomplished, for example, by filtration through sterile filtration membranes. 2. Medication

[0175] In some embodiments, a dose of cells is administered to a subject according to the provided methods and / or using the provided articles of manufacture or compositions. In some embodiments, the size or timing of the dose is determined as a function of the specific disease or condition (e.g., cancer, e.g., B-cell malignancy) in the subject. In some cases, the size or timing of the dose for a specific disease can be empirically determined in light of the provided descriptions.

[0176] In some embodiments, the dose of cells is 4×10 5 or approximately 4 x 10 5 cells / kg ~ 1 x 10 6 or approximately 1 x 10 6 Between 6 x 10 cells / kg or 6 x 10 5 pieces or approximately 6 x 10 5 cells / kg ~ 8 x 10 5 pieces or approximately 8 x 10 5 between 2 x 10 cells / kg, etc. 5 pieces or approximately 2 x 10 5 cells / kg ~ 2 x 10 6 pieces or approximately 2 x 10 6 In some embodiments, the dose of cells comprises between 3 x 10 cells / kg. 5 cells / kg or less or approximately 3 x 10 5 Cells / kg or less, 4×10 5 cells / kg or less or approximately 4 x 10 5 Cells / kg or less, 5×10 5 cells / kg or less or approximately 5 x 10 5Cells / kg or less, 6×10 5 cells / kg or less or approximately 6 x 10 5 Cells / kg or less, 7×10 5 cells / kg or less or approximately 7 x 10 5 Cells / kg or less, 8×10 5 cells / kg or less or approximately 8 x 10 5 Cells / kg or less, 9×10 5 cells / kg or less or approximately 9 x 10 5 Cells / kg or less, 1×10 6 cells / kg or less or approximately 1 x 10 6 cells / kg or less or 2 x 10 6 cells / kg or less or approximately 2 x 10 6 cells / kg or less, e.g., 2 x 10 cells / kg of subject body weight 5 In some embodiments, the dose of cells comprises at least 3 x 10 cells (e.g., antigen-expressing, e.g., CAR-expressing cells) (cells / kg). 5 cells / kg or at least about 3 x 10 5 Cells / kg or its approximation, at least 4 x 10 5 cells / kg or at least about 4 x 10 5 Cells / kg or its approximation, at least 5 x 10 5 cells / kg or at least about 5 x 10 5 At least 6 x 10 cells / kg or its approximation 5 cells / kg or at least about 6 x 10 5 At least 7 x 10 cells / kg or its approximation 5 cells / kg or at least about 7 x 10 5 Cells / kg or its approximation, at least 8 x 10 5 cells / kg or at least about 8 x 10 5 Cells / kg or its approximation, at least 9 x 10 5 cells / kg or at least about 9 x 10 5 Cells / kg or its approximation, at least 1 x 10 6 cells / kg or at least about 1 x 10 6cells / kg or its approximation, or at least 2 x 10 6 cells / kg or at least about 2 x 10 6 At least 2 x 10 cells / kg or its approximation per kilogram of subject body weight 5 or at least about 2×10 5 or an approximate number of cells (e.g., antigen-expressing, e.g., CAR-expressing cells) (cells / kg).

[0177] In certain embodiments, the cells, or individual populations or subtypes of cells, are in the range of about 1 million to about 100 billion cells, and / or that amount of cells per kilogram of body weight, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, or about 1 million cells). The subject may be administered about 100 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases, about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), or any value therebetween, and / or per kilogram of body weight. Dosages may vary depending on the specific characteristics of the disease or disorder and / or patient and / or other treatments.

[0178] In some embodiments, the dose of cells is a flat dose of cells or a fixed dose of cells such that the dose of cells is not related to or based on the body surface area or weight of the subject.

[0179] In some embodiments, for example, when the subject is a human, the dose is about 5×10 8 Fewer than 10 total recombinant receptor (e.g., CAR)-expressing cells, T cells or peripheral blood mononuclear cells (PBMCs), e.g., about 1 x 10 6 ~5×10 8 In the range of 2 x 10 such cells, e.g., 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 2 × 10 8 , 3×10 8 Or 4 x 10 8 In some embodiments, when the subject is a human, the dose is about 1 x 10 such cells. 6 ~3×10 8 Between about 1 x 10 total recombinant receptor (e.g., CAR)-expressing cells, e.g., about 1 x 10 7 ~2×10 8 in the range of 1 x 10 such cells, e.g., 7 , 5×10 7 , 1×10 8 Or 1.5 x 10 8 In some embodiments, the patient is administered multiple doses, and each or the total dose can be within any of the values ​​set forth above. In some embodiments, the dose of cells is 1 x 10 or more. 5 or approximately 1 x 10 5 pieces~5×10 8 pieces or approximately 5 x 10 8 1 x 10 total recombinant receptor (e.g., CAR)-expressing T cells or total T cells 5 ~1×10 8 5 x 10 total recombinant receptor (e.g., CAR)-expressing T cells or total T cells 5 pieces or approximately 5 x 105 pieces~1×10 7 or approximately 1 x 10 7 1 x 10 total recombinant receptor (e.g., CAR)-expressing T cells or total T cells, or 1 x 10 6 or approximately 1 x 10 6 pieces~1×10 7 or approximately 1 x 10 7 This includes administering 100 total recombinant receptor (e.g., CAR)-expressing T cells or total T cells (each including upper and lower limits).

[0180] In some embodiments, the dose of T cells comprises CD4+ T cells, CD8+ T cells, or CD4+ and CD8+ T cells.

[0181] In some embodiments, for example, when the subject is a human, the dose of CD8+ T cells, including in a dose comprising CD4+ and CD8+ T cells, is about 1 x 10 6 ~1×10 8 Between about 5 x 10 total recombinant receptor (e.g., CAR)-expressing CD8+ cells, for example, about 5 x 10 6 ~1×10 8 such cells in the range of 1 x 10 7 , 2.5×10 7 , 5×10 7 , 7.5×10 7 Or 1 x 10 8 In some embodiments, the patient is administered multiple doses, and each or the total dose can fall within any of the values ​​set forth above. In some embodiments, the dose of cells is 1 x 10 or more. 7 ~0.75×10 8 1 x 10 total recombinant receptor-expressing CD8+ T cells or their approximate number 7 ~2.5×10 7 1 x 10 total recombinant receptor-expressing CD8+ T cells 7 ~0.75×10 8 In some embodiments, the dose of cells comprises administering 1 x 10 total recombinant receptor-expressing CD8+ T cells or approximately 1 x 10 total recombinant receptor-expressing CD8+ T cells (each inclusive). 7 , 2.5×107 , 5×10 7 , 7.5×10 7 Or 1 x 10 8 The method includes administering a total number of recombinant receptor-expressing CD8+ T cells.

[0182] In some embodiments, for example, when the subject is a human, the dose of CD4+ T cells, including in the dose comprising CD4+ and CD8+ T cells, is about 1 x 10 6 ~1×10 8 Between 10 and 20 total recombinant receptor (e.g., CAR)-expressing CD4+ cells, e.g., about 5 x 10 6 ~1×10 8 number of such cells, in the range of 1 x 10 such cells 7 , 2.5×10 7 , 5×10 7 , 7.5×10 7 Or 1 x 10 8 In some embodiments, the patient is administered multiple doses, and each or the total dose can be within any of the values ​​set forth above. In some embodiments, the dose of cells is 1 x 10 or more. 7 pieces or approximately 1 x 10 7 pieces~0.75×10 8 pieces or approximately 0.75 x 10 8 1 x 10 total recombinant receptor-expressing CD4+ T cells 7 ~2.5×10 7 1 x 10 total recombinant receptor-expressing CD4+ T cells 7 pieces or approximately 1 x 10 7 pieces~0.75×10 8 pieces or approximately 0.75 x 10 8 In some embodiments, the dose of cells comprises administering 1 x 10 total recombinant receptor-expressing CD4+ T cells (each inclusive). 7 , 2.5×10 7 , 5×10 7 , 7.5×10 7 Or 1 x 10 8 The method includes administering a total number of recombinant receptor-expressing CD4+ T cells.

[0183] In some embodiments, the dose of cells, e.g., recombinant receptor-expressing T cells, is administered to the subject as a single dose, or only once within a period of two weeks, one month, three months, six months, one year or more.

[0184] In the context of adoptive cell therapy, administration of a given "dose" encompasses administration of a given amount or number of cells as a single composition and / or a single uninterrupted administration, e.g., as a single injection or continuous infusion, as well as administration of a given amount or number of cells as divided doses or as multiple compositions provided in multiple individual compositions or infusions over a specified period of time, such as three days or less. Thus, in some situations, a dose is a single or continuous administration of a specified number of cells given or initiated at a single time point. However, in some situations, a dose is administered in multiple injections or infusions over a period of three days or less, such as once daily for three or two days, or by multiple infusions over a one-day period.

[0185] Thus, in some aspects, the dose of cells is administered in a single pharmaceutical composition, hi some embodiments, the dose of cells is administered in multiple compositions that collectively contain the dose of cells.

[0186] In some embodiments, the term "split dose" refers to a dose that is divided so as to be administered over more than one day. This type of administration is encompassed by the present method and is considered to be a single dose.

[0187] Thus, the dose of cells can be administered as split doses, e.g., split doses administered over time. For example, in some embodiments, the dose can be administered to a subject over two or three days. An exemplary method for split dosing includes administering 25% of the dose on day 1 and the remaining 75% of the dose on day 2. In other embodiments, 33% of the dose can be administered on day 1 and the remaining 67% on day 2. In some aspects, 10% of the dose is administered on day 1, 30% of the dose is administered on day 2, and 60% of the dose is administered on day 3. In some embodiments, the split doses are not spread out over more than three days.

[0188] In some embodiments, a dose of cells can be administered by administering multiple compositions or solutions, such as a first and a second, optionally more, each containing a portion of the dose of cells. In some aspects, multiple compositions each containing different populations and / or subtypes of cells are administered separately or independently, optionally within a certain period of time. For example, the populations or subtypes of cells can each be CD8 + and CD4 + The compositions can include CD8+ T cells, and / or enriched populations of CD8+ and CD4+ T cells, respectively, e.g., CD4+ and / or CD8+ T cells, each of which individually comprises cells genetically engineered to express a recombinant receptor. In some embodiments, administering the doses includes administering a first composition comprising a dose of CD8+ T cells or a dose of CD4+ T cells, and administering a second composition comprising the other of the doses of CD4+ T cells and CD8+ T cells.

[0189] In some embodiments, administration of a composition or dose, e.g., administration of multiple cell compositions, involves administration of separate cell compositions. In some aspects, the separate administrations occur simultaneously or sequentially, in any order. In some embodiments, a dose comprises a first composition and a second composition, and the first composition and the second composition are administered 0-12 hours apart, 0-6 hours apart, or 0-2 hours apart. In some embodiments, the initiation of administration of the first composition and the initiation of administration of the second composition occur no more than 2 hours, no more than 1 hour, or no more than 30 minutes apart, and no more than 15 minutes, no more than 10 minutes, or no more than 5 minutes apart. In some embodiments, the initiation and / or completion of administration of the first composition and the completion and / or initiation of administration of the second composition occur no more than 2 hours, no more than 1 hour, or no more than 30 minutes apart, and no more than 15 minutes, no more than 10 minutes, or no more than 5 minutes apart.

[0190] In some embodiments, the first composition and the second composition are mixed before being administered to a subject.In some embodiments, the first composition and the second composition are mixed shortly (for example, within 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1.5 hours, 1 hour or 0.5 hours) before being administered.In some embodiments, the first composition and the second composition are mixed just before being administered.

[0191] In some compositions, the first composition, for example, the first composition of a dose, comprises CD4+ T cells. In some compositions, the first composition, for example, the first composition of a dose, comprises CD8+ T cells. In some embodiments, the first composition is administered before the second composition.

[0192] In some embodiments, the cell dose or composition comprises a defined or target ratio of CD4+ cells expressing the recombinant receptor to CD8+ cells expressing the recombinant receptor, and / or CD4+ cells to CD8+ cells, optionally about 1:1 or between about 1:3 and about 3:1, e.g., about 1:1. In some aspects, administering a composition or dose at a target or desired ratio of different cell populations (such as a CD4+:CD8+ ratio or a CAR+CD4+:CAR+CD8+ ratio, e.g., 1:1) involves administering a cell composition containing one of the populations, followed by a separate cell composition comprising the other of the populations, the administrations being at or about the target or desired ratio. In some aspects, administering a cell dose or composition at a defined ratio results in improved expansion, persistence, and / or anti-tumor activity of T cell therapy.

[0193] In some embodiments, the subject receives multiple doses of cells, for example, two or more doses or multiple consecutive doses. In some embodiments, two doses are administered to the subject. In some embodiments, the subject receives consecutive doses, for example, the second dose, approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days after the first dose. In some embodiments, multiple consecutive doses are administered after the first dose, such that the additional dose is administered after the administration of the consecutive dose. In some aspects, the number of cells administered to the subject in the additional dose is the same or similar to that of the first dose and / or the consecutive dose. In some embodiments, the additional dose is greater than the previous dose.

[0194] In some embodiments, the size of the first and / or subsequent doses is determined based on one or more criteria, such as the subject's response to previous treatment, e.g., chemotherapy; the disease burden in the subject, e.g., tumor burden, bulk, size or extent, extent or type of metastasis, stage; and / or the likelihood or incidence of the subject developing a toxicity outcome, e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and / or the host immune response to the administered cells and / or recombinant receptor.

[0195] In some embodiments, the time between the administration of the first dose and the administration of the subsequent dose is about 9 to about 35 days, about 14 to about 28 days, or 15 to 27 days. In some embodiments, the administration of the subsequent dose occurs more than about 14 days but less than about 28 days after the administration of the first dose. In some embodiments, the time between the first and subsequent doses is about 21 days. In some embodiments, an additional dose, e.g., a subsequent dose, is administered after the administration of the subsequent dose. In some embodiments, the additional subsequent dose is administered at least about 14 days but less than about 28 days after the administration of the previous dose. In some embodiments, the additional dose is administered less than about 14 days after the previous dose, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 days after the previous dose. In some embodiments, a dose is not administered less than about 14 days after the previous dose, and / or a dose is not administered more than about 28 days after the previous dose.

[0196] In some embodiments, the dose of cells, e.g., recombinant receptor-expressing cells, comprises two doses (e.g., a double dose) comprising a first dose of T cells and sequential doses of T cells, wherein one or both of the first and second doses comprises administration of split doses of T cells.

[0197] In some embodiments, the dose of cells is generally large enough to be effective in reducing disease burden.

[0198] In some embodiments, cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell types and / or a desired ratio of cell types. Thus, the dosage of cells is, in some embodiments, based on the total number of cells (or number per kg body weight) and the desired ratio of individual populations or subtypes, for example, the ratio of CD4+ to CD8+. In some embodiments, the dosage of cells is based on the desired total number (or number per kg body weight) of cells in each population or of each cell type. In some embodiments, the dosage is based on a combination of such features, such as the desired number of total cells, the desired ratio, and the desired total number of cells in each population.

[0199] In some embodiments, CD8 + and CD4 + A population or subtype of cells, such as T cells, is administered at a desired dose of total cells, such as a desired dose of T cells, or within an acceptable difference. In some embodiments, the desired dose is a desired number of cells, or a desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells / kg. In some embodiments, the desired dose is at or exceeds a minimum number of cells or a minimum number of cells per unit of body weight. In some embodiments, among the total cells administered at a desired dose, individual populations or subtypes are administered at a desired output ratio (e.g., CD4 + Against CD8 + ) within a certain allowable difference or error in such ratio.

[0200] In some embodiments, cells are administered at a desired dose of one or more of the individual populations or subtypes of cells, such as a desired dose of CD4+ cells and / or a desired dose of CD8+ cells, or within an acceptable difference. In some aspects, the desired dose is the desired number of cells of a subtype or population, or the desired number of such cells per unit of body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is at or exceeds the minimum number of cells of a population or subtype, or the minimum number of cells of a population or subtype per unit of body weight.

[0201] Thus, in some embodiments, dosage is based on a desired fixed dose and desired ratio of total cells and / or based on a desired fixed dose of one or more, e.g., each, of individual subtypes or subpopulations. Thus, in some embodiments, dosage is based on a desired fixed or minimum dose of T cells and CD4 + Against CD8 + Based on the desired ratio of cells and / or CD4 + and / or CD8 + Based on desired fixation or minimum dose of cells.

[0202] In some embodiments, cells are administered at a desired output ratio or within an acceptable range of multiple cell populations or subtypes, such as CD4+ and CD8+ cells or subtypes. In some aspects, the desired ratio can be a specific ratio or a range of ratios. For example, in some embodiments, the desired ratio (e.g., CD4 + Against CD8 + The ratio of cells is between 5:1 or about to 5:1 or about to (or greater than about 1:5 and less than about 5:1), or between 1:3 or about to 3:1 or about to (or greater than about 1:3 and less than about 3:1), for example, between 2:1 or about to 1:5 or about to (or greater than about 1:5 and less than about 2:1), for example, 5:1, 4.5:1, 4:1, 3.5:1, 3:1 , 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5 or an approximation thereof. In some embodiments, acceptable differences are within about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value in between these ranges.

[0203] In certain embodiments, the number and / or concentration of cells refers to the number of recombinant receptor (e.g., CAR)-expressing cells. In other embodiments, the number and / or concentration of cells refers to the number or concentration of administered total cells, T cells, or peripheral blood mononuclear cells (PBMCs).

[0204] In some embodiments, the size of the dose is determined based on one or more criteria such as the subject's response to previous treatment, e.g., chemotherapy; the disease burden in the subject, e.g., tumor burden, volume, size, or extent; the extent or type, stage of metastasis; and / or the likelihood or incidence that the subject will develop a toxic outcome, e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and / or a host immune response, to the cells and / or recombinant receptor being administered.

[0205] In some embodiments, the method also includes administering one or more additional doses of cells expressing a chimeric antigen receptor (CAR) and / or lymphodepletion therapy, and / or one or more steps of the method are repeated. In some embodiments, the one or more additional doses are the same as the initial dose. In some embodiments, the one or more additional doses are different from the initial dose, e.g., higher than the initial dose, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more higher, or lower than the initial dose, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or less. In some embodiments, the administration of one or more additional doses is determined based on the subject's response to the initial treatment or any previous treatments, the disease burden in the subject, e.g., tumor burden, bulk, size or extent, extent or type of metastasis, stage, and / or the likelihood or incidence of the subject developing a toxicity outcome, e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and / or the host immune response to the administered cells and / or recombinant receptor.

