CAR T-cell therapy and immunomodulatory compound combinations 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 of related applications This application claims priority under U.S. Provisional Patent Application No. 63 / 167,599, filed on 29 March 2021, and U.S. Provisional Patent Application No. 63 / 277,134, filed on 8 November 2021, the entire contents of each of these applications being incorporated herein by reference for any purpose.

[0002] Inclusion by referencing the sequence list This application is pending along with an electronically formatted sequence listing. The sequence listing is provided as a file titled 735042024540SeqList.txt, created on March 27, 2022, and is 36 kilobytes in size. The information within the electronically formatted sequence listing is incorporated entirely by reference.

[0003] field This disclosure relates, in some embodiments, to methods, compositions, uses and products of combination therapies for treating subjects suffering from certain diseases and conditions, such as certain B-cell malignancies, including adoptive cell therapies, immunotherapies such as T-cell therapy, and (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione or its enantiomers or mixtures of enantiomers or pharmaceutically acceptable salts, solvates, hydrates, cocrystals, clathrates or polymorphs, as well as related methods, compositions, uses and products. T-cell therapy includes cells expressing recombinant receptors such as chimeric antigen receptors (CARs). In some embodiments, the disease or condition is non-Hodgkin lymphoma (NHL), such as relapsed or refractory NHL or certain NHL subtypes. [Background technology]

[0004] For example, various strategies are available for immunotherapy involving the administration of T cells engineered for adoption therapy. For instance, strategies for manipulating T cells are available that involve expressing genetically engineered antigen receptors such as CARs and administering compositions containing such cells to a target. Improved strategies are needed to enhance the efficacy of the cells, for example, by improving the persistence, activity, and / or proliferation of the cells upon administration to a target. Methods, compositions, kits, and systems are provided to satisfy such needs. [Overview of the project]

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

[0006] In some of the methods provided, cancers that express CD19 are lymphomas.

[0007] In some embodiments, methods for treating lymphoma are also provided herein, comprising the steps of administering to a subject suffering from lymphoma cancer: (i) T-cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds to differentiation antigen group 19 (CD19); and (ii) combination therapy comprising a compound that is (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate or polymorph thereof.

[0008] In some of the methods offered, the administration of the compound is initiated before the administration of T-cell therapy.

[0009] In some embodiments, a method for treating a CD19-expressing cancer is also provided herein, comprising the step of administering a T-cell therapy to a subject with a CD19-expressing cancer, comprising a dose of engineered cells including T cells expressing a cancer-targeting chimeric antigen receptor (CAR), wherein the T-cell therapy is administered in combination with a compound which is (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate or polymorph thereof, wherein the administration of the compound is initiated prior to the administration of the T-cell therapy.

[0010] In some of the methods provided, cancers that express CD19 are lymphomas.

[0011] In some embodiments, a method for treating lymphoma is also provided herein, comprising the step of administering to a subject suffering from lymphoma cancer a T-cell therapy comprising a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds to differentiation antigen group 19 (CD19), wherein the T-cell therapy is administered in combination with a compound which is (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate or polymorph thereof, wherein the administration of the compound is initiated prior to the administration of the T-cell therapy.

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

[0013] In some embodiments, a method for treating a CD19-expressing cancer is also provided herein, comprising the step of administering to a subject with a CD19-expressing cancer a compound which is (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate, or polymorph thereof, wherein the compound is administered in combination with T-cell therapy which includes a dose of engineered cells including T cells expressing a cancer-targeting chimeric antigen receptor (CAR), and the administration of the compound is initiated after the administration of the T-cell therapy.

[0014] In some of the methods provided, cancers that express CD19 are lymphomas.

[0015] In some embodiments, a method for treating lymphoma is also provided herein, comprising the step of administering to a subject suffering from lymphoma, a cancer, a compound which is (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate, or polymorph thereof, wherein the compound is administered in combination with T-cell therapy which includes a dose of engineered cells which are T cells expressing a chimeric antigen receptor (CAR) that binds to differentiation antigen group 19 (CD19), and the administration of the compound is initiated after the administration of the T-cell therapy.

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

[0017] In some of the methods provided, administration of the compound is initiated between day 1 and day 29 of the combination therapy (including upper and lower limits).

[0018] In some of the methods provided, the compound is administered as multiple doses, each dose being 0.1 mg or approximately 0.1 mg to 0.6 mg or approximately 0.6 mg (including upper and lower limits).

[0019] In some of the methods provided, the compound is administered as multiple intermittent doses, administered at least once a week.

[0020] In some embodiments, a method for treating lymphoma, comprising: (i) a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds to differentiation antigen group 19 (CD19), administered to a subject with lymphoma on day 1 of combination therapy; and (ii) the structure described below: [ka] Also provided herein is a method comprising administering a combination therapy comprising the compound having (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, which is administered in an intermittent (i.e., not daily) dosing regimen, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate or polymorph thereof.

[0021] In some embodiments, a method for treating lymphoma, comprising the following structure for a subject suffering from lymphoma: [ka] A method is also provided herein comprising the step of administering a compound having (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate or polymorph thereof, wherein the compound is administered in combination with T-cell therapy comprising a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) bound to differentiation antigen group 19 (CD19), the T-cell therapy being administered on day 1 of the combination therapy, and the compound being administered in an intermittent (i.e., not daily) dosing regimen.

[0022] In some of the methods provided, the compound is administered as multiple intermittent doses. In some of the methods provided, the compound is administered at least once a week.

[0023] In any of the methods provided, each dose of the compound is 0.1 mg or about 0.1 mg to 0.6 mg or about 0.6 mg (including upper and lower limits).

[0024] In some of the methods provided, administration of the compound is initiated between day 1 and day 29 of the combination therapy (including upper and lower limits).

[0025] In some embodiments, a method for treating lymphoma, comprising: (i) a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds to differentiation antigen group 19 (CD19), administered to a subject with lymphoma on day 1 of combination therapy; and (ii) the structure described below: [ka] A method is also provided herein that comprises administering a combination therapy comprising the step of administering a compound having (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate or polymorph thereof, wherein the compound is administered as multiple intermittent doses administered no more than once per week, each dose being 0.1 mg or about 0.1 mg to 0.6 mg or about 0.6 mg (including upper and lower limits), and administration of the compound is initiated between day 1 and day 29 of the combination therapy (including upper and lower limits).

[0026] In some embodiments, a method for treating lymphoma, comprising the following structure for a subject suffering from lymphoma: [ka] A method is also provided herein comprising the step of administering a compound having (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate or polymorph thereof, wherein the compound is administered in combination with T-cell therapy comprising a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) bound to differentiation antigen group 19 (CD19), the T-cell therapy being administered on day 1 of the combination therapy; the compound being administered as multiple intermittent doses administered at least once per week, each dose being 0.1 mg or about 0.1 mg to 0.6 mg or about 0.6 mg (including upper and lower limits), and administration of the compound being initiated between day 1 and day 29 of the combination therapy (including upper and lower limits).

[0027] In some of the methods provided, administration of the compound is initiated between day 1 and day 22 (including upper and lower limits). In some of the methods provided, administration of the compound is initiated between day 1 and day 15 (including upper and lower limits). In some of the methods provided, administration of the compound is initiated between day 8 and day 15 (including upper and lower limits).

[0028] In some of the methods provided, administration of the compound is initiated on day 1 or approximately day 1. In some of the methods provided, administration of the compound is initiated on day 8 or approximately day 8. In some of the methods provided, administration of the compound is initiated on day 15 or approximately day 15.

[0029] In any part of the method provided, each of the multiple intermittent doses is identical.

[0030] In some of the methods provided, the compound is administered once a week. In some of the methods provided, the compound is administered once every 7 days (Q7D). In some of the methods provided, the compound is administered once every 2 weeks. In some of the methods provided, the compound is administered once every 14 days (Q14D).

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

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

[0033] In some of the methods provided, the dose of the compound is 0.3 mg or approximately 0.3 mg to 0.6 mg or approximately 0.6 mg (including upper and lower limits).

[0034] In some of the methods provided, the dose of the compound is 0.2 mg or approximately 0.2 mg to 0.4 mg or approximately 0.4 mg (including upper and lower limits). In some of the methods provided, the dose of the compound is 0.4 mg or approximately 0.4 mg. In some of the methods provided, the dose of the compound is less than 0.4 mg. In some of the methods provided, the dose of the compound is 0.3 mg or approximately 0.3 mg. In some of the methods provided, the dose of the compound is 0.2 mg or approximately 0.2 mg.

[0035] In some embodiments, a method for treating lymphoma, comprising the following structure for a subject suffering from lymphoma: [ka] A method is also provided herein comprising the step of administering a compound having (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate or polymorph thereof, wherein the compound is administered in combination with T-cell therapy comprising a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) bound to differentiation antigen group 19 (CD19), the T-cell therapy being administered on day 1 of the combination therapy; and the compound being administered as a plurality of intermittent doses once every 7 days (Q7D), administered on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85, each dose being 0.3 mg.

[0036] In some embodiments, a method for treating lymphoma, comprising the following structure for a subject suffering from lymphoma: [ka] A method is also provided herein comprising the step of administering a compound having (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate or polymorph thereof, wherein the compound is administered in combination with T cell therapy comprising a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) bound to differentiation antigen group 19 (CD19), the T cell therapy being administered on day 1 of the combination therapy; and the compound being administered as a plurality of intermittent doses administered once every 7 days (Q7D), on days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85, with each dose being 0.3 mg.

[0037] In some embodiments, a method for treating lymphoma, comprising the following structure for a subject suffering from lymphoma: [ka] A method is also provided herein comprising the step of administering a compound having (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate or polymorph thereof, wherein the compound is administered in combination with T-cell therapy comprising a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) bound to differentiation antigen group 19 (CD19), the T-cell therapy being administered on day 1 of the combination therapy; and the compound being administered as a plurality of intermittent doses administered once every 7 days (Q7D), on days 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85, each dose being 0.3 mg.

[0038] In some embodiments, a method for treating lymphoma, comprising the following structure for a subject suffering from lymphoma: [ka] A method is also provided herein comprising the step of administering a compound having (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate or polymorph thereof, wherein the compound is administered in combination with T cell therapy comprising a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) bound to differentiation antigen group 19 (CD19), the T cell therapy being administered on day 1 of the combination therapy; and the compound being administered as a plurality of intermittent doses administered once every 7 days (Q7D), on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85, each dose being 0.4 mg.

[0039] In some embodiments, a method for treating lymphoma, comprising the following structure for a subject suffering from lymphoma: [ka] A method is also provided herein comprising the step of administering a compound having (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate or polymorph thereof, wherein the compound is administered in combination with T-cell therapy comprising a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) bound to differentiation antigen group 19 (CD19), the T-cell therapy being administered on day 1 of the combination therapy; and the compound being administered as a plurality of intermittent doses administered once every 14 days (Q14D), on days 8, 22, 36, 50, 64 and 78, with each dose being 0.3 mg.

[0040] In some embodiments, a method for treating lymphoma, comprising the following structure for a subject suffering from lymphoma: [ka] A method is also provided herein comprising the step of administering a compound having (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate or polymorph thereof, wherein the compound is administered in combination with T cell therapy comprising a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) bound to differentiation antigen group 19 (CD19), the T cell therapy being administered on day 1 of the combination therapy; and the compound being administered as a plurality of intermittent doses administered once every 7 days (Q7D), on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85, each dose being 0.2 mg.

[0041] In some embodiments, a method for treating lymphoma, comprising the following structure for a subject suffering from lymphoma: [ka] A method is also provided herein comprising the step of administering a compound having (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate or polymorph thereof, wherein the compound is administered in combination with T cell therapy comprising a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) bound to differentiation antigen group 19 (CD19), the T cell therapy being administered on day 1 of the combination therapy; and the compound being administered as a plurality of intermittent doses administered once every 7 days (Q7D), on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85, each dose being 0.6 mg.

[0042] In any part of the method provided, the compound is (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt thereof.

[0043] In any part of the method provided, the compound is or comprises a pharmaceutically acceptable salt of (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione. In any part of the method provided, the compound is or comprises a hydrate of (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione. In any part of the method provided, the compound is or comprises the solvate of (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione.

[0044] In any part of the methods provided, at the start of administration of the compound, the subject does not exhibit severe toxicity after administration of T-cell therapy. In any part of the methods provided, severe toxicity is severe cytokine release syndrome (CRS), as appropriate, grade 3 or higher CRS, long-term grade 3 or higher CRS, grade 4 CRS or grade 5 CRS; and / or severe toxicity is severe neurotoxicity, as appropriate, grade 3 or higher neurotoxicity, long-term grade 3 or higher neurotoxicity, grade 4 neurotoxicity or grade 5 neurotoxicity. In some embodiments, severe toxicity is severe CRS. In some embodiments, severe toxicity is severe neurotoxicity.

[0045] In any part of the methods provided, if a subject exhibits toxicity, or hematological toxicity, after administration of the compound, the administration of the compound is withheld, and / or the dose of the compound is modified, or reduced, as appropriate. In any part of the methods provided, if a subject exhibits toxicity after administration of the compound, the administration of the compound is withheld. In any part of the methods provided, if a subject exhibits toxicity after administration of the compound, the dose of the compound is reduced. In any part of the methods provided, toxicity is hematological toxicity. In any part of the methods provided, toxicity is severe thrombocytopenia, or, as appropriate, grade 4 thrombocytopenia or prolonged grade 4 thrombocytopenia. In any part of the methods provided, toxicity is severe neutropenia, or, as appropriate, grade 4 neutropenia or prolonged grade 4 neutropenia or febrile neutropenia, or, as appropriate, grade 3 or higher febrile neutropenia or prolonged grade 3 or higher febrile neutropenia.

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

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

[0048] In any part of the methods provided, lymphoma is a B-cell malignancy. In any part of the methods provided, lymphoma is a relapsed / refractory lymphoma. In any part of the methods provided, lymphoma is an invasive lymphoma. In any part of the methods provided, lymphoma is non-Hodgkin lymphoma (NHL), and where appropriate, NHL includes invasive NHL; diffuse large B-cell lymphoma (DLBCL); DLBCL-NOS, where appropriate, 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), where appropriate, follicular lymphoma grade 3B (FL3B); and / or MYC with DLBCL histological features and high-grade B-cell lymphoma (double / triple hit) with BCL2 and / or BCL6 rearrangements.

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

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

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

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

[0053] In any part of the methods provided, the CAR comprises, in order, a cytoplasmic signaling domain derived from a co-stimulatory molecule, which is a CD19-specific scFv; a spacer; a transmembrane domain; and optionally 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.

[0054] In some parts of the methods provided, the spacer is a polypeptide spacer comprising all or part of an immunoglobulin hinge or a modified version thereof, or comprising about 15 amino acids or less. In some parts of the methods provided, the spacer comprises 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 all or part of an immunoglobulin hinge such as IgG4 or a modified version thereof. In some parts of the methods provided, the spacer comprises all or part of an IgG4 hinge or a modified version thereof. In some parts of the methods provided, the spacer comprises about 15 amino acids or less. In some parts of the methods provided, the spacer is 12 amino acids or about 12 amino acids long. In some parts of the methods provided, the spacer is 12 amino acids or about 12 amino acids long. In any part of the methods provided, the spacer has or consists of the sequence of sequence number 1; the sequences coded by sequence number 2, sequence number 30, sequence number 31, sequence number 32, sequence number 33, sequence number 34; or any of the aforementioned variants having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In any part of the methods provided, the spacer has or consists of the sequence of sequence number 1. In some embodiments, the spacer has the sequence coded by sequence number 2.

[0055] In any part of the methods provided, the cytoplasmic signaling domain derived from the co-stimulatory molecule includes the sequence described 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%, or 99% sequence identity thereto. In any part of the methods provided, the cytoplasmic signaling domain derived from the primary signaling ITAM-containing molecule includes any of the sequences in 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%, or 99% sequence identity thereto.

[0056] In any part of the provided method, the scFv includes 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); as well as 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 any part of the methods provided, scFv includes 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); as well as 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 any part of the methods provided, scFv includes a variable light chain region containing the CDRL1 sequence of FMC63, the CDRL2 sequence of FMC63, and the CDRL3 sequence of FMC63, and a variable heavy chain region containing the CDRH1 sequence of FMC63, the CDRH2 sequence of FMC63, and the CDRH3 sequence of FMC63. In any part of the methods provided, scFv includes the variable heavy chain region of FMC63 and the variable light chain region of FMC63. In some embodiments, scFv includes VH, which contains the amino acid sequence described in SEQ ID NO: 41, and VL, which contains the amino acid sequence described in SEQ ID NO: 42. In any part of the provided method, scFv includes 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 aspects of any of the provided methods, the scFv comprises the variable heavy chain region of FMC63 and the variable light chain region of FMC63. In some aspects of any 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 aspects of any of the provided methods, the scFv has the amino acid sequence set forth in SEQ ID NO: 43.

[0057] In some aspects of any of the provided methods, the dose of engineered cells is 1×10 5 cells, or about 1×10 5 cells to 5×10 8 cells, or about 5×10 8 total CAR-expressing T cells; 1×10 6 cells, or about 1×10 6 cells to 2.5×10 8 cells, or about 2.5×10 8 total CAR-expressing T cells; 5×10 6 cells, or about 5×10 6 cells to 1×10 8 cells, or about 1×10 8 total CAR-expressing T cells; 1×10 7 or about 1×10 7 to 2.5×10 8 cells, or about 2.5×10 8 total CAR-expressing T cells; or 5×10 7 cells, or about 5×10 7 cells to 1×10 8 cells, or about 1×10 8 total CAR-expressing T cells, inclusive of the upper and lower limits, respectively. In some aspects of any of the provided methods, the dose of engineered cells comprises 1×10 5 to 5×10 8 total CAR-expressing T cells, or an approximate number thereof, inclusive of the upper and lower limits. In some aspects of any of the provided methods, the dose of engineered cells is 1×10 6 cells, or about 1×10 6 cells to 2.5×10 8 cells, or about 2.5×10 8It contains a total of CAR-expressing T cells (including upper and lower limits). In any part of the method provided, the dose of manipulated cells is 5 × 10⁶ 6 pieces or approximately 5 x 10 6 pieces~1×10 8 individual or approximately 1 x 10 8 It contains a total of CAR-expressing T cells (including upper and lower limits). In any part of the method provided, the dose of the manipulated cells is 1 × 10⁶ 7 individual or approximately 1 x 10 7 pieces~2.5×10 8 1 or approximately 2.5 x 10 8 It contains a total of CAR-expressing T cells (including upper and lower limits). In any part of the method provided, the dose of manipulated cells is 5 × 10⁶ 7 pieces or approximately 5 x 10 7 pieces~1×10 8 individual or approximately 1 x 10 8 Includes a total of [number] CAR-expressing T cells (including upper and lower limits).

[0058] In any part of the method provided, the dose of the manipulated cells is at least 1 × 10⁶ 5 individual or at least approximately 1 × 10 5 A number of CAR-expressing cells, at least 2.5 × 10⁶ 5 individual or at least approximately 2.5 × 10 5 A number of CAR-expressing cells, at least 5 × 10⁶ 5 individual or at least approximately 5 x 10 5 A number of CAR-expressing cells, at least 1 × 10⁶ 6 individual or at least approximately 1 × 10 6 A number of CAR-expressing cells, at least 2.5 × 10⁶ 6 individual or at least approximately 2.5 × 10 6 A number of CAR-expressing cells, at least 5 × 10⁶ 6 individual or at least approximately 5 x 10 6 A number of CAR-expressing cells, at least 1 × 10⁶ 7 individual or at least approximately 1 × 10 7 A number of CAR-expressing cells, at least 2.5 × 10⁶ 7 individual or at least approximately 2.5 × 10 7A number of CAR-expressing cells, at least 5 × 10⁶ 7 individual or at least approximately 5 x 10 7 A number of CAR-expressing cells, at least 1 × 10⁶ 8 individual or at least approximately 1 × 10 8 A number of CAR-expressing cells, at least 2.5 × 10⁶ 8 individual or at least approximately 2.5 × 10 8 10 CAR-expressing cells, or at least 5 × 10⁶ cells 8 individual or at least approximately 5 x 10 8 Contains individual CAR-expressing cells.