[0206] B. Administration of Compound C In some embodiments of the methods, compositions, combinations, kits, or articles of manufacture provided herein, the combination therapy has the chemical name (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione and / or has Formula I: [ka] (Formula I) or an enantiomer or a mixture of enantiomers thereof, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate or polymorph thereof.

[0207] In some embodiments, compound C is an enantiomer or a mixture of enantiomers of (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph of (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione. In some embodiments, compound C is a solvate of (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione. In some embodiments, compound C is a hydrate of (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione. In some embodiments, compound C is a pharmaceutically acceptable salt of (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione. In some embodiments, compound C is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione. In some embodiments, compound C has the structure of formula I:

[0208] In certain embodiments, Compound C is a solid. In certain embodiments, Compound C is hydrated. In certain embodiments, Compound C is solvated. In certain embodiments, Compound C is anhydrous. In certain embodiments, Compound C is non-hygroscopic.

[0209] In certain embodiments, Compound C is amorphous. In certain embodiments, Compound C is crystalline.

[0210] In some embodiments, Compound C is a pharmaceutically acceptable salt of (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione. As used herein, the term "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable non-toxic acid or base, including inorganic acids and bases and organic acids and bases. Suitable pharmaceutically acceptable base addition salts of Compound C include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc, or organic salts made from lysine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methyl-glucamine), and procaine. Suitable non-toxic acids include inorganic and organic acids such as acetic acid, alginic acid, anthranilic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, formic acid, fumaric acid, furoic acid, galacturonic acid, gluconic acid, glucuronic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, propionic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, sulfuric acid, tartaric acid, and p-toluenesulfonic acid. Others are well known in the art and are described, for example, in Remington's Pharmaceutical Sciences, 18 th eds., Mack Publishing, Easton PA (1990) or Remington: The Science and Practice of Pharmacy, 19 th eds., Mack Publishing, Easton PA (1995).

[0211] In certain embodiments, Compound C is the hydrochloride salt of (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or an enantiomer or a mixture of enantiomers thereof; or a pharmaceutically acceptable solvate, hydrate, co-crystal, clathrate, or polymorph thereof. In certain embodiments, the hydrochloride salt is a solid. In certain embodiments, the hydrochloride salt is anhydrous. In certain embodiments, the hydrochloride salt is non-hygroscopic. In certain embodiments, the hydrochloride salt is amorphous. In certain embodiments, the hydrochloride salt is crystalline.

[0212] As used herein, and unless otherwise indicated, the term "stereoisomer" or "stereomerically pure" means one stereoisomer of a compound that is substantially free of other stereoisomers of that compound. For example, a stereochemically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereochemically pure compound having two chiral centers will be substantially free of other diastereomers of the compound. A typical stereochemically pure compound contains more than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, more than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of other stereoisomers of the compound, more than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of other stereoisomers of the compound, or more than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of other stereoisomers of the compound. The compounds may have chiral centers and may occur as racemates, individual enantiomers or diastereomers, and mixtures thereof. Methods involving the administration of any such isomeric form of Compound C are included within the embodiments provided herein, including the administration of mixtures thereof.

[0213] In some embodiments, Compound C provided herein contains one chiral center and can exist as a mixture of enantiomers, e.g., a racemic mixture. The present disclosure encompasses the use of stereochemically pure forms of such compounds as well as mixtures of these forms. For example, mixtures containing equal or unequal amounts of the enantiomers of Compound C provided herein can be used in the methods and compositions disclosed herein. These isomers can be asymmetrically synthesized or resolved using standard methods, such as chiral columns or chiral resolving agents. See, for example, Jacques, J., et al., Enantiomers, Racemates and Resolutions (Wiley-Interscience, New York, 1981); Wilen, SH, et al., Tetrahedron 33:2725 (1977); Eliel, E. L., Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, SH, Tables of Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., University of Notre Dame Press, Notre Dame, IN, 1972).

[0214] It is understood that the chiral centers of Compound C can undergo epimerization in vivo. As such, one of skill in the art will recognize that, in the event of epimerization in vivo, administration of Compound C in its (R) form may be equivalent to administration of Compound C in its (S) form.

[0215] Optically active (+) and (−), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as chromatography on chiral stationary phases.

[0216] In some embodiments, Compound C is a solvate of (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione. The term "solvate" means a physical association of one or more solvent molecules with a compound, whether organic or inorganic. This physical association includes hydrogen bonding. In certain instances, the solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated in the crystal lattice of a crystalline solid. "Solvate" encompasses both solution-phase and insoluble solvates. Exemplary solvates include hydrates, ethanolate adducts, methanolate adducts, isopropanolate, acetonitrile solvates, and ethyl acetate solvates. In some embodiments, compound C is a hydrate of (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione. Methods of solvation are known in the art.

[0217] "Tautomer" refers to isomeric forms of a compound that are in equilibrium with each other. The concentration of isomeric forms depends on the environment in which the compound is found, and may vary depending on, for example, whether the compound is a solid or in an organic or aqueous solution. For example, in aqueous solution, pyrazole can exhibit the following isomeric forms, referred to as tautomers of each other: [ka]

[0218] As will be readily understood by one of ordinary skill in the art, a wide variety of functional groups and other structures can exhibit tautomerism, and administration of any tautomer of Compound C is within the scope of the methods provided herein.

[0219] It should also be noted that compound C used in the provided methods can contain unnatural proportions of atomic isotopes at one or more of its atoms. For example, compound C can contain, for example, tritium ( 3 H), iodine-125( 125 I), sulfur-35( 35 S) or carbon-14 ( 14 It can be radiolabeled with radioactive isotopes such as C, or deuterium ( 2 H), carbon-13( 13 C) or nitrogen-15( 15 The compound may be isotopically enriched with isotopic species such as deuterium, carbon-13 (N), etc. As used herein, an "isotopic species" is an isotopically enriched compound. The term "isotopically enriched" refers to an atom having an isotopic composition other than the natural isotopic composition of that atom. "Isotopically enriched" can also refer to a compound containing at least one atom having an isotopic composition other than the natural isotopic composition of that atom. The term "isotopic composition" refers to the amount of each isotope found in a given atom. Radiolabeled and isotopically enriched compounds are useful as therapeutic agents, e.g., cancer therapeutic agents, research reagents, e.g., binding assay reagents, and diagnostic agents, e.g., in vivo imaging agents. Methods involving the administration of any isotopic variant of Compound C, whether radioactive or not, are intended to be encompassed within the scope of the methods provided herein. In some embodiments, an isotopic species of Compound C, e.g., deuterium, carbon-13 (N), etc., is used. 13 C) and / or nitrogen-15( 15 Provided herein are methods involving the administration of a compound enriched with hydrogen (H). As used herein, "deuterated" refers to a compound in which at least one hydrogen (H) is replaced by a deuterium (D or N). 2 H), i.e., the compound is enriched with deuterium in at least one position.

[0220] It is understood that, regardless of stereochemistry or isotopic composition, Compound C can be administered in the form of any of the pharmaceutically acceptable salts described herein. At the same time, it is understood that the isotopic composition can vary independently from the stereochemical composition of Compound C. Furthermore, while the isotopic composition is limited to the elements present in Compound C or its salt, it can vary in other respects independently from the choice of pharmaceutically acceptable salt of Compound C.

[0221] It should be noted that if there is a discrepancy between a depicted structure and the name given to that structure, the depicted structure should be given more weight. Additionally, if the stereochemistry of a structure or portion of a structure is not indicated, for example, with bold or dashed lines, the structure or portion of a structure should be interpreted as encompassing all stereoisomers of the structure.

[0222] 1. Compositions and Formulations In some embodiments of the combination therapy methods, compositions, combinations, kits and uses provided herein, the combination therapy can be administered in one or more compositions, e.g., pharmaceutical compositions, containing Compound C.

[0223] In some embodiments, compositions containing Compound C, e.g., pharmaceutical compositions, can include a carrier, such as a diluent, adjuvant, excipient, or vehicle, with which Compound C and / or cells are administered. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of Compound C, generally in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to a patient. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. The pharmaceutical composition may contain any one or more of a diluent, adjuvant, antiadherent, binder, coating, filler, flavoring, coloring, lubricant, glidant, preservative, detergent, adsorbent, emulsifier, pharmaceutical excipient, pH buffer) or sweetener, and combinations thereof. In some embodiments, the pharmaceutical composition may be in liquid, solid, lyophilized powder, gel form, and / or combinations thereof. In some aspects, the choice of carrier is determined in part by the particular inhibitor and / or method of administration.

[0224] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin; The compound C may be a protein such as albumin, gelatin, or immunoglobulin; a hydrophilic polymer such as polyvinylpyrrolidone; an amino acid such as glycine, glutamine, asparagine, histidine, arginine, or lysine; a monosaccharide, disaccharide, and other carbohydrate, including glucose, mannose, or dextrin; a chelating agent such as EDTA; a sugar such as sucrose, mannitol, trehalose, or sorbitol; a salt-forming counterion such as sodium; a metal complex (e.g., a Zn-protein complex); and / or a non-ionic surfactant, stabilizer, and / or preservative, such as polyethylene glycol (PEG). The composition containing Compound C may be lyophilized.

[0225] In some embodiments, the pharmaceutical composition can be formulated for administration by any route known to those skilled in the art, including intramuscular, intravenous, intradermal, intralesional, intraperitoneal injection, subcutaneous, intratumoral, epidural, nasal, oral, vaginal, rectal, topical, local, otic, inhalation, buccal (e.g., sublingual), and transdermal administration, or any route. In some embodiments, other modes of administration are also contemplated. In some embodiments, administration is by bolus injection, by injection, for example, intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjunctival injection, sub-Tenon injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, administration is parenteral, intrapulmonary, and intranasal, and, if desired for localized treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus administration. In some embodiments, it is administered by multiple bolus administrations or continuous infusion administration, for example, over a period of 3 days or less.

[0226] In some embodiments, administration can be local, topical, or systemic depending on the location of treatment. In some embodiments, local administration to the area requiring treatment can be achieved by, for example, but not limited to, local infusion during surgery, topical application, for example, in conjunction with wound dressing after surgery, injection using a catheter, a suppository, or an implant. In some embodiments, the composition can also be administered with other bioactive agents, either sequentially, intermittently, or in the same composition. In some embodiments, administration can also include controlled release systems, including controlled release formulations and device-controlled release, such as with pumps. In some embodiments, administration is oral.

[0227] In some embodiments, Compound C is typically formulated and administered in unit or multiple dosage forms. Each unit dosage contains a predetermined amount of therapeutically active Compound C sufficient to produce the desired therapeutic effect in association with the required pharmaceutical carrier, vehicle, or diluent. In some embodiments, unit dosage forms include, but are not limited to, tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, oral solutions or suspensions, and oil-and-water emulsions containing an appropriate amount of Compound C. Unit dosage forms may be contained in ampoules and syringes or individually packaged tablets or capsules. Unit dosage forms may be administered in fractions or multiples thereof. In some embodiments, multiple dosage forms are multiple identical unit dosage forms packaged within a single container to be administered in segregated unit dosage forms. Examples of multiple dosage forms include vials, bottles of tablets or capsules, or bottles of pints or gallons.

[0228] 2. Medication In some embodiments, the provided combination therapy methods involve initiating administration of Compound C before, after, during, during the course of, simultaneously with, nearly simultaneously with, sequentially with, simultaneously with, and / or intermittently with, initiation of cell therapy, such as T cell therapy (e.g., CAR-expressing T cells). In some embodiments, initiation of administration of Compound C in the provided combination therapy methods occurs after or subsequent to the initiation of administration of T cell therapy.

[0229] In some embodiments, administration of Compound C is initiated after (following) the initiation of cell therapy, such as T cell therapy (e.g., CAR-expressing T cells). In some embodiments, administration of Compound C is initiated at or before peak or maximum levels of cells of the T cell therapy are detectable in the subject's blood.

[0230] In some cases, initiation of administration of Compound C occurs (i) at a time when the peak or maximum level of T cell therapy cells is detectable in the subject's blood; (ii) after becoming detectable in the blood, the number of T cell therapy cells detectable in the blood becomes undetectable or is reduced, optionally reduced compared to an earlier time point after administration of the T cell therapy; (iii) the number of T cell therapy cells detectable in the blood is reduced by 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 10-fold or more than the peak or maximum number of T cell therapy cells detectable in the subject's blood after initiation of administration of the T cell therapy; (iv) at a time after the peak or maximum level of cells of the T cell therapy are detectable in the subject's blood, the number of cells detectable in or derived therefrom in the blood from the subject is less than 10%, less than 5%, less than 1%, or less than 0.1% of the total peripheral blood mononuclear cells (PBMCs) in the subject's blood; (v) the subject exhibits disease progression and / or relapse following remission after treatment with T cell therapy; and / or (iv) is performed at or within one week prior, such as within one, two, or three days prior, the subject exhibits an increased tumor burden compared to the tumor burden prior to or after administration of the cells and prior to the initiation of administration of Compound C. In certain embodiments, the methods provided are performed to enhance, increase, or potentiate T cell therapy in a subject to improve response to T cell therapy, e.g., to reduce the presence of T cells and / or tumor burden.

[0231] In some embodiments, the cell therapy, such as a T cell therapy (e.g., a CAR-expressing T cell), is administered on day 1 of the combination therapy.

[0232] In some embodiments, administration of Compound C is initiated after (or subsequent to) the initiation of cell therapy. In some embodiments, administration of Compound C is initiated after (or subsequent to) the initiation of T cell therapy and on the same day as the initiation of T cell therapy (i.e., on day 1 of combination therapy). In some embodiments, administration of Compound C is initiated between days 1 and 29, inclusive, of combination therapy. In some embodiments, administration of Compound C is initiated between days 1 and 22, inclusive, of combination therapy. In some embodiments, administration of Compound C is initiated between days 1 and 15, inclusive, of combination therapy. In some embodiments, administration of Compound C is initiated between days 2 and 15, inclusive, of combination therapy. In some embodiments, administration of Compound C is initiated between days 3 and 15, inclusive, of combination therapy. In some embodiments, administration of Compound C is initiated between days 4 and 15, inclusive, of combination therapy. In some embodiments, administration of Compound C begins between days 5 and 15 (inclusive) of combination therapy. In some embodiments, administration of Compound C begins between days 6 and 15 (inclusive) of combination therapy. In some embodiments, administration of Compound C begins between days 7 and 15 (inclusive) of combination therapy. In some embodiments, administration of Compound C begins between days 8 and 15 (inclusive) of combination therapy.

[0233] In some embodiments, administration of Compound C begins on or about day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 of the combination therapy. In some embodiments, administration of Compound C begins on or about day 1 of the combination therapy. In some embodiments, administration of Compound C begins on or about day 2 of the combination therapy. In some embodiments, administration of Compound C begins on or about day 3 of the combination therapy. In some embodiments, administration of Compound C begins on or about day 4 of the combination therapy. In some embodiments, administration of Compound C begins on or about day 5 of the combination therapy. In some embodiments, administration of Compound C begins on or about day 5 of the combination therapy. In some embodiments, administration of Compound C begins on or about day 7 of combination therapy. In some embodiments, administration of Compound C begins on or about day 8 of combination therapy. In some embodiments, administration of Compound C begins on or about day 9 of combination therapy. In some embodiments, administration of Compound C begins on or about day 10 of combination therapy. In some embodiments, administration of Compound C begins on or about day 11 of combination therapy. In some embodiments, administration of Compound C begins on or about day 12 of combination therapy. In some embodiments, administration of Compound C begins on or about day 13 of combination therapy. In some embodiments, administration of Compound C begins on or about day 14 of combination therapy. In some embodiments, administration of Compound C begins on or about day 15 of combination therapy. In some embodiments, administration of Compound C begins on or about day 16 of combination therapy. In some embodiments, administration of Compound C begins on or about day 17 of combination therapy. In some embodiments, administration of Compound C begins on or about day 18 of combination therapy. In some embodiments, administration of Compound C begins on or about day 19 of combination therapy. In some embodiments, administration of Compound C begins on or about day 20 of combination therapy.In some embodiments, administration of Compound C begins on or about day 21 of combination therapy. In some embodiments, administration of Compound C begins on or about day 22 of combination therapy.

[0234] In some embodiments, administration of Compound C is initiated prior to administration of cell therapy, e.g., T cell therapy. In some embodiments, administration of Compound C is initiated within one month prior to administration of cell therapy. In some embodiments, administration of Compound C is initiated within four weeks prior to administration of cell therapy. In some embodiments, administration of Compound C is initiated within three weeks prior to administration of cell therapy. In some embodiments, administration of Compound C is initiated within two weeks prior to administration of cell therapy. In some embodiments, administration of Compound C is initiated within one week prior to administration of cell therapy. In some embodiments, administration of Compound C is initiated within six days prior to administration of cell therapy. In some embodiments, administration of Compound C is initiated within five days prior to administration of cell therapy. In some embodiments, administration of Compound C is initiated within four days prior to administration of cell therapy. In some embodiments, administration of Compound C is initiated within three days prior to administration of cell therapy. In some embodiments, administration of Compound C is initiated within two days prior to administration of cell therapy. In some embodiments, administration of Compound C begins within one day prior to administration of the cell therapy.

[0235] In some embodiments, administration of Compound C is initiated one month prior to administration of cell therapy. In some embodiments, administration of Compound C is initiated four weeks prior to administration of cell therapy. In some embodiments, administration of Compound C is initiated three weeks prior to administration of cell therapy. In some embodiments, administration of Compound C is initiated two weeks prior to administration of cell therapy. In some embodiments, administration of Compound C is initiated one week prior to administration of cell therapy. In some embodiments, administration of Compound C is initiated six days prior to administration of cell therapy. In some embodiments, administration of Compound C is initiated five days prior to administration of cell therapy. In some embodiments, administration of Compound C is initiated four days prior to administration of cell therapy. In some embodiments, administration of Compound C is initiated three days prior to administration of cell therapy. In some embodiments, administration of Compound C is initiated two days prior to administration of cell therapy. In some embodiments, administration of Compound C is initiated one day prior to administration of cell therapy.

[0236] In some embodiments, administration of compound C is initiated after administration of lymphodepleting therapy and before administration of cell therapy. In some embodiments, cell therapy is administered 2-7 days after the end of administration of lymphodepleting therapy. In some embodiments, administration of compound C is initiated on the last day of lymphodepleting therapy. In some embodiments, administration of compound C is initiated within 7 days after the end of administration of lymphodepleting therapy. In some embodiments, administration of compound C is initiated within 6 days after the end of administration of lymphodepleting therapy. In some embodiments, administration of compound C is initiated within 5 days after the end of administration of lymphodepleting therapy. In some embodiments, administration of compound C is initiated within 4 days after the end of administration of lymphodepleting therapy. In some embodiments, administration of compound C is initiated within 3 days after the end of administration of lymphodepleting therapy. In some embodiments, administration of compound C is initiated within 2 days after the end of administration of lymphodepleting therapy. In some embodiments, administration of Compound C begins within one day after the end of administration of lymphodepleting therapy.

[0237] In some embodiments, administration of Compound C begins 7 days after the end of administration of lymphodepleting therapy. In some embodiments, administration of Compound C begins 6 days after the end of administration of lymphodepleting therapy. In some embodiments, administration of Compound C begins 5 days after the end of administration of lymphodepleting therapy. In some embodiments, administration of Compound C begins 4 days after the end of administration of lymphodepleting therapy. In some embodiments, administration of Compound C begins 3 days after the end of administration of lymphodepleting therapy. In some embodiments, administration of Compound C begins 2 days after the end of administration of lymphodepleting therapy. In some embodiments, administration of Compound C begins 1 day after the end of administration of lymphodepleting therapy.

[0238] In some embodiments, at the time the subject is first administered Compound C and / or at any subsequent time point after initiation of administration, the subject does not exhibit signs or symptoms of severe toxicity, such as severe cytokine release syndrome (CRS) or severe toxicity. In some embodiments, Compound C is administered at a time when the subject does not exhibit signs or symptoms of severe CRS and / or does not exhibit CRS of Grade 3 or higher, such as prolonged Grade 3 CRS or Grade 4 or 5 CRS. In some embodiments, Compound C is administered at a time when the subject does not exhibit signs or symptoms of severe neurotoxicity and / or does not exhibit neurotoxicity of Grade 3 or higher, such as prolonged Grade 3 neurotoxicity or Grade 4 or 5 neurotoxicity. In some aspects, between the time of initiation of administration of T cell therapy and the time of administration of Compound C, the subject does not exhibit severe CRS and / or does not exhibit CRS of Grade 3 or higher, such as prolonged Grade 3 CRS or Grade 4 or 5 CRS. In some instances, between the time of initiation of administration of the T cell therapy and the time of administration of Compound C, the subject does not exhibit severe neurotoxicity and / or does not exhibit Grade 3 or higher neurotoxicity, such as prolonged Grade 3 neurotoxicity or Grade 4 or 5 neurotoxicity.

[0239] In some embodiments, Compound C is administered in an intermittent (i.e., not daily) dosing regimen. In some embodiments, Compound C is administered as multiple intermittent doses. In some embodiments, each dose of the multiple intermittent doses of Compound C is the same. In other embodiments, the multiple intermittent doses of Compound C can be of different amounts.

[0240] In some embodiments, the dose of Compound C is between 0.1 mg or about 0.1 mg and 1.0 mg or about 1.0 mg, for example, between 0.1 mg or about 0.1 mg and 0.9 mg or about 0.9 mg, between 0.1 mg or about 0.1 mg and 0.8 mg or about 0.8 mg, between 0.1 mg or about 0.1 mg and 0.7 mg or about 0.7 mg, between 0.1 mg or about 0.1 mg and 0.6 mg or about 0.6 mg, between 0.1 mg or about 0.1 mg and 0.5 mg or about 0.5 mg, between 0.1 mg or about 0.1 mg and 0.4 mg or between about 0.4 mg, 0.1 mg or between about 0.1 mg and 0.3 mg or about 0.3 mg, 0.1 mg or between about 0.1 mg and 0.2 mg or about 0.2 mg, 0.2 mg or between about 0.2 mg and 1.0 mg or about 1.0 mg, 0.2 mg or between about 0.2 mg and 0.9 mg or about 0.9 mg, 0.2 mg or between about 0.2 mg and 0.8 mg or about 0.8 mg, 0.2 mg or between about 0.2 mg and 0.7 mg or about 0.7 mg, 0.2 mg or about 0.2 mg and 0.6 mg or about 0.6 mg between 0.2 mg or about 0.2 mg to 0.5 mg or about 0.5 mg, between 0.2 mg or about 0.2 mg to 0.4 mg or about 0.4 mg, between 0.2 mg or about 0.2 mg to 0.3 mg or about 0.3 mg, between 0.3 mg or about 0.3 mg to 1.0 mg or about 1.0 mg, between 0.3 mg or about 0.3 mg to 0.9 mg or about 0.9 mg, between 0.3 mg or about 0.3 mg to 0.8 mg or about 0.8 mg, between 0.3 mg or about 0.3 mg to 0.7 mg or about 0.7 mg, or between about 0.3 mg and 0.6 mg or about 0.6 mg, 0.3 mg or between about 0.3 mg and 0.5 mg or about 0.5 mg, 0.3 mg or between about 0.3 mg and 0.4 mg or about 0.4 mg, 0.4 mg or between about 0.4 mg and 1.0 mg or about 1.0 mg, 0.4 mg or between about 0.4 mg and 0.9 mg or about 0.9 mg, 0.4 mg or between about 0.4 mg and 0.8 mg or about 0.8 mg, 0.4 mg or between about 0.4 mg and 0.7 mg or about 0.7 mg, 0.4 mg or between about 0.4 mg and 0.Between 6 mg or about 0.6 mg, between 0.4 mg or about 0.4 mg to 0.5 mg or about 0.5 mg, between 0.5 mg or about 0.5 mg to 1.0 mg or about 1.0 mg, between 0.5 mg or about 0.5 mg to 0.9 mg or about 0.9 mg, between 0.5 mg or about 0.5 mg to 0.8 mg or about 0.8 mg, between 0.5 mg or about 0.5 mg to 0.7 mg or about 0.7 mg, between 0.5 mg or about 0.5 mg to 0.6 mg or about 0.6 mg, between 0.6 mg or about 0.6 mg to 1.0 mg or about 1.0 mg, between 0.6 mg or about 0.6 mg to 0.9 mg or about 0.9 mg, or about 0.6 mg to 0.8 mg or about 0.8 mg, 0.6 mg or about 0.6 mg to 0.7 mg or about 0.7 mg, 0.7 mg or about 0.7 mg to 1.0 mg or about 1.0 mg, 0.7 mg or about 0.7 mg to 0.9 mg or about 0.9 mg, 0.7 mg or about 0.7 mg to 0.8 mg or about 0.8 mg, 0.8 mg or about 0.8 mg to 1.0 mg or about 1.0 mg, 0.8 mg or about 0.8 mg to 0.9 mg or about 0.9 mg, or 0.8 mg or about 0.8 mg to 1.0 mg or about 1.0 mg (each inclusive). In some embodiments, the dose of Compound C is between 0.1 mg or about 0.1 mg and 0.6 mg or about 0.6 mg. In some embodiments, the dose of Compound C is between or about 0.1 mg and 0.5 mg or about 0.5 mg. In some embodiments, the dose of Compound C is between or about 0.1 mg and 0.4 mg or about 0.4 mg. In some embodiments, the dose of Compound C is between or about 0.1 mg and 0.3 mg or about 0.3 mg. In some embodiments, the dose of Compound C is 0.1 mg or about 0.1 mg. In some embodiments, the dose of Compound C is 0.2 mg or about 0.2 mg. In some embodiments, the dose of Compound C is 0.3 mg or about 0.3 mg. In some embodiments, the dose of Compound C is 0.4 mg or about 0.4 mg. In some embodiments, the dose of Compound C is 0.5 mg or about 0.In some embodiments, the dose of Compound C is 0.6 mg or about 0.6 mg. In any of the preceding embodiments, each dose of the multiple intermittent doses of Compound C is the same.

[0241] In some embodiments, Compound C as part of an intermittent dosing regimen is administered no more than once every 5 days. In some embodiments, Compound C is administered no more than once every 6 days. In some embodiments, Compound C is administered no more than once every 7 days. In some embodiments, Compound C is administered no more than once every 8 days. In some embodiments, Compound C is administered no more than once every 9 days. In some embodiments, Compound C is administered no more than once every 10 days. In some embodiments, Compound C is administered no more than once every 11 days. In some embodiments, Compound C is administered no more than once every 12 days. In some embodiments, Compound C is administered no more than once every 13 days. In some embodiments, Compound C is administered no more than once every 14 days.

[0242] In some embodiments, Compound C as part of an intermittent dosing regimen is administered once every 5 days. In some embodiments, Compound C is administered once every 6 days. In some embodiments, Compound C is administered once weekly. In some embodiments, Compound C is administered once every 7 days (Q7D). In some embodiments, Compound C is administered once every 8 days. In some embodiments, Compound C is administered once every 9 days. In some embodiments, Compound C is administered once every 10 days. In some embodiments, Compound C is administered once every 11 days. In some embodiments, Compound C is administered once every 12 days. In some embodiments, Compound C is administered once every 13 days. In some embodiments, Compound C is administered once every two weeks. In some embodiments, Compound C is administered once every 14 days (Q14D).

[0243] In some embodiments, compound C is administered for a period subsequent to the initiation of administration of cell therapy. In some embodiments, compound C is administered for at least 1 week after the initiation of administration of cell therapy. In some embodiments, compound C is administered for at least 2 weeks after the initiation of administration of cell therapy. In some embodiments, compound C is administered for at least 3 weeks after the initiation of administration of cell therapy. In some embodiments, compound C is administered for at least 4 weeks after the initiation of administration of cell therapy. In some embodiments, compound C is administered for at least 5 weeks after the initiation of administration of cell therapy. In some embodiments, compound C is administered for at least 6 weeks after the initiation of administration of cell therapy. In some embodiments, compound C is administered for at least 7 weeks after the initiation of administration of cell therapy. In some embodiments, compound C is administered for at least 8 weeks after the initiation of administration of cell therapy. In some embodiments, compound C is administered for at least 9 weeks after the initiation of administration of cell therapy. In some embodiments, compound C is administered for at least 10 weeks after the initiation of administration of cell therapy. In some embodiments, compound C is administered for at least 11 weeks after the initiation of administration of cell therapy. In some embodiments, Compound C is administered for at least 12 weeks after initiation of administration of cell therapy. In some embodiments, Compound C is administered for up to 12 weeks after initiation of administration of cell therapy. In any of the preceding embodiments, Compound C is administered for up to 12 weeks after initiation of administration of cell therapy. Thus, referring to a combination therapy in which cell therapy is administered on day 1 of the combination therapy, in some embodiments, Compound C is administered no later than day 85 of the combination therapy.

[0244] In some embodiments, Compound C is administered on days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85 of the combination therapy. In some embodiments, Compound C is administered on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85 of the combination therapy. In some embodiments, Compound C is administered on days 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85 of the combination therapy. In some embodiments, Compound C is administered on days 8, 22, 36, 50, 64, and 78 of the combination therapy.

[0245] In some embodiments, each dose of the multiple intermittent doses of Compound C is 0.3 mg, and Compound C is administered on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85.

[0246] In some embodiments, each dose of the multiple intermittent doses of Compound C is 0.3 mg, and Compound C is administered on days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85.

[0247] In some embodiments, each dose of the multiple intermittent doses of Compound C is 0.3 mg, and Compound C is administered on days 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85.

[0248] In some embodiments, each dose of the multiple intermittent doses of Compound C is 0.4 mg, and Compound C is administered on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85.

[0249] In some embodiments, each dose of the multiple intermittent doses of Compound C is 0.3 mg, and Compound C is administered on days 8, 22, 36, 50, 64, and 78.

[0250] In some embodiments, each dose of the multiple intermittent doses of Compound C is 0.2 mg, and Compound C is administered on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85.

[0251] In some embodiments, each dose of the multiple intermittent doses of Compound C is 0.6 mg, and Compound C is administered on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85.

[0252] In some cases, the cycling regimen may be interrupted at any time and / or for one or more periods. In some cases, if a subject develops one or more adverse events, dose-limiting toxicities (DLTs), neutropenia or febrile neutropenia, thrombocytopenia, cytokine release syndrome (CRS), and / or neurotoxicity (NT), such as those described in Section IV, the cycling regimen is interrupted or modified. In some embodiments, after a subject develops one or more adverse events, dose-limiting toxicities (DLTs), neutropenia or febrile neutropenia, thrombocytopenia, cytokine release syndrome (CRS), and / or neurotoxicity (NT), such as those described in Section IV, the amount of Compound C per administration or per day on a particular day of the week is modified.

[0253] II. Cellular Therapy and Cell Engineering In some embodiments, cell therapy (e.g., T cell therapy) for use in conjunction with the provided combination therapy methods includes administering engineered cells expressing a recombinant receptor designed to recognize and / or specifically bind to an antigen associated with a disease or condition, such as cancer, e.g., a B-cell malignancy. In some embodiments, binding to the antigen results in a response, such as an immune response, to such antigen. In some embodiments, the cells contain or have been engineered to contain an engineered or recombinant receptor, e.g., an engineered antigen receptor, e.g., a chimeric antigen receptor (CAR). Recombinant receptors, such as CARs, generally comprise an extracellular antigen (or ligand) binding domain linked, in some aspects, via a linker and / or transmembrane domain, to one or more intracellular signaling components. In some aspects, the engineered cells are provided in pharmaceutical compositions and formulations suitable for administration to a subject, such as for adoptive cell therapy. Therapeutic methods for administering the cells and compositions to a subject, e.g., a patient, are also provided. In some embodiments, the method is any of those described in Section I.

[0254] Thus, in some embodiments, cells contain one or more nucleic acids introduced by genetic engineering, thereby expressing recombinant or genetically engineered products of such nucleic acids. In some embodiments, gene transfer is achieved by first stimulating the cells, e.g., in combination with a stimulus that induces a response such as proliferation, survival, and / or activation, e.g., as measured by expression of cytokines or activation markers, and then transducing and expanding the activated cells in culture to numbers sufficient for clinical application.

[0255] 1. Chimeric Antigen Receptor In some embodiments of the provided methods and uses, e.g., any of those described in Section I, engineered cells such as T cells express a chimeric receptor, such as a chimeric antigen receptor (CAR), that contains one or more domains that combine a ligand-binding domain (e.g., an antibody or antibody fragment) that provides specificity for a desired antigen (e.g., a tumor antigen) with an intracellular signaling domain. In some embodiments, the intracellular signaling domain is an activation intracellular domain portion, such as a T cell activation domain, that provides a primary activation signal. In some embodiments, the intracellular signaling domain contains, or further contains, a costimulatory signaling domain to facilitate effector function. Upon specific binding to a molecule, e.g., an antigen, the receptor generally delivers an immunostimulatory signal, such as an ITAM transduction signal, into the cell, thereby promoting an immune response targeted to the disease or condition. In some embodiments, the chimeric receptor, when engineered in an immune cell, can modulate T cell activity and, in some cases, modulate T cell differentiation or homeostasis, resulting in engineered cells with improved longevity, survival, and / or persistence in vivo, such as for use in adoptive cell therapy methods.

[0256] Exemplary antigen receptors, including CARs, as well as methods for engineering and introducing such receptors into cells, are described in, for example, International Patent Application Publication Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. US2002131960, US2013287748, US20130149337, U.S. Patent Nos. 6, 451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353 and 8,479,118 and European Patent Application No. EP 2537416, and / or Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some embodiments, the antigen receptor includes a CAR described in U.S. Patent No. 7,446,190 and those described in International Patent Application Publication No. WO / 2014055668A1.Examples of CARs include those disclosed in any of the above-mentioned publications, such as WO2014031687, US8,339,645, US7,446,179, US2013 / 0149337, U.S. Pat. No. 7,446,190, U.S. Pat. No. 8,389,282, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177), the entire contents of each of which are incorporated herein by reference. See also WO2014031687, US8,339,645, US7,446,179, US2013 / 0149337, U.S. Pat. No. 7,446,190 and U.S. Pat. No. 8,389,282.

[0257] Exemplary CAR T cell therapies targeting CD19 include the FDA-approved products BREYANZI® (lisocabtagene·malaleucel), TECARTUS™ (brexucabtagene·autoleucel), KYMRIAH™ (tisacabtagene·siloleucel), and YESCARTA™ (axicabtagene·siloleucel), which are being studied in clinical trials NCT02644655, NCT03744676, NCT01087294, NCT03366350, NCT03790891, NCT03497533, NCT04007029, NCT03960840, NCT04049383, NCT04094766, NC NCT03366324, NCT02546739, NCT03448393, NCT03467256, NCT03488160, NCT04012879, NCT03016377, NCT03468153, NCT03483688, NCT03398967, NCT03229876, NCT03455972, NCT03423706, NCT03497533, and NCT04002401. Exemplary engineered cells include BREYANZI®, TECARTUS™, KYMRIAH™, YESCARTA™, UCART19, and ALLO-501. In some embodiments, the engineered cells include any of the cells described in Marofi et al., Front. Immunol. (2021) 12:681984, which is incorporated by reference in its entirety.

[0258] In some embodiments, engineered cells, such as T cells, express a recombinant receptor, such as a chimeric antigen receptor (CAR), that has specificity for a particular antigen (or marker or ligand), such as an antigen expressed on the surface of a particular cell type. In some embodiments, the antigen targeted by the receptor is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed in disease or condition cells, e.g., tumor or pathogenic cells, compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed in normal cells and / or expressed in engineered cells.

[0259] In some embodiments, the antigen targeted by the receptor comprises an antigen associated with a B cell malignancy, such as any of a number of known B cell markers. In some embodiments, the antigen targeted by the receptor is CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig kappa, Ig lambda, CD79a, CD79b, or CD30. In a specific aspect, the antigen is CD19. In some embodiments, any of these antigens is an antigen expressed on human B cells.