[0059] In any part of the method provided, the dose of the manipulated cells is at least 1 × 10⁶ 5 individual or at least about 1 × 10 5 The method provides a dose of at least 2.5 × 10⁶ CAR-expressing cells. 5 individual or at least about 2.5 × 10 5 The method provides for a number of CAR-expressing cells. In any part of the method provided, the dose of the manipulated cells is at least 5 × 10⁶ 5 individual or at least about 5 × 10 5 The method provides a dose of at least 1 × 10⁶ CAR-expressing cells. 6 individual or at least about 1 × 10 6 The method provides a dose of at least 2.5 × 10⁶ CAR-expressing cells. 6 individual or at least about 2.5 × 10 6 The method provides for a number of CAR-expressing cells. In any part of the method provided, the dose of the manipulated cells is at least 5 × 10⁶ 6 individual or at least about 5 × 10 6 The method provides a dose of at least 1 × 10⁶ CAR-expressing cells. 7 individual or at least about 1 × 10 7 The method provides a dose of at least 2.5 × 10⁶ CAR-expressing cells.7 individual or at least about 2.5 × 10 7 The method provides for a number of CAR-expressing cells. In any part of the method provided, the dose of the manipulated cells is at least 5 × 10⁶ 7 individual or at least about 5 × 10 7 The method provides a dose of at least 1 × 10⁶ CAR-expressing cells. 8 individual or at least about 1 × 10 8 The method provides a dose of at least 2.5 × 10⁶ CAR-expressing cells. 8 individual or at least about 2.5 × 10 8 The method provides for a number of CAR-expressing cells. In any part of the method provided, the dose of the manipulated cells is at least 5 × 10⁶ 8 individual or at least about 5 × 10 8 Contains individual CAR-expressing cells.

[0060] In any of the methods provided, the dose of the manipulated cells is 1 × 10⁻⁶ 8 pieces or approximately 1 x 10 8 Contains CAR-expressing T cells.

[0061] In any part of the methods provided, the dose of the manipulated cells is administered parenterally, or intravenously, as appropriate.

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

[0063] In any part of the method provided, the dose of manipulated cells comprises CAR-expressing CD4+ T cells and CAR-expressing CD8+ T cells, and the dose administration comprises the step of 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.

[0064] In any part of the method provided, the first composition and the second composition are administered at intervals of 0 to 12 hours, 0 to 6 hours, or 0 to 2 hours, or the administration of the first composition and the administration of the second composition are performed on the same day at intervals of approximately 0 to approximately 12 hours, approximately 0 to approximately 6 hours, or approximately 0 to approximately 2 hours; and / or the initiation of the administration of the first composition and the initiation of the administration of the second composition are performed at intervals of approximately 1 minute to approximately 1 hour or approximately 5 minutes to approximately 30 minutes. In any part of the method provided, 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.

[0065] In any part of the method provided, the first composition comprises CD4+ T cells. In any part of the method provided, the first composition comprises CD8+ T cells.

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

[0067] In any part of the methods provided, prior to the administration of T-cell therapy, the subject is preconditioned with lymphocyte depletion therapy, including the administration of fludarabine and / or cyclophosphamide. In any part of the methods provided, the method further includes a step of administering lymphocyte depletion therapy, including the administration of fludarabine and / or cyclophosphamide, to the subject immediately before the administration of T-cell therapy.

[0068] In some aspects of any of the provided methods, the lymphocyte depletion therapy is administered daily over 2 to 4 days, optionally 3 days, at about 200 to 400 mg / m 2 (inclusive of the upper and lower limits), optionally 300 mg / m 2 or about 300 mg / m 2 of cyclophosphamide and / or about 20 to 40 mg / m 2 , optionally 30 mg / m 2 of fludarabine; or the lymphocyte depletion therapy comprises administration of about 500 mg / m 2 of cyclophosphamide. In some aspects of any of the provided methods, the lymphocyte depletion therapy comprises daily administration over 3 days of 300 mg / m 2 or about 300 mg / m 2 of cyclophosphamide and about 30 mg / m 2 of fludarabine; and / or the lymphocyte depletion therapy comprises daily administration over 3 days of 500 mg / m 2 or about 500 mg / m 2 of cyclophosphamide and about 30 mg / m 2 of fludarabine.

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

[0070] In any part of the methods provided, at least 35%, at least 40%, or at least 50% of subjects treated according to the method will achieve a sustained complete response (CR) over 6 months or more, or over 9 months or more, or at least 60, 70, 80, 90, or 95% of subjects achieving CR will achieve a sustained CR; and / or at least 60, 70, 80, 90, or 95% of subjects achieving CR by 6 months will maintain a response, maintain CR, over 3 months or more and / or over 6 months or more and / or over 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) over 6 months or more or over 9 months or more, and where applicable, the OR is sustained or sustained in at least 60, 70, 80, 90, or 95% of subjects achieving the OR by 6 months; and / or at least 60, 70, 80, 90, or 95% of subjects achieving the OR by 6 months maintain the response or survive over 3 months or more and / or over 6 months or more.

[0071] In any part of the methods provided, at the time of or immediately before administration of a dose of the engineered cells, the subject relapses or becomes refractory to one or more prior therapies for NHL, including, as appropriate, one, two, or three prior therapies other than another dose of engineered cells expressing CAR. In any part of the methods provided, at the time of or immediately before administration of a dose of the engineered cells, the subject relapses or becomes refractory to one prior therapy for NHL. In any part of the methods provided, at the time of or immediately before administration of a dose of the engineered cells, the subject relapses or becomes refractory to two prior therapies for NHL. In any part of the methods provided, at the time of or immediately before administration of a dose of the engineered cells, the subject relapses or becomes refractory to three prior therapies for NHL. In any part of the methods provided, one or more prior therapies do not include another dose of engineered cells expressing CAR.

[0072] In any part of the methods provided, at the time of or prior to the administration of the manipulated cell dose, the subject is identified as having or suffering from double / triple-hit lymphoma; the subject is identified as having or suffering from chemotherapy-resistant lymphoma, appropriately, chemotherapy-resistant DLBCL; and / or the subject has not achieved complete remission (CR) in response to prior therapy. In any part of the embodiments provided, the subject has not achieved complete remission (CR) in response to prior therapy.

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

[0074] In any part of the method provided, the administration of the compound is initiated after the administration of T-cell therapy, and after the administration or initiation of the compound, the subject shows recovery or rescue of antigen or tumor-specific activity or function of CAR-expressing T cells in the subject, and, as appropriate, the recovery, rescue, and / or initiation of the administration of the compound is made at a point in time after the CAR-expressing T cells in the subject or in the subject's blood have shown an exhausted phenotype.

[0075] In any part of the methods provided, administration of the compound includes (a) resulting in an increase in antigen-specific or antigen-receptor-driven activity of naive or non-exhausted T cells in a subject, which may include T cells expressing the CAR, following T cell exposure to the CD19 antigen or an antigen-receptor-specific agent, compared to the absence of the administration of the compound; or (b) preventing, inhibiting, or delaying the development of an exhaustion phenotype in naive or non-exhausted T cells in a subject, which may include T cells expressing the CAR, after T cell exposure to the CD19 antigen or an antigen-receptor-specific agent, compared to the absence of the administration of the compound; or (c) administration in an amount, frequency, and / or duration effective in reversing an exhaustion phenotype in exhausted T cells, which may include T cells expressing the CAR, compared to the absence of the administration of the compound.

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

[0077] In any part of the method provided, the T cells in the subject include T cells expressing the CAR, and / or the antigen is CD19, for example, exposure is exposure to the CD19 antigen.

[0078] In any part of the methods provided, the exhaustion phenotype includes, in relation to T cells or a population of T cells, an increase in the level or degree of surface expression of one or more exhaustion markers, optionally two, three, four, five, or six exhaustion markers, on T cells or a population of T cells, compared to a reference T cell population under identical conditions; or a decrease in the level or degree of activity exhibited by the T cells or population of T cells after exposure to a CD19 antigen or an antigen receptor-specific agent, compared to a reference T cell population under identical conditions.

[0079] In any part of the methods provided, an increase in level, degree, or percentage is greater than 1.2 times or about 1.2 times, 1.5 times or about 1.5 times, 2.0 times or about 2.0 times, 3 times or about 3 times, 4 times or about 4 times, 5 times or about 5 times, 6 times or about 6 times, 7 times or about 7 times, 8 times or about 8 times, 9 times or about 9 times, 10 times or about 10 times or more. In any part of the methods provided, a decrease in level, degree, or percentage is less than 1 / 1.2 or about 1 / 1.2 times, 1 / 1.5 or about 1 / 1.5 times, 1 / 2.0 or about 1 / 2.0 times, 1 / 3 or about 1 / 3 times, 1 / 4 or about 1 / 4 times, 1 / 5 or about 1 / 5 times, 1 / 6 or about 1 / 6 times, 1 / 7 or about 1 / 7 times, 1 / 8 or about 1 / 8 times, 1 / 9 or about 1 / 9 times, 1 / 10 or about 1 / 10 times or less.

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

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

[0082] In any part of the methods provided, the activity is the production of one or more proliferative, cytotoxic, or inflammatory cytokines, one or more of which may be selected from the group consisting of IL-2, IFN-gamma, and TNF-alpha.

[0083] In any part of the methods provided, exposure to the CD19 antigen or antigen receptor-specific agent includes incubation with the CD19 antigen or antigen receptor-specific agent, optionally an agent that binds to the antigen-binding domain of the CAR. In any part of the methods provided, exposure to the CD19 antigen or antigen receptor-specific agent includes the step of exposing T cells to CD19 antigen-expressing target cells, optionally B cell malignant lesion cells.

[0084] Provided are T-cell therapies comprising a dose of engineered cells, including T cells expressing a CD19-expressing cancer-targeting chimeric antigen receptor (CAR), for use in methods of treating cancer according to one of the methods provided, and combination therapies comprising a compound that is (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate or polymorph thereof.

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

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

[0087] Also provided is the use of T cell therapy comprising a dose of engineered cells, including T cells expressing a CD19-expressing cancer-targeting chimeric antigen receptor (CAR), for treating cancer according to one of the methods provided, and combination therapy comprising the compound (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate or polymorph thereof.

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

[0089] Also provided is the use of T-cell therapy, which includes a dose of engineered cells, including T cells expressing a CD19-expressing cancer-targeting chimeric antigen receptor (CAR), for treating cancer according to one of the methods provided.

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

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

[0092] Also provided is the use of T-cell therapy, comprising a dose of engineered cells including T cells expressing a CD19-expressing cancer-targeting chimeric antigen receptor (CAR), for the manufacture of a pharmaceutical for treating cancer, according to one of the methods provided.

[0093] In some of the embodiments provided, the CAR binds to differentiation antigen group 19 (CD19). In some embodiments, the cancer expressing CD19 is lymphoma.

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

[0095] [Figure 1] Figure 1 shows the expression levels of Aiolos and Ikaros transcription factors in anti-CD19 CAR T cells after stimulation with CAR-specific anti-idiotype antibodies in the presence of various immunomodulatory compounds. [Figure 2A] Figure 2A shows the percentage of living anti-CD19 CAR T cells after stimulation with CAR-specific anti-idiotype antibodies in the presence of various immunomodulatory compounds. [Figure 2B]Figure 2B shows the number of cell duplicates of anti-CD19 CAR T cells after stimulation with CAR-specific anti-idiotype antibodies in the presence of various immunomodulatory compounds. [Figure 2C] Figure 2C shows the number of cell duplicates of anti-CD19 CAR T cells after stimulation with CAR-specific anti-idiotype antibodies 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 CAR-specific anti-idiotype antibodies 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 CAR-specific anti-idiotype antibodies in the presence of various immunomodulatory compounds. [Figure 2F] Figure 2F shows cytokine levels in anti-CD19 CAR T cells after stimulation with CAR-specific anti-idiotype antibodies in the presence of various immunomodulatory compounds. Log-2 values ​​greater than zero are indicated by a "+". [Figure 2G] Figure 2G shows the cell count of anti-CD19 CAR T cells and RL tumor cells treated with compound C. [Figure 2H] Figure 2H shows the cell count of anti-CD19 CAR T cells and RL tumor cells treated with compound 2. [Figure 3A] Figure 3A shows cytokine production by 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 (concurrent therapy). [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 (concurrent therapy). [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 (concurrent therapy). [Figure 3F] Figure 3F shows the IFNγ concentration in the supernatant 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 concurrently treated with compound C. [Figure 3I] Figure 3I shows a comparison of the effects on gene expression profiles (log-2 change) induced by concurrent treatment with compound C 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 concurrent treatment with compound C. [Figure 4A] Figure 4A shows a representative image of CD19-expressing tumor spheroids on day 9 of co-culture with anti-CD19 CAR T cells in the presence of compound C (rescue therapy). [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 therapy). [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 therapy). [Figure 4D] Figure 4D shows the IFNγ concentration in the supernatant of co-cultured cells. [Figure 4E] Figure 4E shows a volcano plot illustrating differentially expressed genes in chronically stimulated anti-CD19 CAR T cells after rescue therapy with compound C. [Figure 4F] Figure 4F shows a comparison of the effect of rescue treatment with Compound C on gene expression profiles (log2 fold change) induced 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 the number of tumor cells upon re-challenge of chronically stimulated CAR T cells in the presence of Compound C (concurrent treatment). [Figure 5B] Figure 5B shows the number of tumor cells upon re-challenge of CAR T cells in the presence of Compound C (rescue treatment). [Figure 6A] Figure 6A shows the predicted plasma concentration levels of Compound C based on a once every 7 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) of 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) of exposure to Compound C. [Figure 6D] Figure 6D shows predicted absolute neutrophil count (ANC) profiles based on the Q7D dosing regimen of Compound C generated using four different pharmacodynamic models. [Figure 7A] Figure 7A shows the number of tumor cells upon re-challenge of chronically stimulated CAR T cells in the presence of Compound C, including that for chronically stimulated CAR T cells treated with 1 day of high-concentration Compound C followed by 5 days of low-concentration Compound C in the presence of Compound C. [Figure 7B]Figure 7B shows the cytolytic activity and CD27 / CCR7 expression of CAR-T cells chronically stimulated in the presence of Compound C, including those chronically stimulated in the presence of Compound C for 1 day at high concentration of Compound C, followed by 6 days at low concentration of Compound C, respectively. [Figure 7C] Figure 7C shows the cytolytic activity and CD27 / CCR7 expression of CAR-T cells chronically stimulated in the presence of Compound C, including those chronically stimulated in the presence of Compound C for 1 day at high concentration of Compound C, followed by 6 days at low concentration of Compound C, respectively. MODE FOR CARRYING OUT THE INVENTION

[0096] Cell therapy, such as cell therapy containing engineered T cells (e.g., CAR-T cells), for the treatment of a subject suffering from cancer or a proliferative disease, and the following structure: [Chemical Formula] (Formula I) Compounds having (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, tautomers or racemic mixtures thereof (compound C), as well as methods and uses of their compositions. In some embodiments, the cell therapy is T cell therapy. In some embodiments, the cell therapy is adoptive T cell therapy comprising T cells that specifically recognize and / or target antigens associated with cancer or proliferative disorders, such as antigens associated with B cell malignancies, e.g., lymphoma, e.g., non-Hodgkin lymphoma (NHL) or its subtypes. In some embodiments, the cell therapy comprises T cells engineered with a chimeric antigen receptor (CAR) that binds to the antigen, e.g., an antigen-binding domain that specifically binds. In some cases, the antigen targeted by cell therapy is CD19. In some embodiments, cancer, such as lymphoma, expresses CD19. Compositions comprising cell therapy and / or compositions comprising compound C, combinations and products comprising compound C, such as kits, as well as the use of such compositions and combinations for treating or preventing diseases, conditions and disorders, including cancer such as lymphoma, are also provided herein.

[0097] (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (compound C) is an oral cereblon modulator (CELMoD). Cereblon functions as a substrate receptor for CRL4 ubiquitin E3 ligase, and binding of cereblon modulator compounds mediates cellular effects by inducing the recruitment, ubiquitination, and disruption of key target substrates such as Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3, also known as Ikaros and Aiolos, respectively). As described herein, administration of compound C results in even more pronounced degradation of Ikaros and Aiolos 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, which is secondary to neutrophil maturation arrest. Neutropenia may develop as a result of depletion of Ikaros, a transcription factor of critical importance in controlling hematopoiesis, and is caused by the late maturation arrest of neutrophil precursors.

[0098] Cell therapies, such as T cell-based therapies, including adoptive T cell therapies (therapies involving the administration of cells expressing chimeric receptors specific to the target disease or disorder, such as chimeric antigen receptors (CARs) and / or other recombinant antigen receptors, as well as other adoptive immune cell and adoptive T cell therapies), may be effective in treating diseases and disorders such as B cell malignancies. Manipulated expression of recombinant receptors such as chimeric antigen receptors (CARs) on the surface of T cells allows for redirection of T cell specificity. In clinical studies, CAR-T cells, such as anti-CD19 CAR-T cells, have resulted in 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, the approaches available for adoptive cell therapy may not always be entirely satisfactory. For example, while persistent CAR T cells can be detected in many subjects with lymphoma, fewer complete responses (CRs) were observed in subjects with NHL compared to those with ALL. More specifically, higher overall response rates (CR rates of 47%–60%) of up to 80% were 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 complete response (CR) rate of 40% (Schuster, Ann Hematol. 2016 Oct;95(11):1805-10).

[0100] In some aspects, the explanation for this is immunological exhaustion of circulating CAR-expressing T cells and / or changes in the T lymphocyte population. This is because, in some situations, optimal efficacy may depend on the ability of administered cells to avoid or reduce immunosuppressive states in the local disease microenvironment, related to differentiation into, transitioning to, or reprogramming into, persistent, including long-term, specific phenotypic states (such as long-term memory, poor differentiation, and effector states), providing effective and robust recall responses after clearance and re-exposure to target ligands or antigens, and having the ability to avoid or reduce differentiation into exhaustion, anergy, peripheral tolerance, terminal differentiation, and / or suppressive states.

[0101] In some embodiments, the exposure, persistence, and function of manipulated cells are reduced or diminished after administration to the subject. In some embodiments, after prolonged stimulation or exposure to an antigen and / or exposure under conditions in the tumor microenvironment, T cells may become functionally impaired and / or exhibit characteristics associated with exhaustion over time. In some embodiments, this reduces the persistence and efficacy of T cells against the antigen and limits their ability to be effective. Nevertheless, observations indicate that in some cases, administered cells expressing recombinant receptors can be re-enlarged and / or reactivated in vivo (e.g., showing an increased number of cells or duration over time) to improve efficacy and therapeutic outcomes in adoptive cell therapy. There is a need for methods to improve the efficacy and function of CAR T cells, in particular, to minimize, reduce, prevent, or reverse functionally impaired or exhausted states.

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

[0103] In this specification, in addition to enhancing T cell function, it is also found that treatment with compound C can reverse, delay, or prevent T cell exhaustion, including by increasing T cell signaling and / or by altering one or more genes that are differentially regulated after chronic stimulation. In some cases, agents that increase or enhance T cell activity can drive cells into an exhausted state, but in this specification, it is found that the activity of compound C, which exerts an enhancing effect on T cell activity, is decoupled from T cell exhaustion. Furthermore, observations in this specification show that compound C exhibits activity that rescues T cells from T cell exhaustion, such as by restoring or partially restoring the activity of one or more T cells after the cells have exhibited exhaustion characteristics. Notably, the results in this specification show that chronically stimulated T cells exhibiting exhaustion characteristics can recover their activity, or have their activity restored or partially restored, after exposure to compound C. Observations in this specification support the idea that the methods provided can also achieve improved or more durable responses compared to certain alternative methods, such as in specific groups of subjects being treated.