[0260] Chimeric receptors such as CARs generally comprise an extracellular antigen-binding domain that is the antigen-binding portion of an antibody molecule. In some embodiments, the antigen-binding domain is a portion of an antibody molecule, generally the variable heavy (VH) and / or variable light (VL) chain region of an antibody, e.g., an scFv antibody fragment. In some embodiments, the antigen-binding domain is a single domain antibody (sdAb), e.g., an sdFv, nanobody, V H H and V NAR In some embodiments, the antigen-binding fragment comprises antibody variable regions joined by a flexible linker.

[0261] In some embodiments, an antibody or antigen-binding fragment (e.g., an scFv or V Hdomain) specifically recognizes an antigen, such as CD19. In some embodiments, the antibody or antigen-binding fragment is derived from or a variant of an antibody or antigen-binding fragment that specifically binds to CD19.

[0262] In some embodiments, the antigen is CD19. In some embodiments, the scFv is a V derived from an antibody or antibody fragment specific for CD19. H and V L In some embodiments, the antibody or antibody fragment that binds to CD19 is a mouse-derived antibody, such as FMC63 and SJ25C1. In some embodiments, the antibody or antibody fragment is a human antibody, for example, as described in U.S. Patent Publication No. US2016 / 0152723.

[0263] In some embodiments, the antigen-binding domain is a V derived from FMC63, which in some aspects may be an scFv. H and / or V L FMC63 generally refers to a murine monoclonal IgG1 antibody raised against Nalm-1 and -16 cells expressing CD19 of human origin (Ling, NR, et al. (1987). Leucocyte typing III. 302). In some embodiments, the FMC63 antibody comprises CDR-H1 and CDR-H2 set forth in SEQ ID NOs: 38 and 39, respectively, and CDR-H3 set forth in SEQ ID NO: 40 or 54, and CDR-L1 set forth in SEQ ID NO: 35, and CDR-L2 set forth in SEQ ID NO: 36 or 55, and CDR-L3 set forth in SEQ ID NO: 37 or 56. In some embodiments, the FMC63 antibody comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 41. H ), and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 L ) is included.

[0264] In some embodiments, the scFv comprises a variable light chain containing the CDR-L1 sequence of SEQ ID NO: 35, the CDR-L2 sequence of SEQ ID NO: 36, and the CDR-L3 sequence of SEQ ID NO: 37, and / or a variable heavy chain containing the CDR-H1 sequence of SEQ ID NO: 38, the CDR-H2 sequence of SEQ ID NO: 39, and the CDR-H3 sequence of SEQ ID NO: 40, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the scFv comprises the variable heavy chain region of FMC63 set forth in SEQ ID NO:41 and the variable light chain region of FMC63 set forth in SEQ ID NO:42, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the variable heavy chain and variable light chain are connected by a linker. In some embodiments, the linker is set forth in SEQ ID NO:59. In some embodiments, the scFv comprises, in order, V H , linker and V L In some embodiments, the scFv comprises, in order, V L , linker and V H In some embodiments, the scFv is encoded by the sequence of nucleotides set forth in SEQ ID NO: 57, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 57. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 43, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 43. In some embodiments, the anti-CD19 CAR is the CAR of BREYANZI® (lisocabtagene maraleucel). In some embodiments, the T cell therapy is BREYANZI® (lysocbutagen-malaleucel).

[0265] In some embodiments, the antigen binding domain is a V derived from SJ25C1, which in some aspects may be an scFv. H and / or V L SJ25C1 is a murine monoclonal IgG1 antibody raised against Nalm-1 and -16 cells expressing CD19 of human origin (Ling, NR, et al. (1987). Leucocyte typing III. 302). In some embodiments, the SJ25C1 antibody comprises CDR-H1, CDR-H2, and CDR-H3 sequences set forth in SEQ ID NOS: 47-49, respectively, and CDR-L1, CDR-L2, and CDR-L3 sequences set forth in SEQ ID NOS: 44-46, respectively. In some embodiments, the SJ25C1 antibody comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 50. H ), and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 51 L In some embodiments, the scFv comprises a variable light chain containing the CDR-L1 sequence of SEQ ID NO: 44, the CDR-L2 sequence of SEQ ID NO: 45, and the CDR-L3 sequence of SEQ ID NO: 46, and / or a variable heavy chain containing the CDR-H1 sequence of SEQ ID NO: 47, the CDR-H2 sequence of SEQ ID NO: 48, and the CDR-H3 sequence of SEQ ID NO: 49, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the scFv comprises the variable heavy chain region of SJ25C1 set forth in SEQ ID NO:50 and the variable light chain region of SJ25C1 set forth in SEQ ID NO:51, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the variable heavy chain and variable light chain are connected by a linker. In some embodiments, the linker is set forth in SEQ ID NO:52. In some embodiments, the scFv comprises, in order, V H , linker and V LIn some embodiments, the scFv comprises, in order, V L , linker and V H In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO:53, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:53.

[0266] In some embodiments, the anti-CD19 CAR is a TECARTUS™ (brexcavtagene autreucel) CAR. In some embodiments, the T cell therapy is TECARTUS™ (brexcavtagene autreucel).

[0267] In some embodiments, the anti-CD19 CAR is a KYMRIAH™ (tisagenlecleucel) CAR. In some embodiments, the T cell therapy is KYMRIAH™ (tisagenlecleucel).

[0268] In some embodiments, the anti-CD19 CAR is a YESCARTA™ (axicabtagene ciloreucel) CAR. In some embodiments, the T cell therapy is YESCARTA™ (axicabtagene ciloreucel).

[0269] The term "antibody" as used herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, such as antigen-binding fragment (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy chains (V) capable of specifically binding to an antigen, and H ) regions, single chain antibody fragments including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobodies, V H H or V NAR) or fragments. This term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFvs, and tandem tri-scFvs. Unless otherwise stated, the term "antibody" should be understood to encompass functional antibody fragments thereof. This term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD. In some embodiments, the CAR is a bispecific CAR, e.g., containing two antigen-binding domains with different specificities.

[0270] In some embodiments, antigen-binding proteins, antibodies, and antigen-binding fragments thereof specifically recognize the antigen of a full-length antibody. In some embodiments, the heavy and light chains of the antibody can be full-length or can be antigen-binding portions (Fab, F(ab')2, Fv, or single-chain Fv fragment (scFv)). In other embodiments, the antibody heavy chain constant region is selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, particularly, for example, IgG1, IgG2, IgG3, and IgG4, more particularly, IgG1 (e.g., human IgG1). In another embodiment, the antibody light chain constant region is selected from, for example, kappa or lambda, particularly kappa.

[0271] The terms "complementarity-determining region" and "CDR," which are synonymous with "hypervariable region" or "HVR," are known to refer, in some instances, to noncontiguous sequences of amino acids in an antibody variable region that confer antigen specificity and / or binding affinity. Generally, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. The terms "framework region" and "FR" are known, in some instances, to refer to the non-CDR portions of the heavy and light chain variable regions. Generally, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region.

[0272] The precise amino acid sequence boundaries of a given CDR or FR can be determined using the Kabat numbering scheme; Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 Jan;27(1):55-77 ("IMGT" numbering scheme); Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun 8;309(3):657-70 ("Aho" numbering scheme); and Martin et al.These loops can be readily determined using any of a number of well-known schemes, including the scheme described by [End Page 110] J. Med., "Modeling antibody hypervariable loops: a combined algorithm," PNAS, 1989, 86(23):9268-9272 (the "AbM" numbering scheme).

[0273] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural sequence, while the Chothia scheme is based on structural information. The numbering for both the Kabat and Chothia schemes is based on the length of the most common antibody region sequences, with insertions being represented by insertion letters, such as "30a", and deletions occurring in some antibodies. The two schemes place certain insertions and deletions ("indels") in different positions, resulting in differential numbering. The contact scheme is based on the analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM scheme is a compromise between the Kabat and Chothia definitions, based on those used by Oxford Molecular's AbM antibody modeling software.

[0274] Table 2 below lists exemplary boundary positions for CDR-L1, CDR-L2, CDR-L3, and CDR-H1, CDR-H2, and CDR-H3, as identified by Kabat, Chothia, AbM, and contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are positioned between CDRs; for example, FR-L1 is positioned before CDR-L1, FR-L2 is positioned between CDR-L1 and CDR-L2, FR-L3 is positioned between CDR-L2 and CDR-L3, etc. Note that the Kabat numbering scheme shown places the insertion at H35A and H35B, so the end of the Chothia CDR-H1 loop, when numbered using the Kabat numbering convention shown, varies between H32 and H34 depending on the length of the loop. [Table 2]

[0275] Thus, unless otherwise specified, the "CDRs" or "complementary determining regions" of a given antibody or region thereof, e.g., its variable region, or individual designated CDRs (e.g., CDR-H1, CDR-H2, CDR-H3) should be understood to encompass a (or specific) complementary determining region as defined by either the scheme above or other known schemes. For example, if a particular CDR (e.g., CDR-H3) is a CDR of a given V H or V LWhen a variable region amino acid sequence is described as containing the amino acid sequence of a corresponding CDR, it is understood that such CDR has the sequence of the corresponding CDR (e.g., CDR-H3) in the variable region defined by any of the above-mentioned or other known schemes. In some embodiments, specific CDR sequences are specified. Although exemplary CDR sequences of the provided antibodies are described using various numbering schemes, it is understood that the provided antibodies can include CDRs described according to any of the other above-mentioned or other numbering schemes known to those skilled in the art.

[0276] Similarly, unless otherwise specified, a given antibody or region thereof, such as the FRs of its variable regions or individual designated FRs (e.g., FR-H1, FR-H2, FR-H3, FR-H4), is understood to encompass a (or specific) framework region defined by any known scheme. In some instances, a scheme for identifying a particular CDR, FR, or CDR is specified, such as CDRs defined by the Kabat, Chothia, AbM, or contact method or other known scheme. In other cases, the specific amino acid sequence of a CDR or FR is provided.

[0277] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy and light chains (V, respectively) of a native antibody H and V L ) generally have a similar structure, with each domain containing four conserved framework regions (FRs) and three CDRs (see, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). H or V L The V domains can be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen each have a complementary V domain. L or V HTo screen a library of domains, we used V from an antibody that binds to an antigen. H or V L Domains can be used to isolate proteins. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0278] Among the antibodies provided, antibody fragments are particularly included. An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; variable heavy chain (V); H ) domains, single chain antibody molecules, e.g., scFv and single domain V H and multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single-chain antibody fragment comprising a variable heavy chain region and / or a variable light chain region, such as an scFv.

[0279] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains (V, respectively) of a native antibody H and V L ) generally have a similar structure, with each domain containing four conserved framework regions (FRs) and three CDRs (see, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). H or V L The V domains can be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen each have a complementary V domain. L or V H To screen a library of domains, we used V from an antibody that binds to an antigen. H or V LDomains can be used to isolate proteins. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0280] A single domain antibody (sdAb) is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody. In some embodiments, the CAR comprises an antibody heavy chain domain that specifically binds to an antigen, such as a cancer marker or cell surface antigen of a cell or disease to be targeted, such as a tumor cell or cancer cell, such as any of the target antigens described herein or known. Exemplary single domain antibodies include sdFvs, nanobodies, V H H or V NAR Includes.

[0281] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies or production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, such as a fragment having two or more antibody regions or chains joined by a synthetic linker, e.g., a peptide linker, and / or a fragment containing a non-naturally occurring arrangement, such as a fragment that cannot be produced by enzymatic digestion of a naturally occurring intact antibody. In some embodiments, the antibody fragment is an scFv.

[0282] A "humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody refers to a variant of a non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues were derived), e.g., to restore or improve antibody specificity or affinity.

[0283] In some embodiments, the recombinant receptor, e.g., a chimeric antigen receptor, comprises an extracellular portion containing one or more ligand (e.g., antigen) binding domains, such as an antibody or fragment thereof, and one or more intracellular signaling regions or domains (also interchangeably referred to as cytoplasmic signaling domains or regions). In some embodiments, the recombinant receptor, e.g., a CAR, further comprises a spacer and / or a transmembrane domain or portion. In some embodiments, the spacer and / or transmembrane domain can link the extracellular portion containing the ligand (e.g., antigen) binding domains to the intracellular signaling region or domain.

[0284] In some embodiments, a recombinant receptor such as a CAR comprises an immunoglobulin constant region or a variant or modified version thereof, such as a hinge region, e.g., an IgG4 hinge region and / or a C H 1 / C Land / or a spacer which may be or include at least a portion of the Fc region. In some embodiments, the recombinant receptor further comprises a spacer and / or a hinge region. In some embodiments, the constant region or portion is a constant region or portion of human IgG, e.g., IgG4 or IgG1. In some aspects, the portion of the constant region functions as a spacer region between the antigen recognition component, e.g., the scFv, and the transmembrane domain. The spacer may be of a length that results in increased cellular responsiveness after antigen binding compared to the absence of the spacer. In some examples, the spacer is 12 amino acids in length, or about 12 amino acids in length, or 12 or less amino acids in length. Exemplary spacers include spacers having at least about 10-229 amino acids, about 10-200 amino acids, about 10-175 amino acids, about 10-150 amino acids, about 10-125 amino acids, about 10-100 amino acids, about 10-75 amino acids, about 10-50 amino acids, about 10-40 amino acids, about 10-30 amino acids, about 10-20 amino acids, or about 10-15 amino acids, including any integer between any of the endpoints of the listed ranges. In some embodiments, the spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include IgG4 hinge alone, C H 2 and C H IgG4 hinge linked to three domains, or C H The spacer comprises an IgG4 hinge linked to the 3 domains. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, Hudecek et al. (2015) Cancer Immunol Res. 3(2): 125-135, and International Patent Application Publication No. WO2014031687.

[0285] In some embodiments, the spacer contains only the hinge region of an IgG, such as the hinge-only spacer set forth in SEQ ID NO: 1 and encoded by the sequence set forth in SEQ ID NO: 2, such as the hinge-only spacer of IgG4 or IgG1. H 2 and / or C H In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to the C3 domain. In some embodiments, the spacer is a C4 domain, such as set forth in SEQ ID NO: 3. H 2 and C H In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to the C3 domain. In some embodiments, the spacer is a C4 domain, such as set forth in SEQ ID NO: 4. H In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to only three domains. In some embodiments, the spacer is or includes a glycine-serine rich sequence or other flexible linker, e.g., a known flexible linker. In some embodiments, the constant region or portion is that of IgD. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 5. In some embodiments, the spacer has a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 1, 3, 4, and 5.

[0286] In some embodiments, the spacer (a) comprises or consists of all or a portion of an immunoglobulin hinge or a modified version thereof, or comprises about 15 amino acids or less and does not include the CD28 or CD8 extracellular region; (b) comprises or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4 hinge or a modified version thereof, and / or comprises about 15 amino acids or less and does not include the CD28 or CD8 extracellular region; or (c) is 12 amino acids or about 12 amino acids in length, and / or comprises an immunoglobulin hinge, optionally an IgG4 or a modified version thereof. or (d) consisting of or comprising a sequence of amino acids set forth in SEQ ID NO: 1, 3-5, 27-34 or 58, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto; or (e) a polypeptide spacer comprising or consisting of the formula X1PPX2P, where X1 is glycine, cysteine ​​or arginine and X2 is cysteine ​​or threonine.

[0287] In some embodiments, the antigen receptor comprises an intracellular domain directly or indirectly linked to an extracellular domain. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises an ITAM. For example, in some aspects, the antigen recognition domain (e.g., the extracellular domain) is generally linked to one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex in the case of a CAR, and / or signaling via another cell surface receptor. In some embodiments, the chimeric receptor comprises a transmembrane domain linked or fused between the extracellular domain (e.g., an scFv) and the intracellular signaling domain. Thus, in some embodiments, the antigen-binding component (e.g., an antibody) is linked to one or more transmembrane and intracellular signaling domains.

[0288] In one embodiment, the transmembrane domain that naturally associates with one of the domains in receptor (for example, CAR) is used.In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid the binding of this domain to the transmembrane domain of the same or different surface membrane protein, so as to minimize the interaction with other members of the receptor complex.

[0289] The transmembrane domain, in some embodiments, is derived from either a natural or synthetic source. If the source is natural, the domain, in some aspects, is derived from any membrane-bound or transmembrane protein. The transmembrane region comprises (i.e., comprises at least the transmembrane region of) the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 (4-1BB), or CD154. Alternatively, the transmembrane domain, in some embodiments, is synthetic. In some aspects, synthetic transmembrane domains comprise predominantly hydrophobic residues such as leucine and valine. In some aspects, triplets of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain. In some embodiments, the linkage is via a linker, spacer, and / or transmembrane domain. In some embodiments, the transmembrane domain comprises the transmembrane portion of CD28 or its variant. The extracellular domain and the transmembrane can be directly or indirectly linked. In some embodiments, the extracellular domain and the transmembrane are linked by a spacer such as any of those described herein.

[0290] In some embodiments, the transmembrane domain of a receptor, e.g., a CAR, is the transmembrane domain of human CD28 or a variant thereof, e.g., the 27 amino acid transmembrane domain of human CD28 (Accession No. P10747.1), or is a transmembrane domain comprising the sequence of amino acids set forth in SEQ ID NO: 8, or a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 8. In some embodiments, the transmembrane domain containing portion of a recombinant receptor comprises the sequence of amino acids set forth in SEQ ID NO: 9, or a sequence of amino acids that has at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0291] In some embodiments, the recombinant receptor, e.g., a CAR, comprises at least one intracellular signaling component, e.g., an intracellular signaling region or domain. T cell activation, in some aspects, is described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). In some aspects, a CAR comprises one or both of such signaling components. Particularly included among intracellular signaling sequences are intracellular signaling sequences that mimic or approximate signals mediated by natural antigen receptors, signals mediated by such receptors in combination with costimulatory receptors, and / or signals mediated by costimulatory receptors alone. In some embodiments, a short oligo- or polypeptide linker, e.g., a linker between 2 and 10 amino acids in length, e.g., a linker containing glycine and serine, e.g., a glycine-serine doublet, is present to form the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[0292] In some embodiments, upon ligation of the CAR, the cytoplasmic domain or intracellular signaling region of the CAR activates at least one of the normal effector functions or responses of an immune cell, e.g., a T cell, engineered to express the CAR. For example, in some situations, the CAR induces a T cell function, such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an antigen receptor component or intracellular signaling region of a costimulatory molecule is used in place of an intact immunostimulatory chain, e.g., when transmitting an effector function signal. In some embodiments, the intracellular signaling region, e.g., including the intracellular domain, comprises the cytoplasmic sequence of a T cell receptor (TCR), and in some aspects also comprises the cytoplasmic sequence of a co-receptor that, in its natural context, acts in concert with such receptor to initiate signal transduction after antigen receptor engagement, and / or any derivative or variant of such molecule, and / or any synthetic sequence having the same functional capabilities. In some embodiments, the intracellular signaling region, e.g., including the intracellular domain, comprises the cytoplasmic sequence of a region or domain involved in providing a costimulatory signal.