[0104] This specification also shows 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, this specification also shows that higher doses of compound C (e.g., 10 and 100 nM) can induce sustained targeted degradation of Ikaros and Aiolos, resulting in reduced cytolytic function. Such results suggest that excessive and sustained targeted degradation of Ikaros and Aiolos can have adverse effects and lead 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 was hypothesized that a single weekly dose would be sufficient to induce deep but transient degradation of Ikaros and Aiolos, thus potentially allowing for the restoration of Ikaros and / or Aiolos expression between doses. As shown herein, the pharmacokinetic model of compound C at a weekly dose of 0.3 mg (Q7D) is approximately 1 nM of C on day 7. min So, the rapid C of compound C max (Approximately 20 nM) and biphasic efflux were demonstrated. The model also showed that a weekly dose of 0.3 mg would have virtually no effect on neutrophil count. Furthermore, 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 flushing and chronic low concentrations of compound C (1 nM for 5 days). This high-to-low concentration exposure 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 adverse effects on cytolytic function.

[0105] Therefore, based on the results shown herein, cell therapy, such as CAR T-cell therapy, combined with intermittent (e.g., once every 7 days) administration of compound C can provide a useful therapeutic approach to prevent premature exhaustion of CAR T cells, enhance and prolong T-cell functionality, and produce a more pronounced and sustained response in cancers such as lymphoma, while also reducing the patient risk of developing neutropenia secondary to neutrophil maturation arrest.

[0106] In some embodiments, the T cell function (including functions related to T cell enlargement, proliferation, and persistence) of manipulated T cell therapy administered according to the provided method is improved by compound C. In some embodiments, the method is beneficial based on the administration of T cell therapy, such as a composition containing cells for adoptive cell therapy, such as T cell therapy (e.g., CAR-expressing T cells), in combination with compound C. In some embodiments, the provided method and use provide or achieve an improved or more sustained response or efficacy compared to a particular alternative method. In some embodiments, the provided method enhances or modulates 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 CAR T cell activity across B cell malignancies by modulating the tumor microenvironment, thereby improving the sustained antitumor function of CAR T cells.

[0107] In some embodiments, compound C is administered to the subject a sufficient time after receiving lymphocyte depletion therapy so that the myelosuppressive effects of compound C and lymphocyte depletion therapy are minimized.

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

[0109] In some embodiments, the method provided requires the administration of T cell therapy, e.g., CAR T cells, and the initiation of compound C administration at a point in time prior to the CAR T cells exhibiting or potentially exhibiting an exhausted phenotype. In some embodiments, the administration of compound C is initiated at a point in time when the CAR T cells are still increasing or are still capable of increasing. In some embodiments, the administration of compound C is initiated before or at a suspected point in time when a peak number of CAR T cells is present in the subject's blood. In some embodiments, the initiation of compound C administration at this point in time enhances CAR T cell function. In some embodiments, the initiation of compound C administration at this point in time also delays or prevents CAR T cell exhaustion.

[0110] In some embodiments, administration of compound C is initiated before or approximately when peak CAR-T cells are present in the subject's blood, for example, within 21 days after the start of T cell administration, or when it is suspected or possible that they are present. In some cases, peak CAR-T cells are present within 11 to 15 days after CAR T cell administration. In some embodiments, administration of compound C is initiated 1 to 15 days after the start of cell therapy administration, for example, 1 day, 8 days, 15 days, or approximately one of those days. In some embodiments, compound C is administered when the subject does not exhibit severe toxicity after cell therapy administration.

[0111] In some embodiments, the methods provided do not result in a high rate or likelihood of toxicity or toxic outcomes, or reduce the rate or likelihood of toxicity or toxic outcomes, such as neurotoxicity (NT), cytokine release syndrome (CRS), or hematological toxicity, such as neutropenia, compared to certain other cell therapies or immunomodulatory drug regimens.

[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, less than 50% of subjects exhibit neutropenia higher than grade 3, e.g., prolonged grade 3 neutropenia or grade 4 neutropenia, and / or thrombocytopenia higher than grade 3, e.g., grade 3 or grade 4 thrombocytopenia. In some embodiments, at least 50% of subjects treated according to the method (e.g., at least 60%, at least 70%, at least 80%, or at least 90% or more of treated subjects) exhibit severe neutropenia or severe thrombocytopenia of grade 3 or higher.

[0113] In some embodiments, the method does not result in, or does not increase the risk of, severe NT (sNT), severe CRS (sCRS), macrophage activation syndrome, tumor lysis syndrome, fever of at least or about 38 degrees Celsius lasting 3 or more days, and a plasma CRP level of at least or about 20 mg / dL. In some embodiments, greater than or about more than 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, no more than 50% of treated subjects (e.g., at least 60%, at least 70%, at least 80%, at least 90% or more of treated subjects) do not exhibit cytokine release syndrome (CRS) higher than grade 2 and / or neurotoxicity higher 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 treated subjects) do not exhibit severe toxic outcomes (e.g., severe CRS or severe neurotoxicity), for example, do not exhibit grade 3 or higher neurotoxicity, and / or do not exhibit severe CRS, or do not do so within a certain period after treatment, such as within 1 week, 2 weeks, or 1 month after administration of the cells.

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

[0115] In some embodiments, compound C is administered at a time when it can efficiently / effectively enhance or prepare the cells. In some embodiments, administration of compound C is initiated at or before the time when the peak or maximum level of cells in the cell therapy becomes detectable in the subject's blood. In some embodiments, the method provided can enhance T-cell therapy, e.g., CAR-T cell therapy, which in some embodiments can improve treatment outcomes. In some embodiments, the method is particularly advantageous in subjects where the cells of the T-cell therapy show weak enlargement, are exhausted, or exhibit reduced or decreased persistence, and / or in subjects with cancer that is resistant or refractory to other therapies and / or is invasive or high-risk cancer.

[0116] In some embodiments, subjects receiving T-cell therapy, such as CAR-T cells, are monitored for the presence, absence, or level of the T cells in the subject in a biological sample of the subject, such as the subject's blood. In some embodiments, the method provided results in genetically modified cells having increased persistence and / or superior potency in the subject to which it is administered. In some embodiments, the persistence of genetically modified cells, such as CAR-expressing T cells, in the subject is superior to the persistence that would be achieved by alternative methods, such as a method involving the administration of T-cell therapy but in the absence of compound C administration. In some embodiments, the persistence is increased by at least or about 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or more.

[0117] In some embodiments, the degree or extent of persistence of administered cells can be detected or quantified after administration to the subject. For example, in some embodiments, quantitative PCR (qPCR) is used to assess the content of cells expressing recombinant receptors (e.g., CAR-expressing cells) in the subject's blood or serum or organ or tissue (e.g., disease site). In some embodiments, persistence is quantified as the number of receptors per microgram of DNA, e.g., copies of CAR-encoding DNA or plasmid, or as the number of CAR-expressing cells per microliter of sample, e.g., blood or serum, or per microliter of sample, e.g., the number of peripheral blood mononuclear cells (PBMCs), leukocytes, or T cells. In some embodiments, flow cytometry assays can also be performed to detect receptor-expressing cells, commonly using receptor-specific antibodies. Cell-based assays can also be used to detect the number or percentage of functional cells, such as cells of a disease or condition, or cells that bind to and / or neutralize and / or respond to cells expressing antigens recognized by the receptor, e.g., cells capable of inducing a cytotoxic response. In any such embodiment, the degree or level of expression of another marker associated with recombinant receptors (e.g., CAR-expressing cells) can be used to distinguish 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 persistence of the response. In some embodiments, the method provided is based on the observation that subjects who achieve or are in complete remission (CR) at 3 months are more likely to sustain a longer-term response, such as surviving or surviving without progression for 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more after the completion of treatment or administration of combination therapy and the initial achievement of complete response (CR). In some embodiments, the method is performed by administering compound C in a specific intermittent dosing regimen described, etc., over a period of at least 3 months or approximately 3 months after the initiation of T-cell therapy.

[0119] In some embodiments, the methods and uses provided offer or achieve an improved or more sustained response or efficacy in a particular group of subjects treated, etc., compared to certain alternative methods, such as T-cell therapy as monotherapy or a method including the administration of compound C, or a method that does not include administration as a combination therapy as described herein. In some embodiments, the methods are advantageous based on the administration of T-cell therapy, such as 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 diseases, such as high-risk NHL. In some embodiments, the methods treat subjects having forms of invasive and / or poor-prognosis B-cell non-Hodgkin lymphoma (NHL), such as NHL that has relapsed or is refractory to standard therapy (R / R) or has a poor prognosis. In some embodiments, subjects treated according to the methods provided 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 method and / or with the provided product, kit, or composition achieve a complete response (CR). In some embodiments, subjects are in CR and exhibit minimal residual disease (MRD). In some embodiments, subjects are in CR and 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 method and / or with the provided product, kit, or composition achieve an objective response of partial response (PR). In some embodiments, at least 60%, at least 70%, at least 80%, at least 90%, and at least 95% or more of subjects treated according to the provided method and / or with the provided products, kits, or compositions achieve complete response (CR) or partial response (PR) at 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year after the start of administration of cell therapy.

[0121] In some embodiments, by 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more after the start of administration of cell therapy, at least 60%, at least 70%, at least 80%, at least 90%, and at least 95% of subjects treated according to the provided method and / or with the provided product, kit, or composition maintain a response, such as maintaining a complete response (CR) or objective response (OR). In some embodiments, such a response, such as a CR or OR, persists for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more in at least 60%, at least 70%, at least 80%, at least 90%, and at least 95% of subjects treated according to the provided method, or in such subjects that achieve 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 method provided and / or with the provided product, kit or composition, or such subjects achieving CR by 3 months, 4 months, 5 months or 6 months, survive or survive without progression for 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or more or approximate number of months.

[0122] All publications, including patent documents, scientific articles, and databases, referenced in this application are incorporated herein by reference in whole for any purpose to the same extent as each individual publication is incorporated herein by reference individually. If any definition provided herein contradicts or is inconsistent with any definition provided herein in a patent, application, published application, or other publication incorporated herein by reference, the definition provided herein shall prevail over the definition incorporated herein by reference.

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

[0124] I. Combination Therapy Manipulated cells for the treatment of cancer patients, e.g., T cells (e.g., CAR-T cells) and (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or formula I [ka] (Equation I) Methods and uses of the compound or pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, tautomers, or racemic mixtures (compound C) (including compositions thereof) are provided herein. In some embodiments, the methods are for treating subjects suffering from lymphoma. In some embodiments, the lymphoma is a B-cell malignancy. In some embodiments, the lymphoma is non-Hodgkin lymphoma (NHL). In some embodiments, the cancer, e.g., lymphoma, expresses CD19. In some embodiments, the methods and uses provide or achieve, for example, an improved response and / or a more sustained response or efficacy in a particular group of subjects treated, compared to a particular alternative method.

[0125] A method for preparing compound C is described in US2019 / 0322647, which is incorporated herein by reference in its entirety.

[0126] In some embodiments, the method and use include 1) administering a cell therapy to a subject that includes T cells expressing a genetically engineered cell surface receptor (e.g., recombinant antigen receptor), such as a chimeric receptor, such as a chimeric antigen receptor (CAR), which recognizes an antigen associated with and / or specific to a lymphoma (e.g., B-cell malignant lesion such as NHL) and / or a cell type from which the lymphoma originates; and 2) administering compound C to a subject. In some embodiments, the administration of compound C is initiated after (following) the administration of the cell therapy, or after (following) the initiation of the administration of the cell therapy. In some cases, compound C is administered to a subject that has received the cell therapy. The method may involve administering one or more doses of engineered cells and two or more doses of compound C to a subject.

[0127] For example, combination therapies comprising engineered cells expressing recombinant receptors such as chimeric antigen receptors (CARs) and compound C, or compositions comprising engineered cells and / or compound C as described herein, are useful in a variety of therapeutic, diagnostic, and prophylactic indications. For example, the combination is useful in the treatment of a variety of diseases and disorders in a subject. Such methods and uses include therapeutic methods and uses involving the administration of a composition containing engineered cells, compound C, and / or one or both to a subject suffering from a disease, condition, or disorder such as a tumor or cancer. In some embodiments, the composition containing engineered cells, compound C, and / or one or both is administered in an effective amount to result in the treatment of the disease or disorder. Uses include the use of a composition containing engineered cells, compound C, and / or one or both in such methods and therapies, and in the preparation of a medicament for carrying out such therapeutic methods. In some embodiments, the method is 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 method thereby treats the disease, condition, or disorder in the subject. In some embodiments, the manipulated cells are any of those described in Section II.

[0128] In some embodiments, the combination therapy is administered to subjects suffering from lymphoma. In some embodiments, the combination therapy is administered to subjects suffering from specific B-cell malignancies. The B-cell malignancies to be treated may be those in which the expression of an antigen is related to and / or involved in the pathogenesis of the B-cell malignancy, e.g., causing the B-cell malignancy, exacerbating it, or otherwise involved in it. Exemplary B-cell malignancies may include diseases or conditions (e.g., cancer) associated with malignant lesions or transformations of cells. Exemplary antigens, including antigens associated with various B-cell malignancies that can be treated, are described herein. In certain embodiments, the chimeric antigen receptor specifically binds to the antigen associated with the disease or condition. In some embodiments, the antigen targeted by the receptor includes antigens associated with B-cell malignancies, such as one 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 includes B cells expressing CD19. It is understood that any description of the method provided herein in which CAR-expressing T cells are specific to CD19 may also be achieved by targeting another B cell antigen or antigen associated with or expressed on the surface of a T cell malignancy, such as any of those described above.

[0129] In some embodiments, the 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 lymphoblastic (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 acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma (NHL), and diffuse large B-cell lymphoma (DLBCL). In some embodiments, the disease or condition is NHL, which is selected from the group consisting of invasive NHL, diffuse large B-cell lymphoma (DLBCL), NOS (de novo and low-grade to transformed), mediastinal primary B-cell large B-cell lymphoma (PMBCL), T-cell / histocyte-rich large B-cell lymphoma (TCHRBCL), Burkitt lymphoma, mantle cell lymphoma (MCL), and / or follicular lymphoma (FL), and, as appropriate, follicular lymphoma grade 3B (FL3B).

[0130] In some embodiments, the method involves treating a subject suffering from lymphoma, such as non-Hodgkin lymphoma (NHL), by administering antigen receptor-expressing cells (e.g., CAR-expressing cells) and compound C. In some embodiments, compound C is administered after or following the administration of recombinant receptor-expressing cells (e.g., CAR-expressing cells), such as after the initiation of administration of recombinant receptor-expressing cells (e.g., CAR-expressing cells).

[0131] In some embodiments, NHL can be staged based on the Lugano classification (see, for example, Cheson et al., (2014) JCO 32(27):3059-3067; Cheson, BD (2015) Chin Clin Oncol 4(1):5). In some cases, the stages are indicated by Roman numerals I-IV (1-4), and limited stage (I or II) lymphomas affecting non-lymphatic organs (extranodal organs) are indicated by E. Stage I represents one nodule or involvement of a group of adjacent nodules, or a single extranodal lesion without nodular involvement (IE). Stage II represents a stage I or II with nodularity involving two or more nodular groups on the ipsilateral side of the diaphragm, or limited involvement of adjacent extranodal organs. Stage III represents nodule involvement on both sides of the diaphragm, or a nodule above the diaphragm with splenic involvement. Stage IV represents involvement of additional non-proximal extranodes. In addition, the term "bulky disease" can be used to describe a large tumor in the chest, particularly for Stage II. The extent of the disease is determined by positron emission tomography (PET) and computed tomography (CT) for lymphomas with uptake, and by CT for histological findings without uptake.

[0132] In some embodiments, the Eastern Cooperative Oncology Group (ECOG) Performance Status Index can be used to assess or select subjects for treatment, for example, subjects who have performed poorly to previous therapies (see, e.g., Oken et al. (1982) Am J Clin Oncol. 5:649-655). In some embodiments, subjects have an ECOG status of 1 or less. The ECOG Performance Status scale represents a patient's functional level 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 perform normal activities. In some embodiments, a subject with an ECOG Performance Status of 1 shows some limitations in physical activity, but the subject is fully able to walk. In some embodiments, a patient with an ECOG Performance Status of 2 is able to walk more than 50%. In some cases, subjects with an ECOG performance status of 2 may be capable of self-care; see, for example, Sorensen et al., (1993) Br J Cancer 67(4) 773-775. The criteria reflecting ECOG performance status are listed in Table 1 below: [Table 1]

[0133] In some embodiments, subjects have or have been identified as having a double / triple-hit lymphoma or a double / triple-hit molecular subtype of lymphoma. In some embodiments, the lymphoma is a double-hit lymphoma characterized by the presence of MYC (myelocyte neoplasm), 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, for example, Aukema et al., (2011) Blood 117:2319-2331. In some aspects of such embodiments, subjects are ECOG 0-1. In aspects, a therapy is indicated for such subjects, and / or 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 lymphoma can be considered as MYC with DLBCL histological features and high-grade B-cell lymphoma (double / triple-hit) with BCL2 and / or BCL6 rearrangements.

[0134] In some embodiments, the combination therapy is administered to subjects who are, may be, or are expected to be, inadequate respondents to cell therapy (e.g., CAR+T cell therapy), and / or who do not respond, may not respond, and / or are expected to not respond, or do not respond within a certain time and / or to a certain degree. In some embodiments, the combination therapy is administered to subjects who do not, are unlikely to, or are not expected to show a complete or overall response within one, two, or three months, etc., after the start of cell therapy administration. In some embodiments, the combination therapy is administered to subjects who exhibit, may exhibit, or are expected to exhibit a progressive disease (PD) within one, two, or three months, etc., after cell therapy administration. In some embodiments, subjects are likely to or are expected to not respond, or to not show a certain response, based on multiple similar subjects who have been treated in this manner or previously treated with cell therapy.

[0135] In some embodiments, the method provided involves a step of treating a specific group or subset of subjects, e.g., subjects identified as having a high-risk disease, e.g., high-risk NHL. In some embodiments, the method treats subjects having a form of invasive and / or poor-prognosis B-cell non-Hodgkin lymphoma (NHL), such as NHL that is relapsed or refractory (R / R) or has a poor prognosis to standard therapy. In some cases, the overall response rate (ORR) to available therapies, standard therapy, or reference therapy for the disease and / or patient population to which the therapy is indicated is less than 40%, and / or the complete response (CR) is less than 20%. In some embodiments, the overall response rate (ORR) to reference or available therapy or standard therapy in chemotherapy-resistant DLBCL is approximately 26%, and the complete response (CR) is approximately 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 embodiments, the methods, compositions, uses, and products provided achieve improved and superior responses to available therapies.

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

[0137] In some embodiments, the subject has been subjected to one, two, three, four, five, or more than six previous therapies. In some embodiments, the subject has been subjected to one previous therapy. In some embodiments, the subject has been subjected to approximately two to four previous therapies. In some embodiments, the subject has been subjected to approximately five to six previous therapies. In some embodiments, the subject has been subjected to more than six previous therapies.

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

[0139] In some embodiments, the combination therapy is administered to subjects who have progressed with prior treatment. In some embodiments, the combination therapy is administered to subjects who have discontinued their response to prior therapy. In some embodiments, the combination therapy is administered to subjects who have relapsed following remission after prior treatment. In some embodiments, the combination therapy is administered to subjects who are refractory to prior treatment. In some embodiments, the combination therapy is administered to subjects who have not achieved an optimal response (e.g., complete response, partial response, or stable disease) to prior therapy.