[0293] In some embodiments, CAR comprises a primary cytoplasmic signaling sequence that regulates the primary activation of TCR complex.The primary cytoplasmic signaling sequence that acts in a stimulatory manner can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.Examples of ITAMs that contain primary cytoplasmic signaling sequences include ITAMs derived from CD3 zeta chain, FcR gamma, CD3 gamma, CD3 delta and CD3 epsilon.In some embodiments, the cytoplasmic signaling molecule in CAR contains a cytoplasmic signaling domain, a portion thereof or a sequence derived from CD3 zeta.

[0294] In some embodiments, the receptor comprises an intracellular component of the TCR complex, such as a TCR CD3 chain, e.g., the CD3-zeta chain, which mediates T cell activation and cytotoxicity. Thus, in some aspects, the antigen-binding moiety is linked to one or more cell signaling modules. In some embodiments, the cell signaling module comprises a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. In some embodiments, the receptor, e.g., a CAR, further comprises portions of one or more additional molecules, such as Fc receptor gamma, CD8 alpha, CD8 beta, CD4, CD25, or CD16. For example, in some aspects, a CAR or other chimeric receptor comprises a chimeric molecule between CD3-zeta (CD3-zeta) or Fc receptor gamma and CD8 alpha, CD8 beta, CD4, CD25, or CD16.

[0295] In some embodiments, the intracellular (or cytoplasmic) signaling region comprises a human CD3 chain, optionally a CD3 zeta stimulatory signaling domain or a functional variant thereof, such as the 112 AA cytoplasmic domain of isoform 3 of human CD3ζ (Accession No. P20963.2) or the CD3 zeta signaling domain described in U.S. Patent No. 7,446,190 or U.S. Patent No. 8,911,993. In some embodiments, the intracellular signaling region comprises a sequence of amino acids set forth in SEQ ID NO: 13, 14, or 15, or a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13, 14, or 15.

[0296] In the context of natural TCRs, full activation generally requires not only TCR-mediated signaling but also costimulatory signals. Thus, in some embodiments, components for generating secondary or costimulatory signals to promote full activation are also included in the CAR. In other embodiments, the CAR does not include components for generating costimulatory signals. In some aspects, additional CARs are expressed in the same cells to provide components for generating secondary or costimulatory signals.

[0297] In some embodiments, the chimeric antigen receptor contains the intracellular domain of a T cell costimulatory molecule. In some embodiments, the CAR comprises the signaling domain and / or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, OX40 (CD134), CD27, DAP10, DAP12, ICOS, and / or other costimulatory receptors. In some embodiments, the CAR comprises a costimulatory region or domain of CD28 or 4-1BB, such as human CD28 or human 4-1BB.

[0298] In some embodiments, the intracellular signaling region or domain comprises the intracellular costimulatory signaling domain of human CD28 or a functional variant or portion thereof, e.g., the 41 amino acid domain thereof and / or such a domain having an LL to GG substitution at positions 186-187 of the native CD28 protein. In some embodiments, the intracellular signaling domain can comprise the sequence of amino acids set forth in SEQ ID NO: 10 or 11, or a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10 or 11. In some embodiments, the intracellular region comprises the intracellular costimulatory signaling domain of 4-1BB or a functional variant or portion thereof, e.g., the 42 amino acid cytoplasmic domain of human 4-1BB (Accession No. Q07011.1) or a functional variant or portion thereof, e.g., the sequence of amino acids set forth in SEQ ID NO: 12, or a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12.

[0299] In some embodiments, the same CAR comprises both a primary (or activating) cytoplasmic signaling region and a costimulatory signaling component.

[0300] In some embodiments, an activation domain is included within one CAR, while a costimulatory component is provided by another CAR that recognizes a different antigen. In some embodiments, a CAR includes an activating or stimulatory CAR and a costimulatory CAR, both of which are expressed on the surface of the same cell (see WO2014 / 055668). In some aspects, a cell includes one or more stimulatory or activation CARs and / or costimulatory CARs. In some embodiments, a cell further includes an inhibitory CAR (iCAR, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013)), e.g., a CAR that recognizes an antigen other than an antigen associated with and / or specific for a disease or condition, whereby the activation signal delivered via the disease-targeting CAR is reduced or inhibited by binding of the inhibitory CAR to its ligand, e.g., reducing off-target effects.

[0301] In some cases, CARs are referred to as first-, second-, and / or third-generation CARs. In some embodiments, first-generation CARs are CARs that simply provide a CD3 chain-induced signal upon antigen binding; in some embodiments, second-generation CARs are CARs that provide such a signal and a costimulatory signal, for example, CARs that include an intracellular signaling domain derived from a costimulatory receptor such as CD28 or CD137; in some embodiments, third-generation CARs are CARs that, in some embodiments, include multiple costimulatory domains from different costimulatory receptors.

[0302] In some embodiments, the CAR comprises one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., a primary activation domain, in the cytoplasmic portion. Exemplary CARs include the intracellular components CD3-zeta, CD28, and 4-1BB.

[0303] In some embodiments, the antigen receptor further comprises a marker, and / or cells expressing a CAR or other antigen receptor further comprise a surrogate marker, such as a cell surface marker, that can be used to confirm transduction or manipulation of cells to express the receptor. In some aspects, the marker comprises all or a portion (e.g., a truncated form) of CD34, NGFR, or epidermal growth factor receptor, e.g., a truncated version of such a cell surface receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding a cleavable linker sequence, e.g., a linker sequence such as T2A. For example, the marker and, optionally, the linker sequence, can be any of those disclosed in published patent application WO2014031687. For example, the marker can be a truncated EGFR (tEGFR), which may be linked to a linker sequence such as a T2A cleavable linker sequence.

[0304] Exemplary polypeptides for truncated EGFR (e.g., tEGFR) include the sequence of amino acids set forth in SEQ ID NO: 7 or 16, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7 or 16. Exemplary T2A linker sequences include the sequence of amino acids set forth in SEQ ID NO: 6 or 17, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 6 or 17.

[0305] In some embodiments, the marker is a molecule that is not naturally found on or at the surface of T cells, e.g., a cell surface protein, or portion thereof. In some embodiments, the molecule is a non-self molecule or protein, i.e., one that is not recognized as "self" by the immune system of the host into which the cells will be adoptively transferred.

[0306] In some embodiments, the marker does not serve a therapeutic function and / or produce any effect other than its use as a marker for genetic manipulation, e.g., to select successfully manipulated cells. In other embodiments, the marker may be a therapeutic molecule or a molecule that otherwise exerts some desired effect, e.g., a ligand encountered by the cells in vivo, e.g., a costimulatory or immune checkpoint molecule that enhances and / or attenuates the response of the cells upon adoptive transfer and encounter with the ligand.

[0307] In some embodiments, the chimeric antigen receptor comprises an extracellular portion containing an antibody or fragment described herein. In some aspects, the chimeric antigen receptor comprises an extracellular portion containing an antibody or fragment described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment is an scFv or single domain V H The chimeric antigen receptor comprises an antibody, wherein the intracellular domain contains an ITAM. In some aspects, the intracellular signaling domain comprises the signaling domain of the zeta chain of the CD3-zeta (CD3ζ) chain. In some embodiments, the CD3-zeta chain is a human CD3-zeta chain. In some embodiments, the intracellular signaling region further comprises a CD28 and CD137 (4-1BB, TNFRSF9) costimulatory domain linked to the CD3 zeta intracellular domain. In some embodiments, the CD28 is human CD28. In some embodiments, the 4-1BB is human 4-1BB. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain located between the extracellular domain and the intracellular signaling region. In some aspects, the transmembrane domain contains the transmembrane portion of CD28. The extracellular domain and the transmembrane can be linked directly or indirectly. In some embodiments, the extracellular domain and the transmembrane are linked by a spacer, such as any described herein.

[0308] In some embodiments, a CAR contains an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain that contains a signaling portion of CD28 or a functional variant thereof and a signaling portion of CD3 zeta or a functional variant thereof. For example, in some embodiments, a CAR contains an antibody, such as any described above, e.g., an antibody fragment comprising an scFv specific for CD19, a spacer, such as a spacer containing a portion of an immunoglobulin molecule, such as a hinge region and / or one or more constant regions of a heavy chain molecule, such as an Ig hinge-containing spacer, a transmembrane domain that contains all or a portion of a CD28-derived transmembrane domain, a CD28-derived intracellular signaling domain, and a CD3 zeta signaling domain.

[0309] In some embodiments, the CAR contains an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain that contains a signaling portion of 4-1BB or a functional variant thereof and a signaling portion of CD3 zeta or a functional variant thereof. In some such embodiments, the receptor further comprises a spacer containing a portion of an Ig molecule, such as a human Ig molecule, such as an Ig hinge, e.g., an IgG4 hinge, such as a hinge-only spacer. In some embodiments, the CAR comprises an antibody or fragment, such as an scFv specific for CD19, such as any of those described above, a spacer, such as any of the Ig hinge-containing spacers, a CD28-derived transmembrane domain, a 4-1BB-derived intracellular signaling domain, and a CD3 zeta-derived signaling domain.

[0310] B. Nucleic Acids, Vectors and Genetic Engineering Methods In some embodiments, cells, e.g., T cells, are genetically engineered to express a recombinant receptor. In some embodiments, the engineering is achieved by introducing a polynucleotide encoding the recombinant receptor. Polynucleotides encoding the recombinant receptor, as well as vectors or constructs containing such nucleic acids and / or polynucleotides, are also provided.

[0311] In some cases, the nucleic acid sequence encoding the recombinant receptor contains a signal sequence encoding a signal peptide. In some embodiments, the signal sequence can encode a signal peptide derived from a naturally occurring polypeptide. In other embodiments, the signal sequence can encode a heterologous or non-natural signal peptide, such as the exemplary signal peptide of the GMCSFR alpha chain set forth in SEQ ID NO:25 and encoded by the nucleotide sequence set forth in SEQ ID NO:24. In some cases, the nucleic acid sequence encoding the recombinant receptor, e.g., a chimeric antigen receptor (CAR), contains a signal sequence encoding a signal peptide. Non-limiting exemplary examples of signal peptides include, for example, the GMCSFR alpha chain signal peptide set forth in SEQ ID NO:25 and encoded by the nucleotide sequence set forth in SEQ ID NO:24, or the CD8 alpha signal peptide set forth in SEQ ID NO:26.

[0312] In some embodiments, the polynucleotide encoding the recombinant receptor contains at least one promoter operably linked to control expression of the recombinant receptor, hi some instances, the polynucleotide contains two, three, or more promoters operably linked to control expression of the recombinant receptor.

[0313] In certain cases where the nucleic acid molecule encodes two or more different polypeptide chains, e.g., a recombinant receptor and a marker, each polypeptide chain can be encoded by a separate nucleic acid molecule. For example, two separate nucleic acids are provided, each of which can be individually transferred or introduced into a cell for expression in the cell. In some embodiments, the nucleic acid encoding the recombinant receptor and the nucleic acid encoding the marker are operably linked to the same promoter, which may be separated by an internal ribosome entry site (IRES) or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping, such as T2A, P2A, E2A, or F2A, as appropriate. In some embodiments, the nucleic acid encoding the marker and the nucleic acid encoding the recombinant receptor are operably linked to two different promoters. In some embodiments, the nucleic acid encoding the marker and the nucleic acid encoding the recombinant receptor are present or inserted at different locations within the genome of the cell. In some embodiments, the polynucleotide encoding the recombinant receptor is introduced into a composition containing cultured cells, such as by retroviral transduction, transfection, or transformation.

[0314] In some embodiments, such as those in which the polynucleotide contains a first and a second nucleic acid sequence, the coding sequences encoding each of the different polypeptide chains may be operably linked to promoters, which may be the same or different. In some embodiments, the nucleic acid molecule may contain promoters that drive the expression of two or more different polypeptide chains. In some embodiments, such nucleic acid molecules may be multicistronic (either bicistronic or tricistronic, see, e.g., U.S. Patent No. 6,060,273). In some embodiments, a transcription unit can be engineered as a bicistronic unit containing an IRES (internal ribosome entry site) that allows for simultaneous expression of gene products (e.g., encoding a marker and encoding a recombinant receptor) by messages from a single promoter. Alternatively, in some cases, a single promoter can direct expression of RNA containing two or three genes (e.g., encoding a marker and encoding a recombinant receptor) in a single open reading frame (ORF) that are separated from each other by sequences encoding a self-cleaving peptide (e.g., a 2A sequence) or a protease recognition site (e.g., furin). The ORF thus encodes a single polypeptide that is processed into individual proteins either during translation (in the case of 2A) or post-translation. In some cases, a peptide such as T2A can cause the ribosome to skip synthesis of the peptide bond at the C-terminus of the 2A element (ribosomal skipping), causing a separation between the end of the 2A sequence and the next peptide downstream (see, e.g., de Felipe, Genetic Vaccines and Ther. 2:13 (2004) and de Felipe et al. Traffic 5:616-626 (2004). A variety of 2A elements are known.Examples of 2A sequences that can be used in the methods and systems disclosed herein include, but are not limited to, 2A sequences from foot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 21), equine rhinitis A virus (E2A, e.g., SEQ ID NO: 20), Thosea asigna virus (T2A, e.g., SEQ ID NO: 6 or 17), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO: 18 or 19), as described in U.S. Patent Application Publication No. 20070116690.

[0315] Any of the recombinant receptors described herein may be encoded by a polynucleotide containing one or more nucleic acid sequences encoding the recombinant receptor, in any combination or arrangement. For example, one, two, three, or more polynucleotides can encode one, two, three, or more different polypeptides, e.g., recombinant receptors. In some embodiments, one vector or construct contains a nucleic acid sequence encoding a marker, and a separate vector or construct contains a nucleic acid sequence encoding a recombinant receptor, e.g., a CAR. In some embodiments, the nucleic acid encoding the marker and the nucleic acid encoding the recombinant receptor are operably linked to two different promoters. In some embodiments, the nucleic acid encoding the recombinant receptor is downstream of the nucleic acid encoding the marker.

[0316] In some embodiments, the vector backbone contains a nucleic acid sequence encoding one or more markers, hi some embodiments, the one or more markers are transduction markers, surrogate markers and / or selectable markers.

[0317] In some embodiments, the marker is a transduction marker or a surrogate marker. A transduction marker or a surrogate marker can be used to detect cells transfected with a polynucleotide, such as a polynucleotide encoding a recombinant receptor. In some embodiments, the transduction marker can indicate or confirm cellular modification. In some embodiments, the surrogate marker is a protein engineered to be co-expressed on the cell surface with the recombinant receptor, such as a CAR. In certain embodiments, such a surrogate marker is a surface protein modified to have little or no activity. In certain embodiments, the surrogate marker is encoded by the same polynucleotide encoding the recombinant receptor. In some embodiments, the nucleic acid sequence encoding the recombinant receptor is operably linked to the nucleic acid sequence encoding the marker, which may be separated by an internal ribosome entry site (IRES) or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping, such as a 2A sequence, such as T2A, P2A, E2A, or F2A. Exogenous marker genes may be utilized in conjunction with engineered cells to allow for cell detection or selection, in some cases, and to promote cell suicide, in some cases.

[0318] Exemplary surrogate markers can include truncated forms of cell surface polypeptides, such as truncated forms that are non-functional, do not or cannot transmit the signal normally transmitted by the full-length form of the signal or cell surface polypeptide, and / or do not or cannot be internalized. Exemplary truncated cell surface polypeptides include truncated forms of growth factors or other receptors, such as truncated human epidermal growth factor receptor 2 (tHER2), truncated epidermal growth factor receptor (tEGFR, exemplary tEGFR sequences are set forth in SEQ ID NOs: 7 or 16), or prostate-specific membrane antigen (PSMA), or modified forms thereof. tEGFR can contain an epitope recognized by the antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibodies or binding molecules, which can be used to identify or select cells engineered with the tEGFR construct and the encoded exogenous protein and / or to extrude or separate cells expressing the encoded exogenous protein. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. 2016 April; 34(4): 430-434. In some embodiments, the marker, e.g., a surrogate marker, comprises all or a portion (e.g., a truncated form) of CD34, NGFR, CD19, or a truncated CD19, e.g., a truncated non-human CD19, or epidermal growth factor receptor (e.g., tEGFR).

[0319] In some embodiments, the marker is or comprises a fluorescent protein, e.g., green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), e.g., superfold GFP (sfGFP), red fluorescent protein (RFP), e.g., tdTomato, mCherry, mStrawberry, AsRed2, DsRed, or DsRed2, cyan fluorescent protein (CFP), blue-green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), as well as variants thereof, including species variants, monomeric variants, and codon-optimized and / or enhanced variants of fluorescent proteins. In some embodiments, the marker is or comprises an enzyme, e.g., luciferase, the lacZ gene from E. coli, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), or chloramphenicol acetyltransferase (CAT). Exemplary luminescent reporter genes include luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or variants thereof.

[0320] In some embodiments, the marker is a selectable marker. In some embodiments, the selectable marker is or comprises a polypeptide that confers resistance to an exogenous agent or drug. In some embodiments, the selectable marker is an antibiotic resistance gene. In some embodiments, the selectable marker is an antibiotic resistance gene that confers antibiotic resistance to mammalian cells. In some embodiments, the selectable marker is or comprises a puromycin resistance gene, a hygromycin resistance gene, a blasticidin resistance gene, a neomycin resistance gene, a geneticin resistance gene, or a zeocin resistance gene, or modified forms thereof.

[0321] In some embodiments, the molecule is a non-self molecule, e.g., a non-self protein, i.e., one that is not recognized as "self" by the immune system of the host into which the cells are to be adoptively transferred.

[0322] In some aspects, the marker does not serve a therapeutic function and / or produce no effect other than being used as a marker for genetic manipulation, e.g., to select successfully manipulated cells. In other embodiments, the marker can be a therapeutic molecule or a molecule that exerts some desired effect in other ways, e.g., a ligand for cells that will be encountered in vivo, e.g., a costimulatory or immune checkpoint molecule to enhance and / or attenuate the response of cells after adoptive transfer and encounter with the ligand.

[0323] In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding a cleavable linker sequence, such as T2A. For example, the marker, and optionally the linker sequence, can be any of those disclosed in PCT International Publication No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) that may be linked to a linker sequence, such as a T2A cleavable linker sequence. Exemplary polypeptides of truncated EGFR (e.g., tEGFR) include the sequence of amino acids set forth in SEQ ID NO: 7 or 16, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7 or 16.

[0324] In some embodiments, the marker is or comprises a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), e.g., superfold GFP (sfGFP), red fluorescent protein (RFP), e.g., tdTomato, mCherry, mStrawberry, AsRed2, DsRed, or DsRed2, cyan fluorescent protein (CFP), blue-green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), as well as variants thereof, including species variants, monomeric variants, and codon-optimized and / or enhanced variants of fluorescent proteins. In some embodiments, the marker is or comprises an enzyme, such as luciferase, the lacZ gene from E. coli, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), or chloramphenicol acetyltransferase (CAT). Exemplary luminescent reporter genes include luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or variants thereof.

[0325] In some embodiments, the marker is a selectable marker. In some embodiments, the selectable marker is or comprises a polypeptide that confers resistance to an exogenous agent or drug. In some embodiments, the selectable marker is an antibiotic resistance gene. In some embodiments, the selectable marker is an antibiotic resistance gene that confers antibiotic resistance to mammalian cells. In some embodiments, the selectable marker is or comprises a puromycin resistance gene, a hygromycin resistance gene, a blasticidin resistance gene, a neomycin resistance gene, a geneticin resistance gene, or a zeocin resistance gene, or modified forms thereof.

[0326] In some embodiments, the recombinant nucleic acid is transferred into cells using a recombinant infectious viral particle, such as a vector derived from simian virus 40 (SV40), adenovirus, or adeno-associated virus (AAV). In some embodiments, the recombinant nucleic acid is transferred into T cells using a recombinant lentiviral or retroviral vector, e.g., a gamma-retroviral vector (see, e.g., Koste et al. (2014) Gene Therapy, 2014 Apr 3. doi: 10.1038 / gt.2014.25; Carlens et al. (2000) Exp. Hematol., 28(10): 1137-46; Alonso-Camino et al. (2013) Mol. Ther. Nucl. Acids., 2, e93; Park et al., Trends Biotechnol., 2011 November 29(11): 550-557).

[0327] In some embodiments, the vector is an adeno-associated virus (AAV).

[0328] In some embodiments, the retroviral vector has long terminal repeat sequences (LTRs), such as retroviral vectors derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), and spleen focus-forming virus (SFFV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include those derived from any avian or mammalian cell source. Retroviruses are typically amphotropic, meaning they can infect several types of host cells, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol, and / or env sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Patent Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, AD (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109).

[0329] Methods for lentiviral transduction are known. Exemplary methods are described, for example, in Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101: 1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.

[0330] In some embodiments, recombinant nucleic acids are transferred into T cells via electroporation (see, e.g., Chicaybam et al. (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, recombinant nucleic acids are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods for introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

[0331] Other approaches and vectors for the transfer of nucleic acids encoding recombinant products are described, for example, in International Patent Application Publication No. WO2014055668 and U.S. Patent No. 7,446,190.

[0332] In some embodiments, cells, e.g., T cells, can be transfected with, e.g., a T cell receptor (TCR) or a chimeric antigen receptor (CAR) either during or after expansion. This transfection for the introduction of the gene for the desired receptor can be performed, for example, using any suitable retroviral vector. The genetically modified cell population can then be released from the initial stimulation (e.g., anti-CD3 / anti-CD28 stimulation) and subsequently stimulated with a second type of stimulation, e.g., via the de novo introduced receptor. This second type of stimulation can include antigen stimulation in the form of a peptide / MHC molecule, a cognate (crosslinking) ligand of the genetically introduced receptor (e.g., a natural ligand of the CAR), or any ligand (e.g., an antibody) that directly binds within the framework of the new receptor (e.g., by recognizing the constant region within the receptor). See, for example, Cheadle et al., "Chimeric antigen receptors for T-cell based therapy" Methods Mol Biol. 2012; 907:645-66 or Barrett et al., Chimeric Antigen Receptor Therapy for Cancer Annual Review of Medicine Vol. 65: 333-347 (2014).

[0333] In some cases, vectors can be used that do not require cells, such as T cells, to be activated. In some such cases, cells can be selected and / or transduced before activation. Thus, cells can be manipulated before or after culturing the cells, in some cases simultaneously with culturing, or during at least part of the culturing.

[0334] Among the additional nucleic acids, e.g., genes for introduction, are those that improve the efficacy of therapy, for example, by promoting the viability and / or function of the introduced cells; genes that provide genetic markers for cell selection and / or evaluation, for example, to assess in vivo survival or localization; genes that improve safety by making cells susceptible to negative selection, as described, for example, by Lupton SD et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also PCT / US91 / 08442 and PCT / US94 / 05601 publications by Lupton et al., which describe the use of bifunctional selectable fusion genes derived from the fusion of dominant positive selectable markers and negative selectable markers. See, for example, columns 14-17 of U.S. Patent No. 6,040,177 to Riddell et al.

[0335] C. Cells and Cell Preparation for Genetic Manipulation In some embodiments, the nucleic acid is heterologous, i.e., not normally present in the cell or sample obtained from the cell, e.g., obtained from another organism or cell not normally found in the cell being manipulated and / or from an organism from which such cell is not derived. In some embodiments, the nucleic acid is non-naturally occurring, including those that do not occur in nature, e.g., those that contain chimeric combinations of nucleic acids encoding various domains from a variety of different cell types.

[0336] The cells are generally eukaryotic cells, such as mammalian cells, and typically are human cells. In some embodiments, the cells are cells of the immune system, such as myeloid or lymphoid cells, derived from blood, bone marrow, lymph, or lymphoid organs, and include lymphocytes, typically T cells and / or NK cells, including cells of the innate or adaptive immunity. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells are typically primary cells, such as those isolated directly from a subject and / or isolated and frozen from a subject. In some embodiments, the cells are T cells or one or more subsets of other cell types, e.g., the total T cell population, CD4 T cells, or one or more subsets of other cell types, e.g., the total T cell population, CD4 T cells, or CD4 T cells. + cells, CD8 + These methods include cells and their subpopulations, such as those defined by function, activation state, maturity, differentiation potential, expansion, recirculation, localization, and / or persistence, antigen specificity, antigen receptor type, presence in specific organs or compartments, marker or cytokine secretion profile, and / or degree of differentiation. The cells can be allogeneic and / or autologous with respect to the subject to be treated. Some methods include commercially available methods. In some aspects, e.g., with commercially available techniques, the cells are pluripotent and / or multipotent, e.g., stem cells, e.g., induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from a subject, preparing, processing, culturing, and / or manipulating them, and reintroducing them into the same subject before or after cryopreservation.

[0337] T cells and / or CD4 + and / or CD8 + Among the T cell subtypes and subpopulations are naive T (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes, such as stem cell memory T (T SCM ), Central Memory T(T CM ), Effector Memory T(T EM), or well-differentiated effector memory T cells, cell-infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally occurring adaptive regulatory T (Treg) cells, helper T cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells.

[0338] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils.

[0339] In some embodiments, the cells contain one or more nucleic acids introduced via genetic engineering, thereby expressing recombinant or engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., not normally present in the cell or sample obtained from the cell, e.g., from another organism or cell not normally found in the engineered cell and / or from an organism from which such cell is not derived. In some embodiments, the nucleic acids are non-naturally occurring, including those that do not occur in nature, e.g., contain chimeric combinations of nucleic acids encoding various domains from a variety of cell types.

[0340] In some embodiments, the preparation of engineered cells involves one or more culture and / or preparation steps. Cells for introduction of a nucleic acid encoding a transgenic receptor, such as a CAR, can be isolated from a sample, e.g., a biological sample, e.g., a sample obtained from or derived from a subject. In some embodiments, the subject from which the cells are isolated is one suffering from a disease or condition, or one in need of cell therapy, or one to which cell therapy will be administered. In some embodiments, the subject is a human in need of a specific therapeutic intervention, such as adoptive cell therapy, in which cells are isolated, processed, and / or engineered.

[0341] Thus, in some embodiments, the cells are primary cells, for example, primary human cells. Samples include tissues, fluids, and other samples directly collected from a subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic manipulation (e.g., transduction with a viral vector), washing, and / or incubation. Biological samples can be samples obtained directly from biological sources or processed samples. Biological samples include, but are not limited to, bodily fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, tissue and organ samples (including processed samples derived therefrom).

[0342] In some embodiments, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from apheresis or leukapheresis products. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organ, and / or cells derived therefrom. Samples include samples from autologous and allogeneic sources in the context of cell therapy, such as adoptive cell therapy.

[0343] In some embodiments, the cells are derived from a cell line, e.g., a T cell line. The cells are, in some embodiments, obtained from heterologous sources, e.g., mouse, rat, non-human primate, and pig.

[0344] In some embodiments, cell isolation involves one or more preparative and / or non-affinity-based cell separation steps. In some cases, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, e.g., to remove undesired components, enrich for desired components, and lyse or remove cells that are sensitive to a particular reagent. In some cases, cells are isolated based on one or more properties, e.g., density, adhesive properties, size, sensitivity and / or resistance to a particular component.

[0345] In some examples, cells from the subject's circulating blood are obtained, for example, by apheresis or leukapheresis. The sample, in some embodiments, contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some embodiments, cells other than red blood cells and platelets.

[0346] In some embodiments, blood cells collected from a subject are washed, e.g., to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution lacks calcium and / or magnesium and / or many or all divalent cations. In some aspects, the wash step is performed in a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, the wash step is performed by tangential flow filtration (TFF) according to the manufacturer's instructions. In some aspects, the cells are washed with, e.g., Ca ++ / Mg ++ The cells are resuspended in various biocompatible buffers after washing, such as PBS containing no erythrocytes. In certain embodiments, the components of the blood cell sample are removed and the cells are resuspended directly in culture medium.

[0347] In some embodiments, the method comprises a density-based cell separation method, such as the preparation of white blood cells from peripheral blood by lysing red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0348] In some embodiments, the isolation method involves separating different cell types based on the expression or presence in cells of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known method for separation based on such markers can be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, in some aspects, isolation involves separating cells and cell populations based on the cellular expression or expression level of one or more markers, usually cell surface markers, from cells that are not bound by the antibody or binding partner, e.g., by incubation with an antibody or binding partner that specifically binds to such marker, typically followed by a washing step and separation of the antibody- or binding partner-bound cells.

[0349] Such separation steps can be based on positive selection, in which cells bound to the reagent are retained for further use, and / or negative selection, in which cells that are not bound to the antibody or binding partner are retained. In some cases, both fractions are retained for further use. In some embodiments, negative selection is particularly useful when antibodies that specifically identify cell types in a heterogeneous population are not available, so that separation is best performed based on markers expressed by cells other than the desired population.

[0350] Separation does not necessarily result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell, such as one that expresses a marker, refers to increasing the number or percentage of such cells, but does not necessarily result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal or deletion of a particular type of cell, such as one that expresses a marker, refers to reducing the number or percentage of such cells, but does not necessarily result in the complete removal of all such cells.

[0351] In some cases, a series of multiple separation steps are performed, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some cases, a single separation step can simultaneously deplete cells expressing multiple markers, for example, by incubating cells with multiple antibodies or binding partners, each specific to a marker targeted for negative selection. Similarly, multiple cell types can be simultaneously positively selected by incubating cells with multiple antibodies or binding partners expressed on various cell types.

[0352] For example, in some embodiments, specific subpopulations of T cells, such as cells that are positive for or express high levels of one or more surface markers, e.g., CD28 + , CD62L + , CCR7 + , CD27 + , CD127 + , CD4 + , CD8 + , CD45RA + , and / or CD45RO + T cells are isolated by positive or negative selection techniques.

[0353] For example, CD3 + , CD28 +T can be positively selected using anti-CD3 / anti-CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0354] In some embodiments, isolation is achieved by enriching for a particular cell population by positive selection, or by depleting a particular cell population by negative selection. In some embodiments, positive or negative selection is achieved by isolating cells by a marker (a marker) that is expressed on the positively or negatively selected cells, respectively. + ) or expressed at relatively high levels (markers 高 ) by incubation with one or more antibodies or other binding agents that specifically bind to one or more surface markers.

[0355] In some embodiments, T cells are isolated from the PBMC sample by negative selection of markers expressed on non-T cells, e.g., B cells, monocytes, or other white blood cells, e.g., CD14. + or CD8 + The selection process involves CD4 + Helper and CD8 + It is used to isolate cytotoxic T cells. + and CD8 + The population can be further sorted into subpopulations by positive or negative selection for markers expressed or relatively highly expressed on one or more naive, memory, and / or effector T cell subpopulations.

[0356] In some embodiments, CD8 + The cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, e.g., by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, central memory T (T CMEnrichment for TCM-enriched CD8 cells is performed to enhance efficacy, e.g., to improve long-term survival, expansion, and / or engraftment after administration, which in some aspects is particularly robust in such subpopulations. See Terakura et al. (2012) Blood, 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, TCM-enriched CD8 + T cells and CD4 + Combining T cells further enhances efficacy.

[0357] In one embodiment, the memory T cells are CD8 + CD62L on peripheral blood lymphocytes + and CD62 - PBMCs can be analyzed using, for example, anti-CD8 and anti-CD62L antibodies to identify CD62L-CD8 + and / or CD62L + CD8 + The fractions can be enriched or depleted.

[0358] In some embodiments, the central memory (T CM Enrichment for central memory (T) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127, and in some embodiments, central memory (T CM ) cells are based on negative selection for cells that express or highly express CD45RA and / or granzyme B. In some embodiments, T CM Enriched CD8 cells + Isolation of the population is performed by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. CMEnrichment for CD8) cells begins with a negative fraction of cells selected on the basis of CD4 expression, which is subjected to negative selection based on expression of CD14 and CD45RA, and positive selection based on CD62L. Such selections are performed simultaneously in some embodiments, and sequentially in other embodiments, in either order. In some embodiments, both the positive and negative fractions from the CD4-based separation are retained and enriched for CD8+ cells for use in subsequent steps of the method, optionally following one or more additional positive or negative selection steps. + The same CD4 expression-based selection process used in preparing the cell population or subpopulation may also be used to select for CD4 + It may also be used to generate cell populations or subpopulations.

[0359] In certain instances, the sample of PBMCs or other white blood cell sample is CD4 + The cells are subjected to selection, in which negative and positive fractions are retained, and the negative fraction is then subjected to negative selection based on expression of CD14 and CD45RA or CD19, and positive selection based on characteristic markers of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are performed in either order.

[0360] CD4 + T helper cells are classified into naive, central memory, and effector cells by identifying cell populations that possess cell surface antigens. + Lymphocytes can be obtained by standard methods. In some embodiments, naive CD4 + T lymphocytes are CD45RO - , CD45RA + , CD62L + , CD4 + In some embodiments, central memory CD4 + The cells are CD62L + and CD45RO + In some embodiments, the effector CD4 +The cells are CD62L - and CD45RO - is.

[0361] In one example, negative selection resulted in CD4 + To enrich cells, monoclonal antibody cocktails typically contain antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, antibodies or binding partners are bound to a solid support or matrix, such as magnetic or paramagnetic beads, to allow for cell separation for positive and / or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In vivo, p 17-25 Edited by: SA Brooks and U. Schumacher (Copyright) Humana Press Inc., Totowa, NJ).

[0362] In some embodiments, a sample or composition of cells to be separated is incubated with a small magnetizable or magnetically responsive material, e.g., magnetically responsive particles or microparticles, e.g., paramagnetic beads (e.g., Dynalbeads or MACS beads, etc.). The magnetically responsive material, e.g., particles, are generally attached directly or indirectly to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., a surface marker, present on the cells or population of cells desired to be separated, e.g., negatively or positively selected.

[0363] In some embodiments, the magnetic particles or beads comprise a magnetically responsive material bound to a specific binding partner, such as an antibody or other binding partner. There are many well-known magnetically responsive materials that can be used in magnetic separation methods. Suitable magnetic particles include those described in U.S. Patent No. 4,452,773 to Molday and European Patent Specification EP 452342B, which are hereby incorporated by reference. Other examples include colloidal-sized particles, such as those described in U.S. Patent No. 4,795,698 to Owen and U.S. Patent No. 5,200,084 to Liberti et al.

[0364] Incubation is generally carried out under conditions in which the antibody or binding partner, or a molecule that specifically binds to such an antibody or binding partner, e.g., a secondary antibody or other reagent, that is bound to the magnetic particles or beads, will specifically bind to the cell surface molecule if present on cells in the sample.

[0365] In some embodiments, the sample is placed in a magnetic field, and cells with attached magnetically responsive or magnetizable particles are bound to a magnet and separated from unlabeled cells. For positive selection, cells bound to the magnet are retained, and for negative selection, unbound cells (unlabeled cells) are retained. In some embodiments, a combination of positive and negative selection is performed during the same selection step, where positive and negative fractions are retained, further processed, and subjected to additional separation steps.

[0366] In certain embodiments, magnetically responsive particles are coated with a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In certain embodiments, magnetic particles are bound to cells via coating with a primary antibody specific for one or more markers. In certain embodiments, cells, rather than beads, are labeled with a primary antibody or binding partner, and then magnetic particles coated with a cell-type-specific secondary antibody or other binding partner (e.g., streptavidin) are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.