[0140] In some embodiments, the subject is refractory to the last previously administered therapy. In some embodiments, the subject has relapsed to the last previously administered therapy. If the subject achieved less than a partial response to the last previously administered therapy, the condition is refractory. In some embodiments, the subject has received prior chemotherapy. In some embodiments, the subject is chemotherapy-resistant to prior chemotherapy. In some embodiments, the subject is chemosensible to prior therapy. The condition is chemotherapy-resistant, and the subject achieved stable disease (SD) or progressive disease (PD) to the last chemotherapy-containing regimen, or relapsed less than 12 months after autologous stem cell transplantation. Otherwise, the condition is chemosensible.

[0141] In some embodiments, the prior treatment or therapy includes a CD20 targeting agent. In some embodiments, the prior treatment or therapy includes an anthracycline. In some embodiments, the prior 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 products involve or are used to treat subjects that include a step of selecting or identifying a specific group or subset of subjects based on a particular type of disease, diagnostic criteria, prior treatment and / or response to prior treatment, such as subjects in any of the groups described. In some embodiments, the method involves a step of treating subjects that have relapsed or become refractory to one or more prior therapies following remission after treatment with one or more prior therapies; or subjects that have relapsed or become refractory (R / R) to one or more prior therapies, e.g., first-line or higher-order standard therapy, e.g., cell therapy (e.g., CAR+ T cell). In some embodiments, the method involves a step of treating subjects suffering from diffuse large B-cell lymphoma (DLBCL), unspecified (NOS; de novo and low-grade to 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 method involves a step of treating subjects with an East Coast Cancer Clinical Trial Group Performance Status (ECOG) of less than 1, such as 0 to 1. In some embodiments, the method treats a poor-prognosis population or subjects of DLBCL patients who generally do not respond well to therapy or specific reference therapy, such as those with one or more, for example, two or three chromosomal translocations (so-called "double-hit" or "triple-hit" lymphomas, which are high-grade B-cell lymphomas with DLBCL histological features, MYC and BCL2 and / or BCL6 rearrangements; this includes those with the translocated MYC / 8q24 locus, usually combined with the t(14;18)(q32;q21)bcl-2 gene or / and the 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, relapsed as appropriate, and / or those suspected of being chemotherapy-resistant.

[0143] In some embodiments, subjects have germinal center-like (GCB) DLBCL. In some embodiments, subjects have non-germinal center-like (non-GCB) DLBCL. In some embodiments, subjects have double-hit lymphoma (DHL). In some embodiments, subjects have triple-hit lymphoma (THL). In some embodiments, subjects are positive for gene expression indicating responsiveness to treatment with compound C. In some embodiments, subjects are negative for gene expression. 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 NHL. In some embodiments, the antigen associated with the 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 method includes cell therapy and administration of compound C to subjects at risk of or suspected of having B-cell malignancy.

[0146] In some embodiments, the method involves administering cells to subjects selected or identified as having a particular prognosis or risk of NHL. Non-Hodgkin lymphoma (NHL) can be a variable disease. Some subjects with NHL may 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 may be “low-risk,” “moderate-risk,” “high-risk,” and / or “very high-risk,” and patients may 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 the manufactured product or composition are classified or identified based on their risk of NHL. In some embodiments, the subjects are those with high-risk NHL.

[0147] In some embodiments, the subjects to be treated include a group of subjects suffering from invasive NHL, particularly diffuse large B-cell lymphoma (DLBCL), unspecified (NOS; de novo and low-grade to transformed), T-cell / histiocyte-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 MYC with DLBCL histological features, as well as high-grade B-cell lymphoma ("double-hit" or "triple-hit" lymphoma) with BCL2 and / or BCL6 rearrangements. In some embodiments, the disease of the subjects has relapsed or is refractory to at least two previous-line therapies. In some embodiments, the prior therapies include CD20-targeted agents and / or anthracyclines. In some embodiments, the subjects have an ECOG score of 0–1 at screening. In some embodiments, the subjects have positron emission tomography (PET)-positive disease according to the Lugano classification (Cheson, 2014). In some embodiments, the subjects may have been previously treated with allogeneic stem cell transplantation (SCT).

[0148] In some embodiments, the subjects are adults. In some embodiments, the subjects are male. In some embodiments, the subjects are female. In some embodiments, the subjects are at least 40 years old at the time of administration of the combination therapy (e.g., at the time of administration of cell therapy). In some embodiments, the subjects are under 40 years old at the time of administration of the combination therapy (e.g., at the time of administration of cell therapy). In some embodiments, the subjects are approximately 40 to 65 years old at the time of administration of the combination therapy (e.g., at the time of administration of cell therapy). In some embodiments, the subjects are at least 65 years old at the time of administration of the combination therapy (e.g., at the time of administration of cell therapy).

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

[0150] In some embodiments, cells administered for use in or in connection with the provided method contain or are engineered to contain engineered receptors, such as engineered antigen receptors, such as chimeric antigen receptors (CARs) or T cell receptors (TCRs). Among the compositions, pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy, are particularly mentioned. Methods of therapy for administering cells and compositions to a subject, such as a patient, are also provided in conjunction with the provided method and / or using the provided products or compositions.

[0151] Cells generally express recombinant receptors, including functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs), and other antigen-binding receptors such as transgenic T cell receptors (TCRs). Among receptors, other chimeric receptors are particularly noteworthy. Exemplary engineered cells for administration as cell therapy in the provided method are described in Section II.

[0152] In some embodiments, cell therapy, such as adoptive T-cell therapy, is performed by autotransfer, in which case the 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 embodiments, the cells are derived from a subject in need of treatment, such as a patient, and the cells are administered to the same subject after isolation and processing.

[0153] In some embodiments, cell therapy, such as adoptive T-cell therapy, is performed by allogeneic transfer, in which case cells are isolated from and / or otherwise prepared from a subject other than the subject that is to receive or will ultimately receive the 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] T-cell therapy cells can be administered in a composition formulated for administration, or alternatively, in two or more compositions formulated for separate administrations (e.g., two compositions). The cell dose may include a specific number or relative number of cells or engineered cells, and / or two or more subtypes of defined ratios or compositions within the composition, such as CD4 vs CD8 T cells.

[0155] Cells can be administered by any suitable means, for example, by bolus injection, injection, such as intravenous or subcutaneous injection, intraocular injection, periorbital injection, subretinal injection, intravitreous injection, transseptal injection, subscleral injection, intrachoroidal injection, anterior chamber injection, subconjectval injection, subconjunctival injection, sub-Tenon injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, cells are administered parenterally, intrapulmonaryly and intranasally, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intra-arterial, 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 bolus administrations of cells over a period of, for example, three days or less, or by continuous infusion administration of cells. In some embodiments, the administration of cellular doses or any additional therapies, such as lymphocyte depletion therapy, intervention therapy, and / or combination therapy, is carried out by outpatient delivery.

[0156] For the treatment of a disease, the appropriate dosage may depend on the type of disease to be treated, the type of cell or recombinant receptor, the severity and course of the disease, previous therapies, the subject's clinical history and response to the cells, and the discretion of the attending physician. The composition and cells are administered appropriately to the subject, either in a single dose or over a series of treatments, in some embodiments.

[0157] Preconditioning of patients with immunodepletion therapy (e.g., lymphocyte depletion) can, in some embodiments, improve the efficacy of adoptive cell therapy (ACT).

[0158] Therefore, in some embodiments, the method includes administering a preconditioning agent, such as a lymphocyte depletion or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof, to the subject before the initiation of cell therapy. For example, the subject may be administered the preconditioning agent at least two days before the initiation of cell therapy, such as at least three, four, five, six, or seven days before. In some embodiments, the subject is administered the preconditioning agent seven days or less before the initiation of cell therapy, such as six, five, four, three, or two days or less before.

[0159] In some embodiments, subjects are preconditioned with cyclophosphamide in doses between 20 mg / kg and 100 mg / kg, or approximately between 40 mg / kg and 80 mg / kg. In some embodiments, subjects are preconditioned with 60 mg / kg or approximately 60 mg / kg of cyclophosphamide. In some embodiments, cyclophosphamide may be administered in a single dose or in multiple doses, such as daily, every other day, or every three days. In some embodiments, cyclophosphamide is administered once daily over one or two days. In some embodiments, if the lymphocyte depletion agent contains cyclophosphamide, subjects receive 200 mg / m². 2 ~400mg / m 2 Alternatively, 250 mg / m² 2 ~350mg / m 2 100 mg / m² 2 ~500mg / m 2 The patient is administered cyclophosphamide in doses between or approximately between (including upper and lower limits). In some cases, the patient is given approximately 300 mg / m². 2 The patient is administered cyclophosphamide. In some cases, the patient receives approximately 500 mg / m². 2The patient is administered cyclophosphamide. In some embodiments, cyclophosphamide may be administered in a single dose, or in multiple doses, such as daily, every other day, or every three days. In some embodiments, cyclophosphamide is administered daily for 1 to 5 days, for example, 3 to 5 days. In some examples, the patient receives approximately 300 mg / m² daily for 3 days prior to the initiation of cell therapy. 2 The patient is administered cyclophosphamide. In some cases, the patient receives approximately 500 mg / m² daily for three days prior to the initiation of cell therapy. 2 The patient is administered cyclophosphamide.

[0160] In some embodiments, if the lymphocyte depletion agent contains fludarabine, the target dose is 10 mg / m². 2 ~75mg / m 2 , 15 mg / m² 2 ~50mg / m 2 , 20 mg / m² 2 ~40mg / m 2 or 24 mg / m² 2 ~35mg / m 2 Between or approximately between 1 mg / m² 2 ~100mg / m 2 Fludarabine is administered in doses between or approximately between (including upper and lower limits). In some cases, subjects are given approximately 30 mg / m². 2 Fludarabine is administered. In some embodiments, fludarabine may be administered in a single dose, or in multiple doses, such as daily, every other day, or every three days. In some embodiments, fludarabine is administered daily for 1 to 5 days, for example, 3 to 5 days. In some examples, subjects are given approximately 30 mg / m² daily for 3 days prior to the initiation of cell therapy. 2 Fludarabine is administered.

[0161] In some embodiments, the lymphocyte depletion agent includes a combination of drugs, such as a combination of cyclophosphamide and fludarabine. Thus, the drug combination may 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 embodiments, the subject is given 60 mg / kg (approximately 2 g / m²) before the first or subsequent dose. 2 ) Cyclophosphamide and 3-5 doses of 25 mg / m² 2 Fludarabine is administered. In some embodiments, subjects receive 300 mg / m² daily for 3 days prior to the initiation of cell therapy. 2 Cyclophosphamide and 30 mg / m² 2 Fludarabine is administered. In some embodiments, subjects receive 500 mg / m² daily for 3 days prior to the initiation of cell therapy. 2 Cyclophosphamide and 30 mg / m² 2 Fludarabine is administered.

[0162] Following cell administration, the bioactivity of the engineered cell population is measured in some embodiments, for example, by one of numerous known methods. Parameters for evaluation include the specific binding of engineered or native 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 engineered cells to destroy target cells can be measured using any suitable known method, such as the cytotoxicity assays described in, for example, 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 bioactivity of 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 embodiments, bioactivity is measured by evaluating clinical outcomes, such as reduction of tumor burden or burden.

[0163] 1. Compositions and Formulations In some embodiments, the dose of cells for cell therapies such as T-cell therapy, which includes recombinant antigen receptors, for example, cells manipulated by CAR or TCR, is provided as a composition or formulation, such as a pharmaceutical composition or formulation. Such compositions can be used in accordance with the provided method and / or with the provided product or composition, such as in the treatment of B-cell malignancies.

[0164] The term "pharmaceutical preparation" 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 recipient.

[0165] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical preparation other than the active ingredient that is non-toxic to the target. 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 embodiments, the choice of carrier is determined, in part, by the specific cells or drug and / or by the method of administration. Thus, a variety of suitable formulations exist. For example, a pharmaceutical composition may contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The preservative or mixture thereof is typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmacoherent carriers are generally non-toxic to the recipient at the dosage and concentration used, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); and low molecular weight (less than approximately 10 residues) polypeptides. This material includes, but is not limited to, butylene; proteins such as serum albumin, gelatin, or immunoglobulin; 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 dextrin; 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 nonionic surfactants such as polyethylene glycol (PEG).

[0167] Buffers are included in the composition in some embodiments. Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, mixtures of two or more buffers are used. Buffers or mixtures thereof are 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 detail, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0168] The formulation may include an aqueous solution. The formulation or composition may also contain two or more active ingredients useful for a specific indication, disease, or condition being treated by the cells or drug, in which case the activity of each does not adversely affect the others. Such active ingredients are appropriately present in combination in amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further includes chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc., or other pharmaceutically active agents or drugs.

[0169] In some embodiments, the pharmaceutical composition contains cells in an amount effective to treat a disease or condition, such as a therapeutically effective or prophylactically effective dose. Therapeutic effectiveness is monitored in some embodiments by periodic evaluation of the treated subject. For repeated administration over several days or longer periods, treatment is repeated, depending on the condition, 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, multiple bolus, or continuous infusion 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 the storage and administration of compositions. With respect to cells, administration can be autologous or heterologous. For example, immune-responsive cells or precursors can be obtained from a subject and administered to the same subject or different compatible subjects. Peripheral blood-derived immune-responsive cells or their progeny (e.g., in vivo, ex vivo, or in vitro-derived) can be administered by localized injection, systemic injection, intravenous injection, or parenteral administration, including catheter administration. When administering therapeutic compositions (e.g., pharmaceutical compositions containing genetically modified immune-responsive cells), these will generally be formulated in unit-dose injectable forms (solutions, suspensions, emulsions).

[0171] The formulations include formulations for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, percutaneous, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the drug 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 drug or cell population is administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0172] The compositions are provided, in some embodiments and in some aspects, as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which can be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions are somewhat easier to administer, particularly by injection. Viscous compositions, on the other hand, can be formulated within a suitable viscosity range to provide a longer contact period with specific tissues. Liquid or viscous compositions may include, for example, a carrier that can serve as a solvent or dispersion medium containing water, saline solution, phosphate-buffered saline solution, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0173] Sterile injectable solutions can be prepared by incorporating cells into a solvent such as a mixture of sterile water, physiological saline, glucose, dextrose, or other suitable carriers, diluents, or excipients.

[0174] Preparations intended for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile filtration membrane. 2. Medication

[0175] In some embodiments, the dose of cells is administered to the subject in accordance with the method provided and / or using the provided product or composition. In some embodiments, the size or timing of the dose is determined as a function of the specific disease or condition in the subject (e.g., cancer, e.g., B-cell malignancy). In some cases, the size or timing of the dose for a particular disease, considering the provided description, can be determined empirically.

[0176] In some embodiments, the cell dose is 4 × 10 5 individual or approximately 4 x 10 5 cells / kg ~ 1 × 10⁻⁶ 6 individual or approximately 1 x 10 6 Between individual cells / kg or 6 × 10 5 pieces or approximately 6 x 10 5 cells / kg ~ 8 × 10 5 pieces or approximately 8 x 10 5 Between individual cells / kg, etc., 2 × 10 5 pc or approximately 2 × 10 5 cells / kg ~ 2 × 10 6 pc or approximately 2 × 10 6 This includes between individual cells / kg. In some embodiments, the cell dose is 3 × 10⁶ 5 Cells / kg or less, or approximately 3 x 10⁻⁶ 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 10⁻⁶ 5 Cells / kg or less, 7×10 5 Cells / kg or less, or approximately 7 x 10⁻⁶ 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 6 Cells / kg or less or 2 × 10⁻⁶ 6 Cells / kg or less, or approximately 2 x 10⁻⁶ 6 Cells / kg or less, etc., 2 x 10 per kilogram of the subject's body weight 5 It contains a number of cells (e.g., antigen-expressing cells, e.g., CAR-expressing cells) (cells / kg) or less. In some embodiments, the cell dose is at least 3 × 10⁶ 5 cells / kg or at least approximately 3 × 10⁻⁶ 5 cells / kg or its value or approximate number, at least 4 × 10 5 cells / kg or at least approximately 4 × 10⁻⁶ 5 Cells / kg or its value or approximate number, at least 5 × 10 5 cells / kg or at least approximately 5 × 10⁻⁶ 5 Cells / kg or its value or approximate number, at least 6 × 10⁻⁶ 5 cells / kg or at least approximately 6 × 10⁻⁶ 5 Cells / kg or its value or approximate number, at least 7 × 10⁻⁶ 5 cells / kg or at least approximately 7 × 10⁻⁶ 5 cells / kg or its value or approximate number, at least 8 × 10 5 cells / kg or at least approximately 8 × 10⁻⁶ 5 cells / kg or its value or approximate number, at least 9 × 10 5 cells / kg or at least approximately 9 × 10⁶ 5 cells / kg or its value or approximate number, at least 1 × 10⁻⁶ 6 cells / kg or at least about 1 × 10⁻⁶ 6cells / kg or that value or approximate number, or at least 2 × 10⁻⁶ 6 cells / kg or at least about 2 × 10⁻⁶ 6 Cells / kg, or its value or approximate number, etc., at least 2 × 10 per kilogram of the subject's body weight. 5 individual or at least approximately 2 × 10 5 This includes cells (e.g., antigen-expressing cells, e.g., CAR-expressing cells) (cells / kg), which are either individual, a value of that number, or an approximate number of that number.

[0177] In certain embodiments, a cell, or an individual population or subtype of cells, is in the range of about 1 million to about 100 billion cells and / or the amount of cells per kilogram of body weight, for example, 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 aforementioned values), for example, 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) The dosage is administered to the subject in a number of cells (approximately 80 million, 90 million, 10 billion, 25 billion, 50 billion, 75 billion, 90 billion, or a range defined by any two of the aforementioned values), and in some cases, approximately 100 million to 50 billion cells (e.g., approximately 120 million, 250 million, 350 million, 450 million, 650 million, 800 million, 900 million, 3 billion, 30 billion, or 45 billion), or any value within these ranges, and / or per kilogram of body weight. The dosage may vary depending on the specific characteristics of the disease or disorder and / or the patient and / or other treatment.

[0178] In some embodiments, the cell dose is a flat dose or a fixed dose of cells, such that the cell dose 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 human, the dose is approximately 5 × 10 8 Fewer than 1 total recombinant receptor (e.g., CAR) expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs), e.g., about 1 × 10⁶ 6 ~5×10 8 Such cells within a range of 10, for example, 2 × 10 6 , 5×10 6 , 1 x 10 7 , 5×10 7 , 1 x 10 8 , 2×10 8 , 3 x 10 8 Or 4 x 10 8 This includes all of the individual such cells, or a range between any two of the aforementioned values. In some embodiments, if the subject is human, the dose is approximately 1 × 10⁻⁶ 6 ~3×10 8 Total recombinant receptor (e.g., CAR) expressing cells between individuals, e.g., approximately 1 × 10⁶ 7 ~2×10 8 Within the range of a single such cell, for example, 1 × 10 7 , 5×10 7 , 1 x 10 8 Or 1.5 × 10 8 The total number of such cells, or a range between any two of the aforementioned values. In some embodiments, the patient is administered multiple doses, and each dose or the total dose may be within any of the aforementioned values. In some embodiments, the dose of cells is 1 × 10⁻⁶ 5 individual or approximately 1 x 10 5 pieces~5×10 8 pieces or approximately 5 x 10 8 1 × 10¹ total recombinant receptor (e.g., CAR) expressing T cells or total T cells 5 ~1 × 10 8 5 × 10¹ total recombinant receptor (e.g., CAR) expressing T cells or total T cells 5 pieces or approximately 5 x 105 pieces~1×10 7 individual or approximately 1 x 10 7 1 x 10⁶ total recombinant receptor (e.g., CAR) expressing T cells or total T cells, or 1 x 10⁶ 6 individual or approximately 1 x 10 6 pieces~1×10 7 individual or approximately 1 x 10 7 This includes the administration of a total recombinant receptor (e.g., CAR) expressing T cells or total T cells (including upper and lower limits for each).