[0367] In some embodiments, the magnetically responsive particles remain bound to cells that are subsequently incubated, cultured, and / or manipulated, and in some aspects, the particles remain bound to cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of unlabeled antibodies and competition of magnetizable particles or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0368] In some embodiments, affinity-based selection is via magnetically activated cell sorting (MACS) (Miltenyi Biotec, Auburn, California). The magnetically activated cell sorting (MACS) system allows for high-purity selection of cells bound to magnetizable particles. In certain embodiments, MACS operates in a manner in which non-target and target species are sequentially eluted after application of an external magnetic field. That is, cells bound to magnetized particles are maintained in place, while unbound species are eluted. Then, after this first elution step is complete, species captured by the magnetic field and prevented from elution are released in some manner so that they can be eluted and recovered. In certain embodiments, non-target cells are labeled and depleted from a heterogeneous population of cells.

[0369] In some embodiments, the isolation or separation is performed using a system, device, or apparatus that performs one or more of the isolation, cell preparation, separation, processing, incubation, culture, and / or formulation steps of the method. In some aspects, a system is used to perform each of these steps in a closed or sterile environment, e.g., to minimize error, user handling, and / or contamination. In one example, the system is a system described in International Patent Application Publication No. WO2009 / 072003 or US20110003380A1.

[0370] In some embodiments, the system or device performs one or more, e.g., all, of the isolation, processing, manipulation, and formulation steps in an integrated or self-contained system and / or in an automated or programmable manner. In some aspects, the system or device includes a computer and / or computer program in communication with the system or device that allows a user to program, control, evaluate the outcome of, and / or adjust various aspects of the processing, isolation, manipulation, and formulation steps.

[0371] In some embodiments, separation and / or other steps are performed using, for example, a CliniMACS system (Miltenyi Biotec) for automated cell separation at clinical scale in a closed and sterile system. Components may include an integrated microcomputer, a magnetic separation unit, a peristaltic pump, and various pinch valves. The integrated computer, in some embodiments, controls all components of the instrument and guides the system to perform repetitive procedures in a standardized sequence. The magnetic separation unit, in some embodiments, includes a movable permanent magnet and holder for the selected column. The peristaltic pump controls the flow rate through the tubing set and, together with the pinch valve, ensures a controlled flow of buffer through the system and continuous suspension of the cells.

[0372] The CliniMACS system, in some embodiments, uses magnetizable particles coupled to antibodies, which are provided in a sterile, non-pyrogenic solution. In some embodiments, after labeling of the cells with magnetic particles, the cells are washed to remove excess particles. The cell preparation bag is then connected to a tubing set, which is subsequently connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing including a pre-column and a separation column and is for single use only. After initiating the separation program, the system automatically applies the cell sample onto the separation column. Labeled cells are retained in the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, cell populations for use with the methods described herein are unlabeled and not retained in the column. In some embodiments, cell populations for use with the methods described herein are labeled and retained in the column. In some embodiments, cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field and collected in a cell collection bag.

[0373] In certain embodiments, separation and / or other steps are performed using the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system, in some embodiments, is equipped with a cell processing unit that allows for automated cell washing and fractionation by centrifugation. The CliniMACS Prodigy system may also include an integrated camera and image recognition software that determines the optimal cell fractionation endpoint by identifying macroscopic layers of the source cell product. For example, peripheral blood is automatically separated into red blood cells, white blood cells, and plasma layers. The CliniMACS Prodigy system may also include an integrated cell culture chamber for performing cell culture protocols, such as cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for sterile removal and replenishment of media, and cells can be monitored using an integrated microscope. See, for example, Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.

[0374] In some embodiments, the cell populations described herein are collected and enriched (or depleted) via flow cytometry, where cells stained for multiple cell surface markers are transported in a flow stream. In some embodiments, the cell populations described herein are collected and enriched (or depleted) via preparative-scale (FACS) sorting. In certain embodiments, the cell populations described herein are collected and enriched (or depleted) using a microelectromechanical system (MEMS) chip in combination with a FACS-based detection system (see, e.g., WO2010 / 033140; Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376). In both cases, cells can be labeled with multiple markers, allowing for the isolation of clearly defined T cell subsets with high purity.

[0375] In some embodiments, antibodies or binding partners are labeled with one or more detectable markers to facilitate separation for positive and / or negative selection.For example, separation can be based on binding to fluorescently labeled antibodies.In some cases, cells are separated based on the binding of antibodies or other binding partners specific to one or more cell surface markers, and transported in a flow stream by, for example, fluorescence-activated cell sorting (FACS), including, for example, a preparative scale (FACS) and / or a microelectromechanical system (MEMS) chip, in combination with a flow cytometry detection system.Such methods allow for simultaneous positive and negative selection based on multiple markers.

[0376] In some embodiments, the preparation method includes a step involving freezing, e.g., cryopreserving, the cells either before or after isolation, incubation, and / or manipulation. In some embodiments, the freezing followed by thawing removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., after a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters can be used in some aspects. One example involves PBS containing 20% ​​DMSO and 8% human serum albumin (HSA), or other suitable cell freezing medium. This is then diluted 1:1 with medium, resulting in final concentrations of 10% and 4% DMSO and HSA, respectively. The cells are then typically frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[0377] In some embodiments, cells are incubated and / or cultured prior to or in conjunction with genetic manipulation. Incubation steps may include culturing, culturing, stimulating, activating, and / or propagating. Incubation and / or manipulation may occur in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other vessel for culturing or cultivating cells. In some embodiments, the composition or cells are incubated under stimulatory conditions or in the presence of a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in a population, to mimic antigen exposure, and / or to prime cells for genetic manipulation, e.g., introduction of a recombinant antigen receptor.

[0378] The conditions may include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agent designed to activate cells.

[0379] In some embodiments, the stimulatory conditions or agents include one or more agents, e.g., ligands, capable of activating or stimulating the intracellular signaling domain of the TCR complex. In some aspects, the agents activate or initiate the TCR / CD3 intracellular signaling cascade in T cells. Such agents may include antibodies, e.g., those specific for the TCR, e.g., anti-CD3. In some embodiments, the stimulatory conditions include one or more agents, e.g., ligands, e.g., anti-CD28, capable of stimulating a costimulatory receptor. In some embodiments, such agents and / or ligands may be bound to a solid support, such as beads, and / or one or more cytokines. Optionally, the expansion method may further include adding anti-CD3 and / or anti-CD28 antibodies to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulatory agents include IL-2, IL-15, and / or IL-7. In some aspects, the IL-2 concentration is at least about 10 units / mL.

[0380] In some embodiments, the incubation is carried out according to techniques such as those described in U.S. Pat. No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0381] In some embodiments, T cells are expanded by adding feeder cells, e.g., non-dividing peripheral blood mononuclear cells (PBMCs), to the culture starter composition (e.g., so that the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g., for a time sufficient to expand the number of T cells). In some aspects, the non-dividing feeder cells can include gamma-irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with gamma rays in the range of about 3000-3600 rad to prevent cell division. In some aspects, the feeder cells are added to the culture medium prior to the addition of the population of T cells.

[0382] In some embodiments, the stimulatory conditions include a temperature suitable for the growth of human T lymphocytes, e.g., at least about 25 degrees Celsius, typically at least about 30 degrees Celsius, and typically at or about 37 degrees Celsius. Optionally, the incubation may further include adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. The LCL may be irradiated with gamma rays in the range of about 6,000 to 10,000 rad. The LCL feeder cells, in some aspects, are provided in any suitable amount, e.g., at a ratio of LCL feeder cells to primary T lymphocytes of at least about 10:1.

[0383] In one embodiment, antigen-specific T cells, e.g., antigen-specific CD4 + and / or CD8 + T cells can be obtained by stimulating naive or antigen-specific T lymphocytes with an antigen. For example, antigen-specific T cell lines or clones can be generated against a cytomegalovirus antigen by isolating T cells from an infected subject and stimulating the cells in vitro with the same antigen.

[0384] III. Exemplary Treatment Outcomes and How to Assess Them In some embodiments of the methods, compositions, combinations, uses, kits, and articles of manufacture provided herein, the provided combination therapy results in one or more therapeutic outcomes, e.g., characteristics associated with any one or more of the therapies or parameters associated with the therapy, as described below. In some embodiments, the method is any of those described in Section I. In some embodiments, the method further includes assessing the exposure, persistence, and proliferation of T cells, e.g., T cells administered for T cell-based therapy. In some embodiments, cell exposure, or long-term expansion and / or persistence, and / or changes in cell phenotype or cell functional activity of cells in the methods provided herein, e.g., cells administered for immunotherapy, e.g., T cell therapy, can be measured by assessing T cell characteristics in vivo or ex vivo. In some embodiments, such assays can be used to determine or confirm T cell, e.g., T cell therapy, function before, during, or after administration of a combination therapy provided herein.

[0385] In some embodiments, the combination therapy may further include one or more screening steps to identify subjects for treatment with the combination therapy and / or continue the combination therapy, and / or to evaluate the outcome of the treatment and / or to monitor the outcome of the treatment. In some embodiments, the step for evaluating the treatment may include a step for evaluating the treatment and / or monitoring it, and / or to identify subjects for administering further or remaining steps of the therapy and / or for repeat therapy. In some embodiments, the screening step and / or evaluation of the outcome of the treatment can be used to determine the dose, frequency, duration, timing, and / or sequence of the combination therapy provided herein.

[0386] In some embodiments, any of the screening steps and / or assessments of therapeutic outcome described herein can be used before, during, during, or after administration of one or more steps of a provided combination therapy, e.g., administration of T cell therapy (e.g., CAR-expressing T cells) and / or Compound C. In some embodiments, the assessment is performed before, during, during, or after performance of any of the methods provided herein. In some embodiments, the assessment is performed before performance of a method provided herein. In some embodiments, the assessment is performed after performance of one or more steps of a method provided herein. In some embodiments, the assessment is performed before administration of one or more steps of a provided combination therapy, e.g., to screen and identify patients suitable for and / or susceptible to receiving the combination therapy. In some embodiments, the assessment is performed during, during, or after administration of one or more steps of a provided combination therapy, e.g., to evaluate intermediate or final therapeutic outcomes, e.g., to determine the effectiveness of the treatment, and / or to decide whether to continue or repeat treatment, and / or to decide whether to administer remaining steps of the combination therapy.

[0387] In some embodiments, a therapeutic outcome includes improved immune function, e.g., of T cells administered for cell-based therapy and / or of endogenous T cells in the body. In some embodiments, exemplary therapeutic outcomes include, but are not limited to, enhanced T cell proliferation, enhanced T cell functional activity, and changes in immune cell phenotypic marker expression, e.g., such characteristics are associated with engineered T cells, e.g., CAR-T cells, administered to a subject. In some embodiments, exemplary therapeutic outcomes include a reduction in disease burden, e.g., tumor burden, improved clinical outcome, and / or enhanced efficacy of therapy.

[0388] In some embodiments, screening and / or assessing therapeutic outcome includes assessing survival and / or function of T cells administered for cell-based therapy. In some embodiments, screening and / or assessing therapeutic outcome includes assessing cytokine or growth factor levels. In some embodiments, screening and / or assessing therapeutic outcome includes assessing disease burden and / or improvement, e.g., assessing tumor burden and / or clinical outcome. In some embodiments, any of the screening and / or assessing therapeutic outcome may include any of the assessment methods and / or assays described herein and / or known, and may be performed one or more times, e.g., prior to, during, during, or subsequent to administration of one or more steps of a combination therapy. Exemplary parameter sets associated with therapeutic outcome that can be assessed in some embodiments of the methods provided herein include peripheral blood immune cell population profiles and / or tumor burden.

[0389] In some embodiments, the method affects the efficacy of cell therapy in a subject. In some embodiments, the persistence, expansion, and / or presence of recombinant receptor expression, e.g., CAR-expressing cells, in a subject after administration of a dose of cells in the method with Compound C is greater than that achieved via a method without administration of Compound C. In some embodiments, the expansion and / or persistence in a subject of administered T cell therapy, e.g., CAR-expressing T cells, is assessed compared to a method in which the T cell therapy is administered to a subject in the absence of Compound C. In some embodiments, the method results in administered T cells that exhibit increased or prolonged expansion and / or persistence in a subject compared to a method in which the T cell therapy is administered to a subject in the absence of Compound C.

[0390] In some embodiments, administration of Compound C reduces disease burden, e.g., tumor burden, in the subject when compared to a method in which a dose of cells expressing the recombinant receptor is administered to the subject in the absence of Compound C. In some embodiments, administration of Compound C reduces myeloblasts in the subject when compared to a method in which a dose of cells expressing the recombinant receptor is administered to the subject in the absence of Compound C. In some embodiments, administration of Compound C results in improved clinical outcomes, e.g., objective response rate (ORR), progression-free survival (PFS), and overall survival (OS), when compared to a method in which a dose of cells expressing the recombinant receptor is administered to the subject in the absence of Compound C.

[0391] In some embodiments, subjects can be screened before administering one or more steps of combination therapy.For example, subjects can be screened for disease and / or disease burden, such as tumor burden, characteristics before administering combination therapy to determine compatibility, response and / or sensitivity to administering combination therapy.In some embodiments, screening steps and / or evaluation of treatment outcome can be used to determine the dose, frequency, duration, timing and / or sequence of the combination therapy provided herein.

[0392] In some embodiments, subjects can be screened after administration of one of the steps of the combination therapy to determine and identify subjects to receive the remaining steps of the combination therapy and / or to monitor the effectiveness of the therapy. In some embodiments, the number, level or amount of administered T cells and / or the proliferation and / or activity of administered T cells is assessed before and / or after administration of Compound C.

[0393] In some embodiments, the change and / or alteration, e.g., increase, elevation, decrease, or reduction, in the level, value, or measurement of a parameter or outcome is determined or assessed compared to the level, value, or measurement of the same parameter or outcome at a different time point of assessment, under different conditions, at a reference time point, and / or in a different subject. For example, in some embodiments, the fold change, e.g., increase or decrease, in a particular parameter, e.g., the number of engineered T cells in a sample, can be determined compared to the same parameter under a different condition, e.g., before administration of Compound C. In some embodiments, the levels, values, or measurements of two or more parameters are determined, and the relative levels are compared. In some embodiments, the determined level, value, or measurement of a parameter is compared to the level, value, or measurement from a control sample or an untreated sample. In some embodiments, the determined level, value, or measurement of a parameter is compared to the level from a sample from the same subject but at a different time point. The values ​​obtained by quantifying individual parameters can be combined for the purpose of disease assessment by performing arithmetic or logical operations on the parameter levels, values, or measurements, e.g., by using multiparametric analysis. In some embodiments, the ratio of two or more particular parameters can be calculated.

[0394] A. Cell Exposure, Persistence, and Growth In some embodiments, parameters associated with therapy or treatment outcome, including parameters that can be assessed for screening steps and / or evaluation of treatment outcome and / or monitoring treatment outcome, are or include assessment of the exposure, persistence, and proliferation of T cells, e.g., T cells administered for T cell-based therapy. In some embodiments, increased exposure, expansion, and / or prolonged persist...

Claims

1. A therapeutic agent for a cancer expressing cluster of differentiation 19 (CD19), the therapeutic agent being used for administering to a subject suffering from a cancer expressing CD19 in a combination therapy, (i) a T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) against the cancer, the T cell therapy being administered on day 1 of the combination therapy; and (ii) A compound which is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or an enantiomer or a mixture of enantiomers thereof, or a pharma- ceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof. A therapeutic agent comprising a combination therapy comprising:

2. 1. A therapeutic agent for a cancer that expresses CD19, the therapeutic agent comprising a T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) for the cancer, the T cell therapy being administered in combination therapy with a compound that is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or an enantiomer or a mixture of enantiomers thereof, or a pharma- ceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, to a subject suffering from a cancer that expresses CD19, the T cell therapy being administered on day 1 of the combination therapy.

3. 1. A therapeutic agent for a cancer that expresses CD19, the therapeutic agent comprising a compound which is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or an enantiomer or a mixture of enantiomers thereof, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate or polymorph thereof, wherein the compound, or an enantiomer or a mixture of enantiomers thereof, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate or polymorph thereof, is administered to a subject suffering from a cancer that expresses CD19 in combination therapy with a T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) against cancer, wherein the T cell therapy is administered on day 1 of the combination therapy.

4. A therapeutic agent for cancer that is lymphoma, the therapeutic agent being used to be administered in combination therapy to a subject suffering from the cancer, (i) a T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19), the T cell therapy being administered on day 1 of the combination therapy; and (ii) A compound which is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or an enantiomer or a mixture of enantiomers thereof, or a pharma- ceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof. A therapeutic agent comprising a combination therapy comprising:

5. 1. A therapeutic agent for treating a cancer that is lymphoma, the therapeutic agent comprising a T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds to cluster of differentiation 19 (CD19), the T cell therapy being administered to a subject suffering from the cancer in combination therapy with a compound that is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or an enantiomer or a mixture of enantiomers thereof, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, the T cell therapy being administered on day 1 of the combination therapy.

6. 1. A therapeutic agent for a cancer that is lymphoma, the therapeutic agent comprising a compound which is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or an enantiomer or a mixture of enantiomers thereof, or a pharma- ceutically acceptable salt, solvate, hydrate, cocrystal, clathrate, or polymorph thereof, the compound being administered to a subject suffering from the cancer in combination therapy with a T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds to cluster of differentiation 19 (CD19), the T cell therapy being administered on day 1 of the combination therapy.

7. (i) administration of the compound is initiated following administration of the T cell therapy; (ii) administration of the compound is initiated between day 1 and day 29 (inclusive) of the initiation of the combination therapy; (iii) the compound is administered in multiple doses, each dose being from 0.1 mg or about 0.1 mg to 0.6 mg or about 0.6 mg, inclusive; and / or (iv) the compound is administered as multiple intermittent doses administered no more than once a week; The therapeutic agent according to any one of claims 1 to 6.

8. (i) administration of the compound begins between days 1 and 22, inclusive; (ii) administration of the compound begins between days 1 and 15, inclusive; (iii) administration of the compound begins between day 8 and day 15, inclusive; (iv) administration of the compound begins on or about day 1; (v) administration of the compound begins on or about day 8 (vi) The method of any one of claims 1 to 7, wherein administration of the compound is initiated on or about the 15th day.