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

[0181] In some embodiments, for example, when the subject is human, the dose of CD8+ T cells includes CD4+ and CD8+ T cells, with a dose of approximately 1 × 10⁻⁶ CD8+ T cells. 6 ~1 × 10 8 Total recombinant receptor (e.g., CAR) expression CD8+ cells between individuals, e.g., approximately 5 × 10⁻⁶ 6 ~1 × 10 8 Such cells within a range of 1 × 10 7 , 2.5×10 7 , 5×10 7 , 7.5×10 7 Or 1 x 10 8 The total number of such cells may include all of them, or a range between any two of the aforementioned values. In some embodiments, the patient is administered multiple doses, and each dose or the total dose may fall within any of the aforementioned values. In some embodiments, the dose of cells may be 1 × 10⁻⁶ 7 ~0.75 × 10 8 1 × 10¹ or approximately 10¹⁶ total recombinant receptor-expressing CD8+ T cells 7 ~2.5×10 7 1 × 10¹ total recombinant receptor-expressing CD8+ T cells 7 ~0.75 × 10 8 This involves administering a total of 10¹ or an approximate number of recombinant receptor-expressing CD8+ T cells (including upper and lower limits for each). In some embodiments, the cell dose is 1 × 10¹⁶ 7 , 2.5×107 , 5×10 7 , 7.5×10 7 Or 1 x 10 8 This includes administration of one or approximately one total recombinant receptor-expressing CD8+ T cells.

[0182] In some embodiments, for example, when the subject is human, the dose of CD4+ T cells, including CD4+ and CD8+ T cells, is approximately 1 × 10⁻⁶. 6 ~1 × 10 8 Total recombinant receptor (e.g., CAR) expression CD4+ cells between individuals, e.g., approximately 5 × 10⁻⁶ 6 ~1 × 10 8 A number of such cells, within the range of such cells, 1 × 10 7 , 2.5×10 7 , 5×10 7 , 7.5×10 7 Or 1 x 10 8 The total number of such cells, or a range between any two of the aforementioned values. In some embodiments, the patient is administered multiple doses, and each dose or the total dose may be within any of the aforementioned values. In some embodiments, the dose of cells is 1 × 10⁻⁶ 7 pieces or approximately 1 x 10 7 pieces~0.75×10 8 individual or approximately 0.75 × 10 8 1 x 10¹ total recombinant receptor-expressing CD4+ T cells 7 ~2.5×10 7 1 x 10¹ total recombinant receptor-expressing CD4+ T cells 7 pieces or approximately 1 x 10 7 pieces~0.75×10 8 individual or approximately 0.75 × 10 8 This involves administering a total recombinant receptor-expressing CD4+ T cell (including upper and lower limits for each). In some embodiments, the cell dose is 1 × 10⁶ 7 , 2.5×10 7 , 5×10 7 , 7.5×10 7 Or 1 x 10 8 This involves administering one or approximately one total recombinant receptor-expressing CD4+ T cells.

[0183] In some embodiments, the dose of cells, for example recombinant receptor-expressing T cells, is administered to the subject as a single dose, or only once over a period of 2 weeks, 1 month, 3 months, 6 months, 1 year or more.

[0184] In the context of adoptive cell therapy, a given “dose” includes the administration of a given amount or number of cells as a single composition and / or a single uninterrupted administration, for example, as a single injection or continuous infusion, and also includes the administration of a given amount or number of cells as a divided dose 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, the dose is a single or continuous administration of a specified number of cells given or initiated at a single point in time. However, in some situations, the dose is administered in multiple injections or infusions over a period of three days or less, such as by multiple infusions over a period of three days or two days, or over a period of one day.

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

[0186] In some embodiments, the term “divided dose” refers to a dose divided to be administered over more than one day. This type of dosing is encompassed by this method and is considered a single dose.

[0187] Therefore, the dose of cells can be administered as a divided dose, for example, a divided dose administered over time. For example, in some embodiments, the dose may be administered to a subject over two or three days. An exemplary method for divided dosing includes administering 25% of the dose on day 1 and the remaining 75% on day 2. In other embodiments, 33% of the dose may be administered on day 1 and the remaining 67% on day 2. In some embodiments, 10% of the dose is administered on day 1, 30% on day 2, and 60% on day 3. In some embodiments, the divided dose does not extend beyond three days.

[0188] In some embodiments, a dose of cells may be administered by administering a plurality of compositions or solutions, such as a first and second, and optionally more, each containing a portion of the dose of cells. In some embodiments, a plurality of compositions each containing different populations and / or subtypes of cells may be administered separately or independently, as appropriate, within a specific period of time. For example, the populations or subtypes of cells may each contain CD8 + and CD4 + The population may consist of T cells and / or CD8+ and CD4+ enriched populations, respectively, for example, CD4+ and / or CD8+ T cells, each comprising, individually, cells genetically engineered to express recombinant receptors. In some embodiments, dose administration may consist of administration of a first composition comprising a dose of CD8+ T cells or a dose of CD4+ T cells, and administration of a second composition comprising the other of doses of CD4+ T cells and CD8+ T cells.

[0189] In some embodiments, the administration of a composition or dose, for example, the administration of multiple cell compositions, involves the administration of separate cell compositions. In some embodiments, the separate administrations are carried out simultaneously, sequentially, or in any order. In some embodiments, the dose comprises a first composition and a second composition, and the first and second compositions are administered at intervals of 0 to 12 hours, 0 to 6 hours, or 0 to 2 hours. In some embodiments, the initiation of the administration of the first composition and the initiation of the administration of the second composition are carried out at intervals of 2 hours or less, 1 hour or less, or 30 minutes or less, and at intervals of 15 minutes or less, 10 minutes or less, or 5 minutes or less. In some embodiments, the initiation and / or completion of the administration of the first composition and the completion and / or initiation of the administration of the second composition are carried out at intervals of 2 hours or less, 1 hour or less, or 30 minutes or less, and at intervals of 15 minutes or less, 10 minutes or less, or 5 minutes or less.

[0190] In some embodiments, the first and second compositions are mixed before administration to the subject. In some embodiments, the first and second compositions are mixed a short time before administration (e.g., within 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1.5 hours, 1 hour, or 0.5 hours). In some embodiments, the first and second compositions are mixed immediately before administration.

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

[0192] In some embodiments, the dose or composition of cells comprises a defined or targeted ratio of CD4+ cells expressing recombinant receptors versus CD8+ cells expressing recombinant receptors, and / or CD4+ cells versus CD8+ cells, where this ratio is, as appropriate, approximately 1:1 or between approximately 1:3 and approximately 3:1, for example, approximately 1:1. In some embodiments, the administration of a composition or dose of different cell populations in a targeted or desired ratio (CD4+:CD8+ ratio or CAR+CD4+:CAR+CD8+ ratio, for example, 1:1) involves the administration of a cell composition containing one of the populations, followed by the administration of a separate cell composition containing the other of the population, where the administration is exactly or approximately the targeted or desired ratio. In some embodiments, the administration of a dose or composition of cells in a defined ratio results in improved enhancement, persistence, and / or antitumor activity of T-cell therapy.

[0193] In some embodiments, the subject receives multiple doses of cells, e.g., two or more doses or multiple consecutive doses. In some embodiments, two doses are administered to the subject. In some embodiments, the subject receives a consecutive dose, e.g., a 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, so that an additional dose is administered after the administration of the consecutive dose. In some embodiments, the number of cells administered to the subject in the additional dose is the same as or similar to that of the first dose and / or 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 successive doses is determined based on one or more criteria, such as the subject's response to prior treatment, e.g., chemotherapy; the disease burden in the subject, e.g., tumor volume, bulk, size or extent; the extent or type, stage of metastasis; and / or toxic outcomes, e.g., the likelihood or incidence of the subject developing CRS, macrophage activation syndrome, oncolytic syndrome, neurotoxicity; and / or the host immune response to the cells and / or recombinant receptors being administered.

[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 is given 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 after the administration of the previous dose but less than about 28 days after. In some embodiments, the additional dose is administered less than about 14 days after the previous dose, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 days after the previous dose. In some embodiments, the dose is not administered less than about 14 days after the previous dose, and / or the dose is not administered more than about 28 days after the previous dose.

[0196] In some embodiments, the dose of cells, for example recombinant receptor-expressing cells, comprises two doses (e.g., a double dose) including a first dose and a continuous dose of T cells, where one or both of the first and second doses include administration of divided doses of T cells.

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

[0198] In some embodiments, cells are administered in a desired dosage, which in some embodiments 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 of body weight) and a 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 of cells or individual cell types (or number per kg of body weight) in individual populations. In some embodiments, the dosage is based on a combination of such features, such as a desired total number of cells, a desired ratio, and a desired total number of cells in individual populations.

[0199] In some embodiments, CD8 + and CD4 + A population or subtype of cells, such as T cells, is administered within a desired dose of total cells, such as a desired dose of T cells, or within an acceptable difference thereof. 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 recipient, e.g., cells / kg. In some embodiments, the desired dose is the minimum number of cells or the minimum number of cells per unit of body weight, or greater than that. In some embodiments, within the total cells administered at the desired dose, individual populations or subtypes have a desired output ratio (CD4 + vs CD8 + (e.g., a ratio of ) or near such a ratio, for example, existing within a certain allowable difference or error of such a ratio.

[0200] In some embodiments, cells are administered in desired doses of one or more 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 between them. In some embodiments, the desired dose is a desired number of cells of a subtype or population, or a desired number of such cells per unit of body weight of the person to whom the cells are administered, e.g., cells / kg. In some embodiments, the desired dose is 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, or greater than that.

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

[0202] In some embodiments, cells are administered in 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 embodiments, the desired ratio may be a specific ratio or a range of ratios. For example, in some embodiments, the desired ratio (e.g., CD4+) + vs CD8 + The cell ratio is between 5:1 or an approximate number and 5:1 or an approximate number (or greater than approximately 1:5 but less than approximately 5:1), or between 1:3 or an approximate number and 3:1 or an approximate number (or greater than approximately 1:3 but less than approximately 3:1), for example, between 2:1 or an approximate number and 1:5 or an approximate number (or greater than approximately 1:5 but less than approximately 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 approximate number thereof. In some embodiments, the acceptable difference is within approximately 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, and 50% of the desired ratio, and includes any value within 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 total cells, T cells, or peripheral blood mononuclear cells (PBMCs) administered.

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

[0205] In some embodiments, the method also includes the step of administering one or more additional doses of cells expressing a chimeric antigen receptor (CAR), and / or lymphocyte depletion therapy, and / or one or more steps of the method are repeated. In some embodiments, one or more additional doses are the same as the initial dose. In some embodiments, one or more additional doses are different from the initial dose, for example, higher than the initial dose, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher, or lower than the initial dose, for example, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9 or 1 / 10 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 prior treatment, the disease burden in the subject, e.g., tumor volume, bulk, size or extent, degree or type, stage of metastasis, and / or toxic outcomes, e.g., the subject's likelihood or incidence of developing CRS, macrophage activation syndrome, oncolytic syndrome, neurotoxicity, and / or the host immune response to the cells and / or recombinant receptors being administered.

[0206] B. Administration of compound C In some embodiments of the methods, compositions, combinations, kits, or products provided herein, the combination therapy has the chemical name (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione and / or formula I: [ka] (Equation I) The process includes administering compound C having the structure, or its enantiomer or mixture of enantiomers, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate or polymorph thereof.

[0207] In some embodiments, compound C is an enantiomer or mixture of enantiomers of (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate or polymorph of (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione. In some embodiments, compound C is the solvate of (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione. In some embodiments, compound C is the hydrate of (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione. In some embodiments, compound C is a pharmaceutically acceptable salt of (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione. In some embodiments, compound C is (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-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 an anhydride. In certain embodiments, compound C is nonhygroscopic.

[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-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-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 as well as organic acids and bases. Suitable pharmaceutically acceptable base addition salts of compound C include metal 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, ethenesulfonic acid, formic acid, fumaric acid, furic 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, mucinic 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, for example, Remington's Pharmaceutical Sciences, 18 th eds., Mack Publishing, Easton PA (1990) or Remington: The Science and Practice of Pharmacy, 19 th See eds., Mack Publishing, Easton PA (1995).

[0211] In certain embodiments, compound C is the hydrochloride salt of (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or its enantiomer or mixture of enantiomers; or a pharmaceutically acceptable solvate, hydrate, cocrystal, clathrate or polymorph thereof. In certain embodiments, the hydrochloride salt is solid. In certain embodiments, the hydrochloride salt is anhydrous. In certain embodiments, the hydrochloride salt is nonhygroscopic. 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 terms “stereoisomer” or “stereomerically pure” mean one stereoisomer of a compound that substantially contains no other stereoisomers of the compound. For example, a stereochemically pure compound having one chiral center would substantially contain no opposite enantiomer of the compound. A stereochemically pure compound having two chiral centers would substantially contain no other diastereomers of the compound. Typical stereochemically pure compounds include more than about 80 wt% of one stereoisomer of the compound and less than about 20 wt% of other stereoisomers of the compound, more than about 90 wt% of one stereoisomer of the compound and less than about 10 wt% of other stereoisomers of the compound, more than about 95 wt% of one stereoisomer of the compound and less than about 5 wt% of other stereoisomers of the compound, or more than about 97 wt% of one stereoisomer of the compound and less than about 3 wt% of other stereoisomers of the compound. The compound may have a chiral center and may arise as a racemic mixture, individual enantiomers or diastereomers, and mixtures thereof. Methods involving the administration of any such isomeric form of compound C are included in the embodiments provided herein, including the administration of a mixture thereof.

[0213] In some embodiments, compound C provided herein contains a single chiral center and may exist as a mixture of enantiomers, for example, a racemic mixture. This disclosure encompasses the use of mixtures of such forms, as well as the use of stereochemically pure forms of such compounds. For example, a mixture containing equal or unequal amounts of enantiomers of compound C provided herein may 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. For example, see Jacques, J., et al, Enantiomers, Racemates and Resolutions (Wiley-Interscience, New York, 1981); Wilen, SH, et al, Tetrahedron 33:2725 (1977); Eliel, EL, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, SH, Tables of Resolving Agents and Optical Resolutions p. 268 (E L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972).

[0214] It should be understood that the chiral center of compound C can undergo epimerization in vivo. As such, those skilled in the art will recognize that, in the case of in vivo epimerization, 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 can be prepared using chiral synthons or chiral reagents, or they can be separated using conventional techniques such as chromatography in a chiral stationary phase.

[0216] In some embodiments, compound C is the solvate of (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione. The term “solvate” refers to the physical bond between a compound and one or more solvent molecules, whether organic or inorganic. This physical bond includes hydrogen bonds. In certain specific examples, for example, if one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate will be isolated. “Solvate” encompasses both solution-phase solvates and insoluble solvates. Exemplary solvates include hydrates, ethanol adducts, methanol adducts, isopropanol adducts, acetonitrile solvate, and ethyl acetate solvate. In some embodiments, compound C is the hydrate of (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindorin-1,3-dione. Methods of solvation are known in the art.

[0217] A "tautomer" refers to an isomer of a compound that is in equilibrium with itself. The concentration of each isomer depends on the environment in which the compound is found, and may differ depending on whether the compound is in a solid state or in an organic solution or aqueous solution. For example, in aqueous solution, pyrazole can exhibit the following isomers, which are called tautomers of each other: [ka]

[0218] As will be readily apparent to those skilled in the art, a wide variety of functional groups and other structures can exhibit tautomerism, and the administration of any tautomer of compound C falls within the scope of the methods provided herein.

[0219] It should also be noted that compound C used in the provided method may contain one or more atomic isotopes in unnatural proportions. For example, compound C may contain tritium ( 3 H), Iodine-125( 125 I), Sulfur-35( 35 S) or carbon-14 ( 14 It can be radioactively labeled with radioactive isotopes such as C, or deuterium ( 2 H), carbon-13 ( 13 C) or nitrogen-15 15 It can be isotope-enriched with N), etc. As used herein, “isotope species” refers to an isotope-enriched compound. The term “isotope-enriched” refers to an atom having an isotope composition other than the natural isotope composition of the atom in question. “Isotope-enriched” may also refer to a compound containing at least one atom having an isotope composition other than the natural isotope composition of the atom in question. The term “isotope composition” refers to the amount of each isotope found in a given atom. Radiolabeled and isotope-enriched compounds are useful as therapeutic agents, e.g., cancer treatment agents, research reagents, e.g., binding assay reagents and diagnostic agents, e.g., in vivo contrast agents. Methods involving the administration of any isotope variant of compound C, whether radioactive or not, are intended to be included within the scope of the methods provided herein. In some embodiments, isotope species of compound C, e.g., deuterium, carbon-13( 13 C) and / or nitrogen-15( 15 N) A method involving the administration of an enrichment compound is provided herein. As used herein, “deuterated” means that at least one hydrogen (H) is deuterized (D or 2 This means a compound in which a deuterium (indicated by H) is replaced, i.e., the compound is enriched with deuterium at at least one position.

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

[0221] It should be noted that if there is a discrepancy between the described structure and the name given to that structure, the described structure should be given more weight. In addition, if the stereochemistry of a structure or part of a structure is not indicated, for example, in bold or with a dashed line, the structure or part 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 may be administered in one or more compositions containing compound C, for example, pharmaceutical compositions.

[0223] In some embodiments, a composition containing compound C, such as a pharmaceutical composition, may include a carrier such as a diluent, adjuvant, excipient, or vehicle, together with which compound C and / or cells are administered. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by EW Martin. Such a composition would generally contain a therapeutically effective amount of compound C in a purified form, together with a suitable amount of carrier, so as to provide a form for appropriate administration to a patient. Such pharmaceutical carriers can be sterile liquids such as water and oil, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Saline solutions and dextrose water and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. The pharmaceutical composition may contain one or more of the following: diluents, adjuvants, antiadherents, binders, coatings, fillers, fragrances, colorants, lubricants, flow enhancers, preservatives, detergents, adsorbents, emulsifiers, pharmaceutical excipients, pH buffers, or sweeteners, or combinations thereof. In some embodiments, the pharmaceutical composition may be in liquid, solid, lyophilized powder, gel, and / or combinations thereof. In some embodiments, the choice of carrier is determined in part by the specific inhibitor and / or method of administration.

[0224] Pharmacochemically acceptable carriers are generally non-toxic to the recipient at the dosage and concentration used, and include buffers such as phosphoric acid, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than approximately 10 residues) polypeptides; serum The composition may include, but is not limited to, proteins such as lubumin, gelatin, or immunoglobulin; 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 dextrin; 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 nonionic surfactants such as polyethylene glycol (PEG), stabilizers, and / or preservatives. The composition containing compound C may be freeze-dried.

[0225] In some embodiments, the pharmaceutical composition may 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, ear, inhalation, buccal (e.g., sublingual) and transdermal administration or any of these routes. In some embodiments, other mechanisms of administration are also considered. In some embodiments, administration is made by bolus injection, by injection, for example, by intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, anterior chamber injection, subconjectval injection, subconjunctival injection, sub-Tenon injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, administration is made parenterally, intrapulmonaryly, and intranasally, and, if desired for local treatment, intra-focal administration. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus. In some embodiments, it is administered by multiple bolus doses or by continuous infusion over a period of, for example, three days or less.