9. 9. The method of claim 7 or 8, wherein each dose of the multiple intermittent doses is the same.

10. (i) the compound is administered (a) once weekly and / or (b) once every seven days (Q7D); or (ii) the compound is administered (a) once every two weeks and / or (b) once every 14 days (Q14D); The therapeutic agent according to any one of claims 1 to 9.

11. (i) the compound is administered for at least 12 weeks following administration of the T cell therapy; or (ii) The method of any one of claims 1 to 10, wherein the compound is administered for up to 12 weeks following administration of the T cell therapy.

12. (i) the compound is administered on days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85; (ii) the compound is administered on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85; (iii) the compound is administered on days 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85; or (iv) The method of any one of claims 1 to 11, wherein the compound is administered on days 8, 22, 36, 50, 64 and 78.

13. (i) the dose of the compound is at or about 0.3 mg to at or about 0.6 mg, inclusive; (ii) the dose of the compound is at or about 0.6 mg; (iii) the dose of the compound is at or about 0.2 mg to at or about 0.4 mg, inclusive; (iv) the dose of the compound is at or about 0.4 mg; (v) the dose of the compound is less than 0.4 mg; (vi) the dose of the compound is at or about 0.3 mg; or (vii) The method of any one of claims 1 to 12, wherein the dose of the compound is 0.2 mg or about 0.2 mg.

14. The therapeutic agent according to any one of claims 1 to 13, wherein the compound is a pharma- ceutically acceptable salt or polymorph of (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione.

15. The therapeutic agent according to any one of claims 1 to 13, wherein the compound is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione.

16. (i) at the start of administration of the compound, the subject does not exhibit severe toxicity following administration of the T cell therapy; (a) the severe toxicity is severe cytokine release syndrome (CRS), optionally Grade 3 or higher CRS, prolonged Grade 3 or higher CRS, Grade 4 CRS or Grade 5 CRS; and / or (b) the severe toxicity is severe neurotoxicity, optionally Grade 3 or greater neurotoxicity, prolonged Grade 3 or greater neurotoxicity, Grade 4 neurotoxicity or Grade 5 neurotoxicity; and / or (ii) if the subject exhibits toxicity, optionally hematological toxicity, following administration of the compound, administration of the compound is withheld and / or the dose of the compound is modified, optionally reduced; (a) the toxicity is severe thrombocytopenia, optionally Grade 4 thrombocytopenia or prolonged Grade 4 thrombocytopenia; or (b) The therapeutic agent of any one of claims 1 to 15, wherein the toxicity is severe neutropenia, optionally Grade 4 neutropenia, prolonged Grade 4 neutropenia or febrile neutropenia, optionally Grade ≥ 3 febrile neutropenia or prolonged Grade ≥ 3 febrile neutropenia.

17. 17. The method of claim 16, wherein administration of the compound is resumed after the subject no longer exhibits toxicity.

18. (i) the cancer is a B cell malignancy; and / or (ii) The therapeutic agent of any one of claims 1 to 17, wherein the cancer is non-Hodgkin's lymphoma (NHL), optionally NHL comprises aggressive NHL; diffuse large B-cell lymphoma (DLBCL); DLBCL-NOS, optionally transformed low-grade; EBV positive DLBCL-NOS; T-cell / histiocyte-rich large B-cell lymphoma; primary mediastinal large B-cell lymphoma (PMBCL); follicular lymphoma (FL), optionally follicular lymphoma grade 3B (FL3B); and / or high-grade B-cell lymphoma with MYC and BCL2 and / or BCL6 rearrangements with DLBCL histology (double / triple hit).

19. (i) the CD19 is human CD19, and / or (ii) The therapeutic agent according to any one of claims 1 to 18, wherein the chimeric antigen receptor (CAR) comprises an extracellular antigen recognition domain that specifically binds to CD19, and an intracellular signaling domain comprising ITAM.

20. (i) the intracellular signaling domain comprises the signaling domain of the CD3-zeta (CD3ζ) chain, suitably the human CD3-zeta chain; and / or (ii) The therapeutic agent of claim 19, wherein the chimeric antigen receptor (CAR) further comprises a costimulatory signaling region, said costimulatory signaling region comprising the signaling domain of CD28 or 4-1BB, suitably human CD28 or human 4-1BB.

21. (i) the CAR comprises an scFv specific for CD19; a transmembrane domain; optionally a cytoplasmic signalling domain derived from a costimulatory molecule which is or comprises 4-1BB, optionally human 4-1BB; optionally a cytoplasmic signalling domain derived from a primary signalling ITAM containing molecule which is or comprises a CD3 zeta signalling domain, optionally a human CD3 zeta signalling domain; optionally the CAR further comprises a spacer between the transmembrane domain and the scFv, (ii) said CAR comprises, in order: an scFv specific for CD19; a transmembrane domain; optionally a cytoplasmic signaling domain derived from a costimulatory molecule, which is or comprises a 4-1BB signaling domain, optionally a human 4-1BB signaling domain; and optionally a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which is a CD3 zeta signaling domain, optionally a human CD3 zeta signaling domain; optionally the CAR further comprises a spacer between the transmembrane domain and the scFv; or (iii) The therapeutic agent of any one of claims 1 to 20, wherein the CAR comprises, in order, an scFv specific for CD19; a spacer; a transmembrane domain; and, optionally, a cytoplasmic signaling domain derived from a costimulatory molecule, which is a 4-1BB signaling domain; and, optionally, a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which is or includes a CD3 zeta signaling domain.

22. (i) the spacer comprises or consists of all or part of an immunoglobulin hinge or a modified version thereof, or is a polypeptide spacer comprising about 15 amino acids or less; (ii) the spacer comprises or consists of all or part of an immunoglobulin hinge, optionally an IgG4 hinge or a modified version thereof, and / or comprises no more than about 15 amino acids; (iii) the spacer is at or about 12 amino acids in length and / or comprises or consists of all or part of an immunoglobulin hinge, optionally IgG4 or a modified version thereof; (iv) the spacer has or consists of the sequence of SEQ ID NO:1; a sequence encoded by SEQ ID NO:2, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34; or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto; (v) the cytoplasmic signaling domain from the costimulatory molecule comprises a sequence set forth in SEQ ID NO: 12, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto; (vi) a cytoplasmic signaling domain from said primary signaling ITAM-containing molecule comprises any of SEQ ID NOs: 13-15, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto; (vii) the scFv comprises a CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 35), a CDRL2 sequence of SRLHSGV (SEQ ID NO: 36), and / or a CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 37); and / or (viii) The therapeutic agent of claim 21, wherein the scFv comprises a CDRH1 sequence of DYGVS (sequence number 38), a CDRH2 sequence of VIWGSETTYYNSALKS (sequence number 39), and / or a CDRH3 sequence of YAMDYWG (sequence number 40).

23. The scFv is the variable heavy chain region of FMC63 and the variable light chain region of FMC63; and / or CDRL1 sequence of FMC63, CDRL2 sequence of FMC63, CDRL3 sequence of FMC63, CDRH1 sequence of FMC63, CDRH2 sequence of FMC63 and CDRH3 sequence of FMC63 Including; Optionally, the scFv comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:41 and a VL comprising the amino acid sequence set forth in SEQ ID NO:

42.

24. The method of any one of claims 21 to 23, wherein the scFv has an amino acid sequence set forth in SEQ ID NO:

43.

25. (i) the dose of the engineered cells is greater than or equal to 1×10 ５ 1 piece or about 1 x 10 ５ pieces ~ 5×10 ８ Pieces or about 5 x 10 ８ Total CAR-expressing T cells, 1 x 10 ６ 1 piece or about 1 x 10 ６ pieces ~ 2.5×10 ８ pcs or about 2.5 x 10 ８ Total CAR-expressing T cells, 5 x 10 ６ Pieces or about 5 x 10 ６ pieces ~ 1×10 ８ 1 piece or about 1 x 10 ８ Total CAR-expressing T cells, 1 x 10 ７ 1 piece or about 1 x 10 ７ pieces ~ 2.5×10 ８ pcs or about 2.5 x 10 ８ total CAR-expressing T cells or 5 x 10 ７ Pieces or about 5 x 10 ７ pieces ~ 1×10 ８ 1 piece or about 1 x 10 ８ total CAR-expressing T cells (inclusive), (ii) the dose of the engineered cells is at least 1×10 ５ or at least about 1×10 ５ CAR-expressing cells, at least 2.5 x 10 ５ or at least about 2.5×10 ５ CAR-expressing cells, at least 5×10 ５ or at least about 5×10 ５ CAR-expressing cells, at least 1 x 10 ６ or at least about 1×10 ６ CAR-expressing cells, at least 2.5 x 10 ６ or at least about 2.5×10 ６ CAR-expressing cells, at least 5×10 ６ or at least about 5×10 ６ CAR-expressing cells, at least 1 x 10 ７ or at least about 1×10 ７ CAR-expressing cells, at least 2.5 x 10 ７ or at least about 2.5×10 ７ CAR-expressing cells, at least 5×10 ７ or at least about 5×10 ７ CAR-expressing cells, at least 1 x 10 ８ or at least about 1×10 ８ CAR-expressing cells, at least 2.5 x 10 ８ or at least about 2.5×10 ８ CAR-expressing cells or at least 5 x 10 ８ or at least about 5×10 ８ CAR-expressing cells, (iii) the dose of the engineered cells is greater than or equal to 1×10 ８ pcs or about 1 x 10 ８ and / or (iv) The method of any one of claims 1 to 24, wherein the dose of engineered cells is administered parenterally, optionally intravenously.

26. (i) the T cells are: (a) primary T cells obtained from the subject; and / or (b) autologous to the subject; or (ii) the T cells are allogeneic to the subject;

27. 27. The therapeutic agent of any one of claims 1-26, wherein the dose of engineered cells comprises CD4+ T cells expressing a CAR and CD8+ T cells expressing a CAR, and administering the dose comprises administering a plurality of separate compositions, the plurality of separate compositions comprising a first composition comprising one of the CD4+ T cells and CD8+ T cells and a second composition comprising the other of the CD4+ T cells and CD8+ T cells.

28. (i) the first composition and the second composition are administered 0 to 12 hours apart, 0 to 6 hours apart, or 0 to 2 hours apart, or the administration of the first composition and the administration of the second composition occur on the same day and are administered about 0 to about 12 hours apart, about 0 to about 6 hours apart, or about 0 to about 2 hours apart; (ii) the initiation of administration of the first composition and the initiation of administration of the second composition occur about 1 minute to about 1 hour apart or about 5 minutes to about 30 minutes apart; and / or (iii) The method of claim 27, wherein the first composition and the second composition are administered at intervals of 2 hours or less, 1 hour or less, 30 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less.

29. (i) the first composition comprises (a) CD4+ T cells or (b) CD8+ T cells; and / or 29. The method of claim 27 or 28, wherein (ii) the first composition is administered before the second composition.

30. The method of any one of claims 1 to 29, wherein prior to administration of the T cell therapy, the subject is preconditioned with a lymphodepleting therapy comprising administration of fludarabine and / or cyclophosphamide.

31. The method of any one of claims 1 to 29, further comprising administering to the subject a lymphodepleting therapy comprising administration of fludarabine and / or cyclophosphamide immediately prior to administration of the T cell therapy.

32. The lymphodepletion therapy comprises administering about 200-400 mg / m daily for 2-4 days, optionally 3 days. ２ (including upper and lower limits), as appropriate, 300 mg / m ２ Or about 300 mg / m ２ of cyclophosphamide and / or about 20-40 mg / m ２ , as appropriate, 30 mg / m ２ or said lymphodepleting therapy comprises administration of about 500 mg / m ２ 32. The method of claim 30 or 31, comprising administering cyclophosphamide at a dose of 1:1 or 1:

1.

33. The therapeutic agent according to any one of claims 1 to 32, wherein the subject is a human.

34. At least 35%, at least 40%, or at least 50% of the treated subjects achieve a complete response (CR) that is durable for 6 months or more, or for 9 months or more, or a CR that is durable in at least 60, 70, 80, 90, or 95% of subjects who achieve a CR; and / or At least 60, 70, 80, 90, or 95% of subjects who achieve a CR by month 6 remain in response, remain in CR, and / or remain alive or progress-free for 3 months or more and / or 6 months or more and / or 9 months or more; and / or at least 50%, at least 60%, or at least 70% of subjects treated according to the method achieve an objective response (OR), and optionally the OR is sustained for 6 months or more, or for 9 months or more, or is sustained in at least 60, 70, 80, 90, or 95% of subjects who achieve an OR; and / or 34. The therapeutic agent of any one of claims 1-33, wherein at least 60, 70, 80, 90, or 95% of subjects who achieve OR by 6 months remain in response or survive for 3 months or more and / or 6 months or more.

35. 35. The therapeutic agent of any one of claims 1-34, wherein at the time of or immediately prior to administration of the dose of engineered cells, the subject has relapsed following remission following treatment with one or more prior therapies for lymphoma, optionally one, two or three prior therapies other than another dose of engineered cells expressing a CAR, or has become refractory thereto.

36. At or prior to administration of the dose of engineered cells, The subject is identified as having or afflicted with double / triple hit lymphoma; The subject is identified as having or suffering from chemotherapy-resistant lymphoma, optionally chemotherapy-resistant DLBCL; and / or The method of any one of claims 1 to 35, wherein the subject has not achieved a complete response (CR) in response to a previous therapy.

37. Administering the compound comprises: reversing an exhaustion phenotype in CAR-expressing T cells in the subject; preventing, inhibiting, or delaying the development of an exhaustion phenotype in CAR-expressing T cells in the subject; reducing the level or degree of exhaustion phenotype in CAR-expressing T cells in the subject; or The therapeutic agent of any one of claims 1 to 36, which reduces the percentage or total number of CAR-expressing T cells in a subject with an exhausted phenotype.

38. 38. The therapeutic agent of any one of claims 1-37, wherein initiation of administration of the compound occurs after administration of the T cell therapy, and following administration or initiation of the compound, the subject exhibits restoration or rescue of antigen- or tumor-specific activity or function of CAR-expressing T cells in the subject, and optionally, said restoration, rescue, and / or initiation of administration of the compound occurs at a time after CAR-expressing T cells in the subject or in the subject's blood exhibit an exhausted phenotype.

39. Administering the compound comprises: (a) results in an increase in antigen-specific or antigen receptor-driven activity of naive or non-exhausted T cells in the subject, which may include T cells expressing said CAR, following exposure of the T cells to a CD19 antigen or antigen receptor-specific agent compared to the absence of said administration of said compound; (b) prevents, inhibits, or delays the development of an exhausted phenotype in naive or non-exhausted T cells in a subject, which may include T cells expressing said CAR, following exposure of the T cells to a CD19 antigen or antigen receptor-specific agent compared to the absence of said administration of said compound; and / or (c) administration of said compound in an amount, frequency and / or duration effective to reverse an exhausted phenotype in exhausted T cells, which may comprise T cells expressing said CAR, in a subject compared to the absence of said administration.

40. 40. The method of claim 39, wherein T cells in the subject comprise T cells that express the CAR and / or the exposure is exposure to a CD19 antigen.

41. the exhaustion phenotype is associated with a T cell or population of T cells, an increase in the level or degree of surface expression on T cells, or the percentage of the population of said T cells exhibiting surface expression, of one or more exhaustion markers, optionally 2, 3, 4, 5 or 6 exhaustion markers, compared to a reference population of T cells under the same conditions, wherein said increase in level, degree or percentage is greater than or equal to 1.2-fold, 1.5-fold or about 1.5-fold, 2.0-fold or about 2.0-fold, 3-fold or about 3-fold, 4-fold or about 4-fold, 5-fold or about 5-fold, 6-fold or about 6-fold, 7-fold or about 7-fold, 8-fold or about 8-fold, 9-fold or about 9-fold, 10-fold or about 10-fold or more; or 41. The therapeutic agent of any one of claims 37 to 40, comprising a decrease in the level or degree of activity exhibited by said T cell or population of T cells following exposure to a CD19 antigen or antigen receptor specific agent compared to a reference T cell population under the same conditions, optionally wherein said level, degree or percentage decrease is less than or equal to 1.2 fold, 1.5 fold or about 1.5 fold, 2.0 fold or about 2.0 fold, 3 fold or about 3 fold, 4 fold or about 4 fold, 5 fold or about 5 fold, 6 fold or about 6 fold, 7 fold or about 7 fold, 8 fold or about 8 fold, 9 fold or about 9 fold, 10 fold or about 10 fold or less.

42. (i) the reference T cell population is a population of T cells known to have a non-exhausted phenotype, a population of naive T cells, a population of central memory T cells, or a population of stem central memory T cells, as appropriate, from the same subject or species as the subject from which the T cells having an exhausted phenotype are derived; (ii) the reference T cell population is a subject-matched population comprising (a) bulk T cells isolated from the blood of the subject from which the T cells with the exhausted phenotype are derived, where optionally the bulk T cells do not express a CAR, and / or (b) are obtained from the subject from which the T cells with the exhausted phenotype are derived prior to receiving a dose of T cells expressing a CAR, and / or 42. The therapeutic agent of claim 41 , wherein (iii) the reference T cell population is a composition comprising a sample of T cells expressing a CAR or a pharmaceutical composition comprising T cells expressing a CAR prior to its administration to a subject, optionally wherein the composition is a cryopreserved sample. a

43. (i) the one or more exhaustion markers are inhibitory receptors; and / or (ii) the one or more exhaustion markers are selected from among PD-1, CTLA-4, TIM-3, LAG-3, BTLA, 2B4, CD160, CD39, VISTA and TIGIT.

44. 44. The method of any one of claims 41 to 43, wherein the activity is one or more of proliferation, cytotoxicity, or production of one or a combination of inflammatory cytokines, optionally wherein the one or a combination of cytokines is selected from the group consisting of IL-2, IFN-gamma, and TNF-alpha.

45. (i) said exposure to a CD19 antigen or antigen receptor-specific agent comprises incubation with a CD19 antigen or antigen receptor-specific agent, optionally an agent that binds to the antigen-binding domain of the CAR; and / or (ii) the exposure to a CD19 antigen or antigen receptor specific agent comprises exposing T cells to CD19 antigen expressing target cells, optionally cells of a B cell malignancy.

Images