[0226] In some embodiments, administration may be local, topical, or systemic, depending on the location of treatment. In some embodiments, local administration to the area requiring treatment may be achieved, for example but not limited to, local injection during surgery, topical application in conjunction with postoperative wound dressing, or injection using a catheter, suppository, or implant. In some embodiments, the composition may be administered sequentially, intermittently, or in the same composition, together with other bioactive agents. In some embodiments, administration may include a controlled release system, including controlled release formulations and device-controlled release using a pump or the like. 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 dose contains compound C with a predetermined content of therapeutic activity sufficient to produce the desired therapeutic effect, in relation to 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, and oral solutions or suspensions, and oil-water emulsions containing a suitable content of compound C. Unit dose forms may be contained in ampoules and syringes or in individually packaged tablets or capsules. Unit dose forms may be administered in fractions or multiples thereof. In some embodiments, multiple dose forms are multiple identical unit dosage forms packaged in a single container to be administered in isolated unit dose forms. Examples of multiple dose forms include vials, bottles of tablets or capsules, or pint or gallon bottles.

[0228] 2. Medication In some embodiments, the provided combination therapy method involves the step of initiating the administration of compound C before, after, during, concurrently with, almost simultaneously with, sequentially with, concurrently with and / or intermittently with, the initiation of a cell therapy such as T cell therapy (e.g., CAR-expressing T cells). In some embodiments, the initiation of the administration of compound C in the provided combination therapy method is made after or following the initiation of the administration of T cell therapy.

[0229] In some embodiments, the administration of compound C is initiated after (subsequently) the initiation of cell therapy, such as T cell therapy (e.g., CAR-expressing T cells). In some embodiments, the administration of compound C is initiated when or before the peak or maximum level of cells in the T cell therapy becomes detectable in the target blood.

[0230] In some cases, the initiation of compound C administration occurs when (i) the peak or maximum level of T-cell therapy cells becomes detectable in the subject's blood; (ii) after detection in the blood, the number of detectable T-cell therapy cells in the blood becomes undetectable or reduced, as appropriate, compared to a preceding point in time after T-cell therapy administration; (iii) the number of detectable T-cell therapy cells in the blood is reduced to 1 / 1.5, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10 or less of the peak or maximum number of T-cell therapy cells detectable in the subject's blood after the initiation of T-cell therapy administration; iv) At the time after the peak or maximum level of T-cell therapy cells becomes detectable in the subject's blood, the number of detectable cells or cells derived from the subject in the subject's blood 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) the subject exhibits an increased tumor burden compared to the tumor burden before or after the administration of cells and before the commencement of administration of compound C, performed at that time or within one week prior, such as within one, two, or three days prior. In certain embodiments, the method provided is performed to enhance, increase, or intensify T-cell therapy in a subject to improve the response to T-cell therapy, e.g., the presence of T cells and / or reduction of tumor burden.

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

[0232] In some embodiments, the administration of compound C is initiated after (subsequently) the initiation of cell therapy. In some embodiments, the administration of compound C is initiated after (subsequently) the initiation of T cell therapy and on the same day as the initiation of T cell therapy (i.e., on day 1 of the combination therapy). In some embodiments, the administration of compound C is initiated between day 1 and day 29 of the combination therapy (including upper and lower limits). In some embodiments, the administration of compound C is initiated between day 1 and day 22 of the combination therapy (including upper and lower limits). In some embodiments, the administration of compound C is initiated between day 1 and day 15 of the combination therapy (including upper and lower limits). In some embodiments, the administration of compound C is initiated between day 2 and day 15 of the combination therapy (including upper and lower limits). In some embodiments, the administration of compound C is initiated between day 3 and day 15 of the combination therapy (including upper and lower limits). In some embodiments, the administration of compound C is initiated between day 4 and day 15 of the combination therapy (including upper and lower limits). In some embodiments, administration of compound C is initiated between day 5 and day 15 of the combination therapy (including upper and lower limits). In some embodiments, administration of compound C is initiated between day 6 and day 15 of the combination therapy (including upper and lower limits). In some embodiments, administration of compound C is initiated between day 7 and day 15 of the combination therapy (including upper and lower limits). In some embodiments, administration of compound C is initiated between day 8 and day 15 of the combination therapy (including upper and lower limits).

[0233] In some embodiments, administration of compound C is initiated on 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, or an approximate number thereof. In some embodiments, administration of compound C is initiated on day 1 or about day 1 of the combination therapy. In some embodiments, administration of compound C is initiated on day 2 or about day 2 of the combination therapy. In some embodiments, administration of compound C is initiated on day 3 or about day 3 of the combination therapy. In some embodiments, administration of compound C is initiated on day 4 or about day 4 of the combination therapy. In some embodiments, administration of compound C is initiated on day 5 or about day 5 of the combination therapy. In some embodiments, administration of compound C is initiated on day 5 or about day 5 of the combination therapy. In some embodiments, administration of compound C is initiated on day 7 or approximately day 7 of the combination therapy. In some embodiments, administration of compound C is initiated on day 8 or approximately day 8 of the combination therapy. In some embodiments, administration of compound C is initiated on day 9 or approximately day 9 of the combination therapy. In some embodiments, administration of compound C is initiated on day 10 or approximately day 10 of the combination therapy. In some embodiments, administration of compound C is initiated on day 11 or approximately day 11 of the combination therapy. In some embodiments, administration of compound C is initiated on day 12 or approximately day 12 of the combination therapy. In some embodiments, administration of compound C is initiated on day 13 or approximately day 13 of the combination therapy. In some embodiments, administration of compound C is initiated on day 14 or approximately day 14 of the combination therapy. In some embodiments, administration of compound C is initiated on day 15 or approximately day 15 of the combination therapy. In some embodiments, administration of compound C is initiated on day 16 or approximately day 16 of the combination therapy. In some embodiments, administration of compound C is initiated on day 17 or approximately day 17 of the combination therapy. In some embodiments, administration of compound C is initiated on day 18 or approximately day 18 of the combination therapy. In some embodiments, administration of compound C is initiated on day 19 or approximately day 19 of the combination therapy. In some embodiments, administration of compound C is initiated on day 20 or approximately day 20 of the combination therapy.In some embodiments, administration of compound C is initiated on day 21 or approximately day 21 of the combination therapy. In some embodiments, administration of compound C is initiated on day 22 or approximately day 22 of the combination therapy.

[0234] In some embodiments, the administration of compound C is initiated before the administration of cell therapy, such as T-cell therapy. In some embodiments, the administration of compound C is initiated within one month before the administration of cell therapy. In some embodiments, the administration of compound C is initiated within four weeks before the administration of cell therapy. In some embodiments, the administration of compound C is initiated within three weeks before the administration of cell therapy. In some embodiments, the administration of compound C is initiated within two weeks before the administration of cell therapy. In some embodiments, the administration of compound C is initiated within one week before the administration of cell therapy. In some embodiments, the administration of compound C is initiated within six days before the administration of cell therapy. In some embodiments, the administration of compound C is initiated within five days before the administration of cell therapy. In some embodiments, the administration of compound C is initiated within four days before the administration of cell therapy. In some embodiments, the administration of compound C is initiated within three days before the administration of cell therapy. In some embodiments, the administration of compound C is initiated within two days before the administration of cell therapy. In some embodiments, administration of compound C is initiated within one day prior to the administration of cell therapy.

[0235] In some embodiments, the administration of compound C is started one month before the administration of cell therapy. In some embodiments, the administration of compound C is started four weeks before the administration of cell therapy. In some embodiments, the administration of compound C is started three weeks before the administration of cell therapy. In some embodiments, the administration of compound C is started two weeks before the administration of cell therapy. In some embodiments, the administration of compound C is started one week before the administration of cell therapy. In some embodiments, the administration of compound C is started six days before the administration of cell therapy. In some embodiments, the administration of compound C is started five days before the administration of cell therapy. In some embodiments, the administration of compound C is started four days before the administration of cell therapy. In some embodiments, the administration of compound C is started three days before the administration of cell therapy. In some embodiments, the administration of compound C is started two days before the administration of cell therapy. In some embodiments, the administration of compound C is started one day before the administration of cell therapy.

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

[0237] In some embodiments, administration of compound C is initiated 7 days after the end of lymphocyte depletion therapy. In some embodiments, administration of compound C is initiated 6 days after the end of lymphocyte depletion therapy. In some embodiments, administration of compound C is initiated 5 days after the end of lymphocyte depletion therapy. In some embodiments, administration of compound C is initiated 4 days after the end of lymphocyte depletion therapy. In some embodiments, administration of compound C is initiated 3 days after the end of lymphocyte depletion therapy. In some embodiments, administration of compound C is initiated 2 days after the end of lymphocyte depletion therapy. In some embodiments, administration of compound C is initiated 1 day after the end of lymphocyte depletion therapy.

[0238] In some embodiments, at the time of initial administration of compound C, and / or at any subsequent point after the start 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, administration of compound C is made at a time when the subject does not exhibit signs or symptoms of severe CRS and / or does not exhibit Grade 3 or higher CRS, such as long-term Grade 3 CRS or Grade 4 or 5 CRS. In some embodiments, administration of compound C is made at a time when the subject does not exhibit signs or symptoms of severe neurotoxicity and / or does not exhibit Grade 3 or higher neurotoxicity, such as long-term Grade 3 neurotoxicity or Grade 4 or 5 neurotoxicity. In some embodiments, between the start of T-cell therapy administration and the administration of compound C, the subject does not exhibit severe CRS and / or does not exhibit Grade 3 or higher CRS, such as long-term Grade 3 CRS or Grade 4 or 5 CRS. In some cases, subjects did not exhibit severe neurotoxicity and / or long-term grade 3 neurotoxicity or grade 4 or 5 neurotoxicity between the start of T-cell therapy and the administration of compound C.

[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 of the multiple intermittent doses of compound C is identical. In other embodiments, the multiple intermittent doses of compound C may 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, for example, between 0.1 mg or about 0.1 mg and 0.9 mg, between 0.1 mg or about 0.1 mg and 0.8 mg, between 0.1 mg or about 0.1 mg and 0.7 mg, between 0.1 mg or about 0.1 mg and 0.6 mg, between 0.1 mg or about 0.1 mg and 0.5 mg, or between 0.1 mg or about 0.4 mg. Or between approximately 0.4 mg, 0.1 mg, or between approximately 0.1 mg and 0.3 mg, or between approximately 0.3 mg, 0.1 mg, or between approximately 0.1 mg and 0.2 mg, or between approximately 0.2 mg and 1.0 mg, or between approximately 1.0 mg, 0.2 mg, or between approximately 0.2 mg and 0.9 mg, or between approximately 0.9 mg, 0.2 mg, or between approximately 0.2 mg and 0.8 mg, or between approximately 0.8 mg, 0.2 mg, or between approximately 0.2 mg and 0.7 mg, or between approximately 0.7 mg, 0.2 mg, or between approximately 0.2 mg and 0.6 mg, or between approximately 0.6 mg Between 0.2mg or approximately 0.2mg to 0.5mg or approximately 0.5mg, between 0.2mg or approximately 0.2mg to 0.4mg or approximately 0.4mg, between 0.2mg or approximately 0.2mg to 0.3mg or approximately 0.3mg, between 0.3mg or approximately 0.3mg to 1.0mg or approximately 1.0mg, between 0.3mg or approximately 0.3mg to 0.9mg or approximately 0.9mg, between 0.3mg or approximately 0.3mg to 0.8mg or approximately 0.8mg, between 0.3mg or approximately 0.3mg to 0.7mg or approximately 0.7mg, and 0.3mg if The amounts are approximately 0.3mg to 0.6mg or between approximately 0.6mg, 0.3mg or between approximately 0.3mg and 0.5mg or between approximately 0.5mg, 0.3mg or between approximately 0.3mg and 0.4mg or between approximately 0.4mg, 0.4mg or between approximately 0.4mg and 1.0mg or between approximately 1.0mg, 0.4mg or between approximately 0.4mg and 0.9mg or between approximately 0.9mg, 0.4mg or between approximately 0.4mg and 0.8mg or between approximately 0.8mg, 0.4mg or between approximately 0.4mg and 0.7mg or between approximately 0.7mg, 0.4mg or between approximately 0.4mg and 0.Between 6 mg or approximately 0.6 mg, between 0.4 mg or approximately 0.4 mg to 0.5 mg or approximately 0.5 mg, between 0.5 mg or approximately 0.5 mg to 1.0 mg or approximately 1.0 mg, between 0.5 mg or approximately 0.5 mg to 0.9 mg or approximately 0.9 mg, between 0.5 mg or approximately 0.5 mg to 0.8 mg or approximately 0.8 mg, between 0.5 mg or approximately 0.5 mg to 0.7 mg or approximately 0.7 mg, between 0.5 mg or approximately 0.5 mg to 0.6 mg or approximately 0.6 mg, between 0.6 mg or approximately 0.6 mg to 1.0 mg or approximately 1.0 mg, between 0.6 mg or approximately 0.6 mg to 0.9 mg or approximately 0.9 mg, 0 The amounts are 0.6 mg or between approximately 0.6 mg and 0.8 mg or approximately 0.8 mg, 0.6 mg or between approximately 0.6 mg and 0.7 mg or approximately 0.7 mg, 0.7 mg or between approximately 0.7 mg and 1.0 mg or approximately 1.0 mg, 0.7 mg or between approximately 0.7 mg and 0.9 mg or approximately 0.9 mg, 0.7 mg or between approximately 0.7 mg and 0.8 mg or approximately 0.8 mg, 0.8 mg or between approximately 0.8 mg and 1.0 mg or approximately 1.0 mg, 0.8 mg or between approximately 0.8 mg and 0.9 mg or approximately 0.9 mg, or 0.8 mg or approximately 0.8 mg and 1.0 mg or approximately 1.0 mg (including upper and lower limits for each). In some embodiments, the dose of compound C is between 0.1 mg or approximately 0.1 mg and 0.6 mg or approximately 0.6 mg. In some embodiments, the dose of compound C is 0.1 mg or between approximately 0.1 mg and 0.5 mg. In some embodiments, the dose of compound C is between 0.1 mg or between approximately 0.1 mg and 0.4 mg. In some embodiments, the dose of compound C is between 0.1 mg or between approximately 0.1 mg and 0.3 mg. In some embodiments, the dose of compound C is 0.1 mg or approximately 0.1 mg. In some embodiments, the dose of compound C is 0.2 mg or approximately 0.2 mg. In some embodiments, the dose of compound C is 0.3 mg or approximately 0.3 mg. In some embodiments, the dose of compound C is 0.4 mg or approximately 0.4 mg. In some embodiments, the dose of compound C is 0.5 mg or approximately 0.The dose is 5 mg. In some embodiments, the dose of compound C is 0.6 mg or about 0.6 mg. In any of the preceding embodiments, each of the multiple intermittent doses of compound C is identical.

[0241] In some embodiments, compound C, as part of an intermittent dosing regimen, is administered at least once every 5 days. In some embodiments, compound C is administered at least once every 6 days. In some embodiments, compound C is administered at least once every 7 days. In some embodiments, compound C is administered at least once every 8 days. In some embodiments, compound C is administered at least once every 9 days. In some embodiments, compound C is administered at least once every 10 days. In some embodiments, compound C is administered at least once every 11 days. In some embodiments, compound C is administered at least once every 12 days. In some embodiments, compound C is administered at least once every 13 days. In some embodiments, compound C is administered at least once every 14 days.

[0242] In some embodiments, compound C is administered once every 5 days as part of an intermittent dosing regimen. In some embodiments, compound C is administered once every 6 days. In some embodiments, compound C is administered once a week. 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 2 weeks. In some embodiments, compound C is administered once every 14 days (Q14D).

[0243] In some embodiments, compound C is administered over a period following the initiation of cell therapy administration. In some embodiments, compound C is administered for at least one week after the initiation of cell therapy administration. In some embodiments, compound C is administered for at least two weeks after the initiation of cell therapy administration. In some embodiments, compound C is administered for at least three weeks after the initiation of cell therapy administration. In some embodiments, compound C is administered for at least four weeks after the initiation of cell therapy administration. In some embodiments, compound C is administered for at least five weeks after the initiation of cell therapy administration. In some embodiments, compound C is administered for at least six weeks after the initiation of cell therapy administration. In some embodiments, compound C is administered for at least seven weeks after the initiation of cell therapy administration. In some embodiments, compound C is administered for at least eight weeks after the initiation of cell therapy administration. In some embodiments, compound C is administered for at least nine weeks after the initiation of cell therapy administration. In some embodiments, compound C is administered for at least ten weeks after the initiation of cell therapy administration. In some embodiments, compound C is administered for at least eleven weeks after the initiation of cell therapy administration. In some embodiments, compound C is administered for at least 12 weeks after the start of administration of cell therapy. In some embodiments, compound C is administered for up to 12 weeks after the start of administration of cell therapy. In any of the preceding embodiments, compound C is administered for up to 12 weeks after the start of administration of cell therapy. Therefore, referring to a combination therapy in which cell therapy is administered on day 1 of the combination therapy, in some embodiments, compound C is not administered 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 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 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 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 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 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 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 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 point in time and / or over one or more periods of time. In some cases, the cycling regimen is interrupted or modified if the subject develops one or more adverse events, dose-restrictive toxicity (DLT), neutropenia or febrile neutropenia, thrombocytopenia, cytokine release syndrome (CRS), and / or neurotoxicity (NT), such as those described in Section IV. In some embodiments, the daily dose of compound C per administration or on a particular day of the week is modified after the subject develops one or more adverse events, dose-restrictive toxicity (DLT), neutropenia or febrile neutropenia, thrombocytopenia, cytokine release syndrome (CRS), and / or neurotoxicity (NT), such as those described in Section IV.

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

[0254] Therefore, in some embodiments, the 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, for example, by combining this with a stimulus that induces a response such as proliferation, survival, and / or activation, as measured by the expression of cytokines or activation markers, and then transducing the activated cells and increasing their numbers in culture to a number sufficient for clinical application.

[0255] 1. Chimeric antigen receptor In the methods and uses provided, for example, in any of the embodiments described in Section I, engineered cells such as T cells express a chimeric receptor, such as a chimeric antigen receptor (CAR), which contains one or more domains that combine a ligand-binding domain (e.g., an antibody or antibody fragment) that provides specificity to a desired antigen (e.g., a tumor antigen) with an intracellular signaling domain. In some embodiments, the intracellular signaling domain is an activated 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 co-stimulatory signaling domain to facilitate effector function. Upon specific binding to a molecule, for example, an antigen, the receptor generally delivers an immunostimulatory signal into the cell, such as an ITAM signaling signal, thereby promoting an immune response targeted to a disease or condition. In some embodiments, when the chimeric receptor is genetically engineered in immune cells, it can modulate T cell activity, and in some cases modulate T cell differentiation or homeostasis, thereby resulting in genetically 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, and methods for manipulating and introducing such receptors into cells, are, 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 Application Publication Nos. 6, The patents described in Nos. 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, as well as those described in European Patent Application No. EP2537416, and / or Sadelain This includes those described by 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; and Wu et al., Cancer, 2012 March 18(2): 160-75. In some embodiments, the antigen receptor includes the CAR described in U.S. Patent No. 7,446,190 and the one described in International Patent Application Publication No. WO / 2014055668A1.Examples of CARs include those disclosed in any of the aforementioned publications, such as WO2014031687, US8,339,645, US7,446,179, US2013 / 0149337, US Patent No. 7,446,190, US Patent 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 these are incorporated herein by reference. See also WO2014031687, US8,339,645, US7,446,179, US2013 / 0149337, US No. 7,446,190, and US No. 8,389,282.

[0257] Exemplary CAR T-cell therapies targeting CD19 include the FDA-approved products BREYANZI® (lisocabtagene / maluloucel), TECARTUS® (brexucabtagene / autoleucel), KYMRIAH® (tisagenlecleucel), and YESCARTA® (axicabutagene / siloloucel), as well as clinical trials NCT02644655, NCT03744676, NCT01087294, NCT03366350, NCT03790891, NCT03497533, NCT04007029, NCT03960840, NCT04049383, NCT04094766, and NC. This includes therapies investigated or under investigation in T03366324, NCT02546739, NCT03448393, NCT03467256, NCT03488160, NCT04012879, NCT03016377, NCT03468153, NCT03483688, NCT03398967, NCT03229876, NCT03455972, NCT03423706, NCT03497533 and NCT04002401. Exemplary manipulated cells include BREYANZI®, TECARTUS®, KYMRIAH®, YESCARTA®, UCART19 and ALLO-501. In some embodiments, the manipulated cells include any of the cells described in Marofi et al., Front. Immunol. (2021) 12:681984, which are incorporated herein by reference in their entirety.

[0258] In some embodiments, engineered cells, such as T cells, express recombinant receptors, such as chimeric antigen receptors (CARs), which have specificity for specific antigens (or markers or ligands), such as antigens expressed on the surface of specific cell types. In some embodiments, the antigen targeted by the receptor is a polypeptide. In some embodiments, this is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed in diseased or diseased cells, such as tumor or pathogenic cells, compared to normal or untargeted cells or tissues. In other embodiments, the antigen is expressed in normal cells and / or engineered cells.

[0259] The antigens targeted by the receptor include, in some embodiments, antigens associated with B-cell malignancies, such as any of the many known B-cell markers. In some embodiments, the antigens targeted by the receptor are CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig kappa, Ig lambda, CD79a, CD79b, or CD30. In certain embodiments, the antigen is CD19. In some embodiments, any of these antigens are antigens expressed in human B cells.

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

[0261] In some embodiments, an antibody or antigen-binding fragment (e.g., scFv or V) is used. HThe domain specifically recognizes antigens such as CD19. In some embodiments, the antibody or antigen-binding fragment is derived from or a variant thereof 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 derived from an antibody or antibody fragment specific to CD19. H and V L It contains. 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 V derived from FMC63, which may be scFv in some aspects. H and / or V L FMC63 generally refers to mouse monoclonal IgG1 antibodies produced against Nalm-1 and -16 cells expressing human-derived CD19 (Ling, NR, et al. (1987). Leucocyte typing III. 302). In some embodiments, the FMC63 antibody includes the CDR-H1 and CDR-H2 sequences described in SEQ ID NOs. 38 and 39, and the CDR-H3 sequence described in SEQ ID NOs. 40 or 54, and the CDR-L1 sequence described in SEQ ID NOs. 35, and the CDR-L2 sequence described in SEQ ID NOs. 36 or 55, and the CDR-L3 sequence described in SEQ ID NOs. 37 or 56. In some embodiments, the FMC63 antibody includes a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NOs. 41. H ), and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 42 L ) includes.

[0264] In some embodiments, scFv includes 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 any of the aforementioned variants having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of sequence identity thereto. In some embodiments, scFv includes the variable heavy chain region of FMC63 described in SEQ ID NO: 41 and the variable light chain region of FMC63 described in SEQ ID NO: 42, or any of the aforementioned variants having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% 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 described in SEQ ID NO: 59. In some embodiments, scFv is, in order, V H , linker and V L Includes. In some embodiments, scFv is V in order. L , linker and V H This includes. In some embodiments, the scFv is coded by the nucleotide sequence described in SEQ ID NO: 57, or a sequence exhibiting 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 includes the amino acid sequence described in SEQ ID NO: 43, or a sequence exhibiting 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® (lysocabtagene / malaluucelle). In some embodiments, the T-cell therapy is BREYANZI® (lysocabtagene maralucell).

[0265] In some embodiments, the antigen-binding domain is V derived from SJ25C1, which may be scFv in some aspects. H and / or V L It includes. SJ25C1 is a mouse monoclonal IgG1 antibody produced against Nalm-1 and -16 cells expressing human-derived CD19 (Ling, NR, et al. (1987). Leucocyte typing III. 302). In some embodiments, the SJ25C1 antibody includes the CDR-H1, CDR-H2, and CDR-H3 sequences described in SEQ ID NOs. 47-49, and the CDR-L1, CDR-L2, and CDR-L3 sequences described in SEQ ID NOs. 44-46, respectively. In some embodiments, the SJ25C1 antibody includes a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO. 50. H ), and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 51 L ) includes. In some embodiments, scFv includes 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 any of the aforementioned variants having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of sequence identity thereto. In some embodiments, scFv includes the variable heavy chain region of SJ25C1 described in SEQ ID NO: 50 and the variable light chain region of SJ25C1 described in SEQ ID NO: 51, or any of the aforementioned variants having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% 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 shown in SEQ ID NO: 52. In some embodiments, scFv is, in order, V H , linker and V LIncludes. In some embodiments, scFv is V in order. L , linker and V H This includes. In some embodiments, scFv includes the amino acid sequence described in SEQ ID NO: 53, or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 53.

[0266] In some embodiments, the anti-CD19 CAR is a CAR from TECARTUS® (brexcabutagen autolucel). In some embodiments, the T-cell therapy is TECARTUS® (brexcabutagen autolucel).

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

[0268] In some embodiments, the anti-CD19 CAR is YESCARTA® (axicabutagen / silolucel). In some embodiments, the T-cell therapy is YESCARTA® (axicabutagen / silolucel).

[0269] In this specification, the term “antibody” is used in its broadest sense and includes polyclonal and monoclonal antibodies comprising intact antibodies and functional (antigen-binding) antibody fragments, wherein the fragments include antigen-binding fragments (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, and variable heavy chains (V) capable of specifically binding to antigens. H ) region, single-chain antibody fragments containing single-chain variable fragments (scFv), and single-domain antibodies (e.g., sdAb, sdFv, nanobody, V) H H or V NARThis term includes immunoglobulins in genetically engineered and / or otherwise modified forms, e.g., intrabodies, peptidebodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, polyspecific antibodies, e.g., bispecific antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, and tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to include its functional antibody fragment. This term also includes intact or full-length antibodies containing antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD. In some embodiments, a CAR is a bispecific CAR containing, for example, two antigen-binding domains with different specificities.

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

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

[0272] The precise amino acid sequence boundaries of a given CDR or FR are as follows: 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.This can be easily determined using one of many well-known schemes, including the scheme described in “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 can 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. Numbering for both the Kabat and Chothia schemes is based on the length of the most common antibody region sequence, with insertions corresponding by insertion letters, e.g., "30a", and deletions occurring in some antibodies. These 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 ways to the Chothia numbering scheme. The AbM scheme is a compromise between the Kabat and Chothia definitions, based on the one used by Oxford Molecular's AbM antibody modeling software.

[0274] Table 2 below lists exemplary positional boundaries of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3, as identified by Kabat, Chothia, AbM, and contact schemes, respectively. For CDR-H1, residue numbering is listed using both 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, and so on. It should be noted that because the Kabat numbering scheme shown places insertions at H35A and H35B, the end of the Chothia CDR-H1 loop will vary between H32 and H34 depending on the loop length, if numbered using the Kabat numbering convention shown. [Table 2]

[0275] Therefore, unless otherwise specified, a given antibody or its region, for example, its variable region's "CDR" or "complementary determination region," or individual designated CDRs (e.g., CDR-H1, CDR-H2, CDR-H3), should be understood to encompass a certain (or specific) complementary determination region defined by the scheme described above or any other known scheme. For example, a particular CDR (e.g., CDR-H3) is a given V H or V LWhen it is described that a regional amino acid sequence contains the amino acid sequence of a corresponding CDR, it is understood that such a CDR has the sequence of a corresponding CDR (e.g., CDR-H3) within a variable region defined by either the scheme described above or any other known scheme. In some embodiments, a specific CDR sequence is specified. While exemplary CDR sequences of the antibodies provided are described using various numbering schemes, it is understood that the antibodies provided may contain CDRs described according to any other numbering scheme described above or any other numbering scheme known to those skilled in the art.

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

[0277] The term "variable region" or "variable domain" refers to a domain in the antibody heavy or light chain that is involved in the binding of an antibody to an antigen. (V) H and V L ) generally have a similar structure, and each domain contains four conserved framework regions (FRs) and three CDRs (see, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). H or V L The domains can be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to specific antigens each have complementary V L or V HTo screen the domain library, V derived from antibodies that bind to the antigen, H or V L It can be isolated using a domain. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0278] Among the antibodies offered, antibody fragments are particularly noteworthy. An "antibody fragment" refers to a molecule other than the intact antibody, containing the 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; diabody; linear antibody; variable heavy chain (V H ) region, single-chain antibody molecule, e.g., scFv and single-domain V H This includes, but is not limited to, single antibodies and polyspecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single-chain antibody fragment containing a variable heavy chain region and / or a variable light chain region, such as scFv.

[0279] The term "variable region" or "variable domain" refers to a domain in the antibody heavy or light chain that is involved in the binding of an antibody to an antigen. (V) H and V L ) generally have a similar structure, and each domain contains four conserved framework regions (FRs) and three CDRs (see, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). H or V L The domains can be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to specific antigens each have complementary V L or V H To screen the domain library, V derived from antibodies that bind to the antigen, H or V LIt can be isolated using a domain. See, for example, 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 containing all or part of the heavy chain variable domain or all or part 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 contains an antibody heavy chain domain that specifically binds to cells to be targeted, such as tumor cells or cancer cells, or to an antigen such as a cancer marker or cell surface antigen of a disease, such as any of the target antigens described herein or known target antigens. Exemplary single-domain antibodies include sdFv, nanobody, and V. H H or V NAR Includes.

[0281] Antibody fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells. In some embodiments, the antibody is a recombinant fragment, such as a fragment having two or more antibody regions or chains linked by a synthetic linker, e.g., a peptide linker, and / or a fragment containing a configuration that does not occur in nature, such as a fragment that cannot be produced by enzymatic digestion of naturally occurring intact antibodies. In some embodiments, the antibody fragment is 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 contain at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of a non-human antibody refers to a variant of a non-human antibody that has been humanized to reduce immunogenicity, typically against humans, while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues derived from a non-human antibody (e.g., the antibody from which the CDR residues were obtained) to restore or improve antibody specificity or affinity, for example.

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

[0284] In some embodiments, recombinant receptors such as CARs include an immunoglobulin constant region or a variant or modified version thereof, e.g., a hinge region, e.g., an IgG4 hinge region and / or C H 1 / C LThe recombinant receptor further includes a spacer which may be or may include at least a portion of the Fc region. In some embodiments, the recombinant receptor further includes a spacer and / or a hinge region. In some embodiments, the constant region or portion is the constant region or portion of human IgG, e.g., IgG4 or IgG1. In some embodiments, the portion of the constant region functions as a spacer region between the antigen-recognition components, e.g., 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 long, or about 12 amino acids long, or 12 or fewer amino acids long. 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, and including any integer between any endpoint of the listed range. 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 3 domains, or C H The spacer includes an IgG4 hinge linked to three 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 IgG, such as only the hinge spacer of IgG4 or IgG1, such as only the hinge spacer of IgG4 or IgG1, such as only the hinge spacer of IgG4 or IgG1, etc. In some embodiments, the spacer is C H 2 and / or C H A three-domain linked Ig hinge, for example, an IgG4 hinge. In some embodiments, the spacer is C, as described in Sequence ID No. 3. H 2 and C H A three-domain linked Ig hinge, for example, an IgG4 hinge. In some embodiments, the spacer is C, as described in Sequence ID No. 4. H The Ig hinge is linked to only three domains, for example, the IgG4 hinge. In some embodiments, the spacer is or includes a glycine-serine rich sequence or other mobile linker, for example, a known mobile linker. In some embodiments, the constant region or portion is that of IgD. In some embodiments, the spacer has the sequence described in SEQ ID NO: 5. In some embodiments, the spacer has an amino acid sequence that exhibits at least or 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 is (a) comprising or consisting of all or part of an immunoglobulin hinge or a modified version thereof, or comprising about 15 amino acids or less and not comprising the CD28 extracellular region or the CD8 extracellular region; (b) comprising or consisting of all or part of an immunoglobulin hinge, optionally an IgG4 hinge or a modified version thereof, and / or comprising about 15 amino acids or less and not comprising the CD28 extracellular region or the CD8 extracellular region; or (c) being 12 amino acids or about 12 amino acids in length, and / or an immunoglobulin hinge, optionally IgG4 or its (d) A polypeptide spacer comprising or consisting of all or part of a modified version; or comprising or consisting of any of the aforementioned variants having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; or comprising or consisting of formula X1PPX2P (wherein X1 is glycine, cysteine, or arginine, and X2 is cysteine ​​or threonine).

[0287] In some embodiments, the antigen receptor includes an intracellular domain directly or indirectly linked to an extracellular domain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some embodiments, the intracellular signaling domain includes an ITAM. For example, in some embodiments, the antigen recognition domain (e.g., the extracellular domain) is generally linked to one or more intracellular signaling components, such as a signaling component that mimics activation via 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 includes a transmembrane domain linked or fused between an extracellular domain (e.g., 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, a transmembrane domain is used that naturally associates with one of the domains in a receptor, such as a CAR. In some examples, the transmembrane domain is selected to avoid binding to the transmembrane domains of the same or different surface membrane proteins, or is modified by amino acid substitutions to do so, in order to minimize interaction with other members of the receptor complex.

[0289] In some embodiments, the transmembrane domain originates from either a natural or synthetic source. If the source is natural, the domain, in some embodiments, originates from any membrane-bound or transmembrane protein. The transmembrane region includes a transmembrane region derived from 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 (i.e., at least its transmembrane region). Alternatively, in some embodiments, the transmembrane domain is synthetic. In some embodiments, the synthetic transmembrane domain predominantly contains hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain. In some embodiments, linkage is by linkers, spacers, and / or transmembrane domains. In some embodiments, the transmembrane domain contains a transmembrane portion of CD28 or a variant thereof. 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 one of those described herein.

[0290] In some embodiments, the transmembrane domain of the receptor, for example, CAR, is the transmembrane domain of human CD28 or a variant thereof, for example, the 27-amino acid transmembrane domain of human CD28 (accession number P10747.1), or a transmembrane domain containing the amino acid sequence described in SEQ ID NO: 8 or an amino acid sequence exhibiting at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of sequence identity with respect to SEQ ID NO: 8. In some embodiments, the transmembrane domain containing a portion of the recombinant receptor contains the amino acid sequence described in SEQ ID NO: 9, or an amino acid sequence exhibiting at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of sequence identity with respect to it.

[0291] In some embodiments, recombinant receptors, e.g., CARs, include at least one intracellular signaling component, e.g., an intracellular signaling region or domain. T cell activation is described in some embodiments as being mediated by two classes of cytoplasmic signaling sequences: a class that initiates antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequence) and a class that acts in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequence). In some embodiments, the CAR includes one or both of such signaling components. Among the intracellular signaling regions, particularly noteworthy are intracellular signaling regions that mimic or approximate signaling mediated by innate antigen receptors, signaling mediated by such receptors in combination with co-stimulatory receptors, and / or signaling mediated by co-stimulatory receptors alone. In some embodiments, there are short oligo or polypeptide linkers, e.g., linkers with a length of 2 to 10 amino acids, e.g., linkers containing glycine and serine, e.g., a glycine-serine doublet, which form a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[0292] In some embodiments, ligation of a CAR causes the cytoplasmic domain or intracellular signaling region of the CAR to activate at least one of the normal effector functions or responses of immune cells engineered to express the CAR, such as T cells. For example, in some situations, the CAR induces T cell function, such as cytolytic activity or T-helper activity, such as the secretion of cytokines or other factors. In some embodiments, a cleaved portion of the intracellular signaling region of an antigen receptor component or co-stimulatory molecule is used in place of an intact immunostimulatory chain, for example, when transmitting effector function signals. In some embodiments, the intracellular signaling region, for example including an intracellular domain, includes the cytoplasmic sequence of a T cell receptor (TCR), and in some embodiments, also includes the cytoplasmic sequence of a co-receptor that, in its natural context, acts in coordination with such a receptor to initiate signaling after antigen receptor engagement, and / or derivatives or variants of any such molecule, and / or any synthetic sequence having identical functional properties. In some embodiments, the intracellular signaling region, for example including an intracellular domain, includes the cytoplasmic sequence of a region or domain involved in providing co-stimulatory signals.

[0293] In some embodiments, the CAR contains a primary cytoplasmic signaling sequence that modulates the primary activation of the TCR complex. The primary cytoplasmic signaling sequence acting in a stimulating manner may contain a signaling motif known as an immunoreceptor-activated tyrosine motif or ITAM. Examples of ITAMs containing a primary cytoplasmic signaling sequence include ITAMs derived from the CD3 zeta chain, FcR gamma, CD3 gamma, CD3 delta, and CD3 epsilon. In some embodiments, the cytoplasmic signaling molecule in the CAR contains a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3 zeta.

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

[0295] In some embodiments, the intracellular (or cytoplasmic) signaling region includes a human CD3 chain, optionally a CD3 zeta-stimulated signaling domain or a functional variant thereof, for example, the 112AA cytoplasmic domain of isoform 3 of human CD3ζ (accession number P20963.2) or a 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 includes the amino acid sequence described in SEQ ID NOs. 13, 14, or 15, or an amino acid sequence exhibiting at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of sequence identity with SEQ ID NOs. 13, 14, or 15.

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

[0297] In some embodiments, the chimeric antigen receptor contains the intracellular domain of a T cell costimulatory molecule. In some embodiments, the CAR includes 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 includes the 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 includes the intracellular costimulatory signaling domain of human CD28 or a functional variant or portion thereof, for example, such a domain having its 41 amino acid domain and / or an LL-to-GG substitution at positions 186-187 of the native CD28 protein. In some embodiments, the intracellular signaling domain may include the amino acid sequence described in SEQ ID NO: 10 or 11, or an amino acid sequence exhibiting at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of sequence identity to SEQ ID NO: 10 or 11. In some embodiments, the intracellular region includes the intracellular costimulatory signaling domain of 4-1BB or a functional variant or portion thereof, for example, the 42-amino acid cytoplasmic domain of human 4-1BB (accession number Q07011.1) or a functional variant or portion thereof, for example, the amino acid sequence described in SEQ ID NO: 12, or an amino acid sequence exhibiting at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of sequence identity with respect to SEQ ID NO: 12.

[0299] In some embodiments, the same CAR includes both a primary (or activated) cytoplasmic signaling region and a co-stimulatory signaling component.

[0300] In some embodiments, an activating domain is contained within one CAR, while a co-stimulatory component is provided by another CAR that recognizes a different antigen. In some embodiments, the CAR comprises an activating or stimulating CAR and a co-stimulatory CAR, both expressed on the surface of the same cell (see WO2014 / 055668). In some embodiments, the cell contains one or more stimulating or activating CARs and / or co-stimulatory CARs. In some embodiments, the cell further includes inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013)), for example, CARs that recognize antigens other than those associated with and / or specific to a disease or condition, thereby reducing or inhibiting the activation signal delivered via the disease-targeting CAR by binding of the inhibitory CAR to its ligand, for example, 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 signals and co-stimulatory signals, for example, CARs that include an intracellular signaling domain derived from a co-stimulatory receptor such as CD28 or CD137; and in some embodiments, third-generation CARs are CARs that include multiple co-stimulatory domains from different co-stimulatory receptors.

[0302] In some embodiments, the CAR contains one or more, for example, two or more co-stimulatory and activating domains, such as a primary activating domain, in its cytoplasmic portion. An exemplary CAR includes intracellular components of 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 comprises a surrogate marker, such as a cell surface marker, which can be used to confirm the transduction or manipulation of cells to express the receptor. In some embodiments, the marker comprises all or part of CD34, NGFR, or epidermal growth factor receptor (e.g., a cleaved form), e.g., a cleaved version of such a cell surface receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably ligated to a cleavable linker sequence, e.g., a polynucleotide encoding a linker sequence such as T2A. For example, the marker and, as appropriate, the linker sequence can be any of those disclosed in published patent application number WO2014031687. For example, the marker can be a cleavable EGFR (tEGFR), which may be ligated to a linker sequence such as a T2A cleavable linker sequence.

[0304] Exemplary polypeptides for cleavage-type EGFR (e.g., tEGFR) include amino acid sequences described in SEQ ID NO: 7 or 16, or amino acid sequences exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 7 or 16. Exemplary T2A linker sequences include amino acid sequences described in SEQ ID NO: 6 or 17, or amino acid sequences exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 6 or 17.

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

[0306] In some embodiments, the marker does not perform a therapeutic function and / or has no effect other than being used as a marker for genetic manipulation, for example, to select cells that have been successfully manipulated. In other embodiments, the marker may be a therapeutic molecule or, in some cases, a molecule that exerts several desired effects, such as a ligand that cells encounter in vivo, such as a co-stimulatory or immune checkpoint molecule that enhances and / or weakens the cellular response upon encounter with adoptive transfer and ligand.

[0307] In some embodiments, the chimeric antigen receptor comprises an extracellular component containing the antibody or fragment described herein. In some embodiments, the chimeric antigen receptor comprises an extracellular component containing the antibody or fragment described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment is scFv or a single-domain V H The antibody is contained, and the intracellular domain contains ITAM. In some embodiments, the intracellular signaling domain contains 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 includes CD28 and CD137 (4-1BB, TNFRSF9) costimulatory domains linked to the CD3 zeta intracellular domain. In some embodiments, CD28 is human CD28. In some embodiments, 4-1BB is human 4-1BB. In some embodiments, the chimeric antigen receptor includes a transmembrane domain positioned between the extracellular domain and the intracellular signaling region. In some embodiments, the transmembrane domain contains the transmembrane portion of CD28. The extracellular domain and the transmembrane domain may be linked directly or indirectly. In some embodiments, the extracellular domain and the transmembrane domain are linked by a spacer such as one of those described herein.

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

[0309] In some embodiments, the CAR comprises an antibody, e.g., an antibody fragment, a transmembrane domain which is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain which 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 which contains a portion of an Ig molecule, such as a hinge-only spacer, an Ig hinge, e.g., an IgG4 hinge, or a human Ig molecule. In some embodiments, the CAR comprises an antibody or fragment such as any of the above, e.g., a CD19-specific scFv, a spacer which is or contains 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. Methods relating to nucleic acids, vectors, and gene manipulation In some embodiments, cells, such as T cells, are genetically engineered to express recombinant receptors. In some embodiments, the engineering is carried out by introducing polynucleotides encoding recombinant receptors. Polynucleotides encoding recombinant receptors, 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 a recombinant receptor contains a signal sequence encoding a signal peptide. In some embodiments, the signal sequence may encode a signal peptide derived from a native polypeptide. In other embodiments, the signal sequence may encode a heterologous or non-native signal peptide, such as the exemplary GMCSFR alpha chain signal peptide described in SEQ ID NO: 25 and encoded by the nucleotide sequence described in SEQ ID NO: 24. In some cases, the nucleic acid sequence encoding a recombinant receptor, such as a chimeric antigen receptor (CAR), contains a signal sequence encoding a signal peptide. Non-limiting and exemplary examples of signal peptides include, for example, the GMCSFR alpha chain signal peptide described in SEQ ID NO: 25 and encoded by the nucleotide sequence described in SEQ ID NO: 24, or the CD8 alpha signal peptide described in SEQ ID NO: 26.

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

[0313] In certain cases, where a nucleic acid molecule encodes two or more different polypeptide chains, such as a recombinant receptor and a marker, each polypeptide chain may be encoded by a separate nucleic acid molecule. For example, two separate nucleic acids may be provided, each individually transferred or introduced into cells for expression in the cells. 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 a nucleic acid encoding an intrasequence ribosome entry site (IRES) or, as appropriate, a self-cleaving peptide or a peptide that induces ribosome skipping, such as T2A, P2A, E2A, or F2A. 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 located at or inserted at different positions within the cell's genome. In some embodiments, the polynucleotide encoding the recombinant receptor is introduced into a composition containing cultured cells by retroviral transduction, transfection, or transformation, etc.

[0314] In some embodiments, such as those in which the polynucleotide contains first and second nucleic acid sequences, the coding sequences encoding each of the different polypeptide chains may be operably linked to promoters that 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 a nucleic acid molecule may be multicistronic (bicistronic or tricistronic; see, for example, U.S. Patent No. 6,060,273). In some embodiments, the transcription unit may be operated as a bicistronic unit containing an IRES (intrasequence ribosome entry site) that enables the simultaneous expression of gene products (e.g., encoding a marker and a recombinant receptor) via a message from a single promoter. Alternatively, in some cases, a single promoter may induce the expression of RNA containing two or three genes (e.g., encoding a marker and a recombinant receptor) separated from each other by a sequence encoding a self-cleaving peptide (e.g., a 2A sequence) or a protease recognition site (e.g., furin) in a single open reading frame (ORF). Thus, the ORF encodes a single polypeptide that is processed into individual proteins either in translation (in the case of 2A) or post-translation. In some cases, peptides such as T2A can cause ribosomes to skip the synthesis of the peptide bond at the C-terminus of the 2A element (ribosome skipping), causing separation between the end of the 2A sequence and the next peptide downstream (e.g., de Felipe, Genetic Vaccines and Ther. 2:13 (2004) and de Felipe et al. Traffic). See 5:616-626 (2004). Various 2A elements are known.Examples of 2A sequences that can be used in the methods and systems disclosed herein include, without limitation, 2A sequences derived 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 rhinitis virus-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 may 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 separate vectors or constructs contain 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 nucleic acid sequences encoding one or more markers. In some embodiments, one or more markers are transduction markers, surrogate markers, and / or selection markers.

[0317] In some embodiments, the marker is a transduction marker or a surrogate marker. The transduction marker or surrogate marker can be used to detect cells transduced by a polynucleotide, for example, a polynucleotide encoding a recombinant receptor. In some embodiments, the transduction marker may indicate or confirm cellular modification. In some embodiments, the surrogate marker is a protein constructed to be co-expressed on the cell surface together with a recombinant receptor, for example, 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 may be operably ligated to a nucleic acid sequence encoding a marker, which may be separated by an intrasequence ribosome entry site (IRES) or by a nucleic acid encoding a self-cleaving peptide or a peptide that induces ribosome skipping, such as a 2A sequence like T2A, P2A, E2A, or F2A. Exogenous marker genes may be used in conjunction with cells that have been manipulated to enable detection or selection of cells, and in other cases to promote cell suicide.

[0318] Exemplary surrogate markers may include cleavage forms of cell surface polypeptides, such as cleavage forms that are non-functional and do not or cannot transmit signals normally transmitted by the signal or the full-length form of the cell surface polypeptide, and / or do not or cannot internalize. Exemplary cleavage cell surface polypeptides include cleavage forms of growth factors or other receptors, such as cleavage human epidermal growth factor receptor 2 (tHER2), cleavage epidermal growth factor receptor (tEGFR, with an exemplary tEGFR sequence described in SEQ ID NO: 7 or 16), or prostate-specific membrane antigen (PSMA) or modified forms thereof. tEGFR may contain an epitope recognized by the antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibodies or binding molecules, which can be used for the identification or selection of cells manipulated with tEGFR constructs and the encoded exogenous protein, and / or for the efflux or isolation of 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., surrogate marker, includes all or part of CD34, NGFR, CD19 or cleaved CD19, e.g., cleaved non-human CD19, or epidermal growth factor receptor (e.g., tEGFR) (e.g., cleaved form).

[0319] In some embodiments, the marker is or includes fluorescent proteins, such as green fluorescent protein (GFP), high-sensitivity green fluorescent protein (EGFP), such as super-fold GFP (sfGFP), red fluorescent protein (RFP), such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue-green fluorescent protein (BFP), high-sensitivity blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), as well as variants thereof, including species variants, monomer variants, and codon-optimized and / or enhanced variants of fluorescent proteins. In some embodiments, the marker is or includes enzymes, such as luciferase, the lacZ gene from E. coli, alkaline phosphatase, secretory embryonic alkaline phosphatase (SEAP), and chloramphenicol acetyltransferase (CAT). Exemplary luminescence reporter genes include luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or variants thereof.

[0320] In some embodiments, the marker is a selection marker. In some embodiments, the selection marker is or includes a polypeptide that confers resistance to an exogenous drug or other drug. In some embodiments, the selection marker is an antibiotic resistance gene. In some embodiments, the selection marker is an antibiotic resistance gene that confers antibiotic resistance to mammalian cells. In some embodiments, the selection marker is or includes a puromycin resistance gene, a hygromycin resistance gene, a blastosidine resistance gene, a neomycin resistance gene, a genethecin resistance gene, or a zeosin resistance gene, or a modified form thereof.

[0321] In some embodiments, the molecule is a non-self molecule, such as a non-self protein, i.e., one that is not recognized as "self" by the host's immune system into which the cell is adopted.

[0322] In some embodiments, the marker does not perform a therapeutic function and / or has no effect other than being used as a marker for genetic engineering, for example, to select successfully engineered cells. In other embodiments, the marker can be a therapeutic molecule or a molecule that exerts a desired effect in other ways, such as a ligand for cells that will be encountered in vivo, or a co-stimulatory or immune checkpoint molecule to enhance and / or attenuate the cellular response after adoptive transfer and encounter with the ligand.

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

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

[0325] In some embodiments, the marker is a selection marker. In some embodiments, the selection marker is or includes a polypeptide that confers resistance to an exogenous drug or other drug. In some embodiments, the selection marker is an antibiotic resistance gene. In some embodiments, the selection marker is an antibiotic resistance gene that confers antibiotic resistance to mammalian cells. In some embodiments, the selection marker is or includes a puromycin resistance gene, a hygromycin resistance gene, a blastosidine resistance gene, a neomycin resistance gene, a genethecin resistance gene, or a zeosin resistance gene, or a modified form thereof.

[0326] In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious viral particles, such as vectors derived from simian virus 40 (SV40), adenovirus, or adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, for example, 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 a terminal repeat sequence (LTR) derived from a retroviral vector such as Moloney's mouse leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), or splenic fociforming virus (SFFV). The majority of retroviral vectors are derived from mouse retroviruses. In some embodiments, the retrovirus may be derived from any avian or mammalian cell source. Retroviruses are typically amphotropic, meaning they can infect several host cells, including human cells. In one embodiment, the gene to be expressed replaces the retroviral gag, pol, and / or env sequences. Numerous descriptive retroviruses have been described (e.g., U.S. Patent No. 5,219,740; U.S. Patent No. 6,207,453; U.S. Patent No. 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 to 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 to T cells via translocation (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-enhanced microparticle impaction (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

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

[0332] In some embodiments, cells, such as T cells, may be transfected with, for example, a T cell receptor (TCR) or a chimeric antigen receptor (CAR) either during or after growth. This transfection for gene introduction of the desired receptor may be carried out, for example, using any suitable retroviral vector. The genetically modified cell population can then be released from an initial stimulus (e.g., anti-CD3 / anti-CD28 stimulus) and subsequently stimulated with a second type of stimulus, for example, via the de novo-introduced receptor. This second type of stimulus may include antigenic stimulation in the form of a peptide / MHC molecule, a homologous (crosslinking) ligand of the gene-introduced receptor (e.g., a native ligand of the CAR) or any ligand (e.g., an antibody) that binds directly within the framework of the novel receptor (e.g., by recognizing a constant region within the receptor). For example, see 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 the activation of cells, such as T cells. In some such cases, cells can be selected and / or transduced before activation. Thus, cells can be manipulated before or after cell culture, and in some cases, concurrently with or during culture.

[0334] Further nucleic acids, such as genes for transfection, may improve the efficacy of therapy by, for example, promoting the viability and / or function of the transfected cells; for example, genes that provide genetic markers for cell selection and / or evaluation to assess in vivo survival or localization; for example, genes that improve safety by making cells more susceptible to negative selection, as described by Lupton SD et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); also see publications PCT / US91 / 08442 and PCT / US94 / 05601 by Lupton et al., describing 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 Riddell et al., U.S. Patent No. 6,040,177.

[0335] C. Cells and cell preparation for genetic manipulation In some embodiments, the nucleic acids are heterogeneous, i.e., not typically present in cells or cell-derived samples, such as those obtained from another organism or cell not normally found in the cells being manipulated, and / or from an organism from which such cells do not originate. In some embodiments, the nucleic acids are non-natural nucleic acids, including, for example, those comprising chimeric combinations of nucleic acids encoding various domains from a wide variety of cell types.

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

[0337] T cells and / or CD4 + and / or CD8 + Among the subtypes and subpopulations of T cells, there are naive T cells (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes, for example, 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, mucosa-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 granule cells, 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 through genetic engineering, thereby expressing recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterogeneous, i.e., not normally present in cells or samples derived from cells, such as those obtained from another organism or cell not normally found in the cells being engineered, and / or from an organism from which such cells do not originate. In some embodiments, the nucleic acids are non-natural nucleic acids, including, for example, those containing chimeric combinations of nucleic acids encoding various domains from a wide variety of cell types.

[0340] In some embodiments, the preparation of the manipulated cells includes one or more culture and / or preparation steps. Cells for the introduction of nucleic acids encoding transgenic receptors such as CARs 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 someone suffering from a disease or condition, or someone who requires cell therapy, or who will be administered cell therapy. In some embodiments, the subject is a human being who requires a specific therapeutic intervention, such as adoptive cell therapy, from which the cells are isolated, processed, and / or manipulated.

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

[0342] In some embodiments, the sample from which cells are derived or isolated is blood or a blood-derived sample, or an apheresis or leukocyte apheresis product, or derived therefrom. Exemplary samples include whole blood, peripheral blood monocytes (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumors, leukemia, lymphoma, lymph nodes, intestinal lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lungs, stomach, intestines, colon, kidneys, pancreas, chest, bone, prostate, cervix, testes, ovaries, tonsils, or other organs, and / or cells derived therefrom. Samples may include autologous and allogeneic source samples in the context of cell therapy, such as adoptive cell therapy.

[0343] In some embodiments, the cells are derived from cell lines, such as T cell lines. In some embodiments, the cells are obtained from heterologous sources, such as mice, rats, non-human primates, and pigs.

[0344] In some embodiments, cell isolation involves one or more preparation and / or non-affinity-based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents to lyse or remove cells that are highly sensitive to a particular reagent, for example, by removing undesirable components and enriching with desired components. In some examples, cells are isolated based on one or more properties, such as density, adhesion properties, size, sensitivity to and / or resistance to a particular component.

[0345] In some cases, the cells derived from the circulating blood of interest are obtained, for example, by apheresis or leukocyte apheresis. In some embodiments, the sample contains lymphocytes including T cells, monocytes, granule cells, B cells, other nucleated leukocytes, erythrocytes, and / or platelets, and in some embodiments, it contains cells other than erythrocytes and platelets.

[0346] In some embodiments, blood cells collected from the subject are washed to remove, for example, the plasma fraction and place the cells in a suitable buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the washing solution is deficient in calcium and / or magnesium and / or many or all divalent cations. In some embodiments, the washing step is carried out in a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions for use. In some embodiments, the washing step is carried out by tangential flow filtration (TFF) according to the manufacturer's instructions for use. In some embodiments, the cells are deficient in, for example, Ca ++ / Mg ++ The cells are resuspended in various biocompatible buffers, such as PBS, after washing, which do not contain [specific components]. In certain embodiments, components of the blood cell sample are removed, and the cells are directly resuspended in culture medium.

[0347] In some embodiments, the method includes density-based cell separation methods such as lysing erythrocytes and preparing leukocytes from peripheral blood by centrifugation using a Percoll or Ficoll gradient.

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

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

[0350] Isolation 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 those expressing a marker, refers to an increase in 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 those expressing a marker, refers to a decrease in 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 positive or negatively selected fractions from one step are subjected to further separation steps, such as 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 with respect to 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, a specific subpopulation of T cells, such as T cells, cells that are positive for one or more surface markers or express them at high levels, e.g., CD28 + CD62L + , CCR7 + CD27 + CD127 + CD4 + CD8 + CD45RA + , and / or CD45RO + T cells are isolated using positive or negative selection techniques.

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

[0354] In some embodiments, isolation is performed by enriching a specific cell population through positive selection, or by depleting a specific cell population through negative selection. In some embodiments, positive or negative selection is performed on cells that have been positively or negatively selected, respectively, by expressing (markers) on the selected cells. + ) or expressed at a relatively high level (marker 高 This is done by incubating with one or more antibodies or other conjugates that specifically bind to one or more surface markers.

[0355] In some embodiments, T cells are isolated from PBMC samples by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other leukocytes, such as CD14. In some embodiments, CD4 + or CD8 + The selection process is CD4 + Helper and CD8 + Used to isolate cytotoxic T cells. Such CD4 + and CD8 + The population may be further selected into subpopulations by positive or negative selection for one or more markers expressed or relatively highly expressed on naive, memory, and / or effector T cell subpopulations.

[0356] In some embodiments, CD8 + Cells are further enriched or depleted with respect to naive, central memory, effector memory, and / or central memory stem cells by positive or negative selection based, for example, on surface antigens associated with each subpopulation. In some embodiments, central memory T(T) CMEnrichment of cells is performed to enhance efficacy, for example, to improve long-term survival, growth, and / or engraftment after administration, and in some embodiments, it 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 is enriched with CD8 + T cells and CD4 + Combining it with T cells further enhances its effectiveness.

[0357] In one embodiment, memory T cells are CD8 + CD62L in peripheral blood lymphocytes + and CD62 - It is present in both subsets. PBMCs can be used, for example, with anti-CD8 and anti-CD62L antibodies to identify CD62L-CD8 + and / or CD62L + CD8 + With respect to the fraction, it can be enriched or depleted.

[0358] In some embodiments, the central memory (T CM Enrichment of 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 The enrichment of cells is based on negative selection of cells that express or highly express CD45RA and / or granzyme B. In some embodiments, T CM CD8 enriched in cells + Population isolation is performed by depletion of cells expressing CD4, CD14, and CD45RA, and positive selection or enrichment of cells expressing CD62L. In one embodiment, central memory (T CMThe enrichment of cells begins with a negative fraction of cells selected based on CD4 expression, which is then subjected to negative selection based on CD14 and CD45RA expression, and positive selection based on CD62L. Such selections are performed simultaneously in some embodiments and sequentially in other embodiments. In some embodiments, both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the method, in one or more subsequent positive or negative selection steps as appropriate, such as CD8 + The same CD4 expression-based selection process used when preparing a cell population or subpopulation is similar to CD4 + It is also used to generate cell populations or subpopulations.

[0359] In certain cases, PBMC samples or other leukocyte samples are CD4 + The cells are subjected to selection, where negative and positive fractions are retained. The negative fraction is then subjected to negative selection based on the expression of CD14 and CD45RA or CD19, and positive selection based on a characteristic marker of central memory T cells, such as CD62L or CCR7, where positive and negative selection 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. CD4 + Lymphocytes can be obtained by standard methods. In some embodiments, naive CD4 + T lymphocytes are CD45RO - CD45RA + CD62L + CD4 + These are T cells. In some embodiments, central memory CD4 + The cells are CD62L + and CD45RO + In some embodiments, the effector CD4 +The cells are CD62L - and CD45RO - That is the case.

[0361] In one example, negative selection leads to CD4 + To enrich cells, monoclonal antibody cocktails typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as magnetic or paramagnetic beads, to enable cell separation with respect to 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 (as outlined in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In vivo, pp. 17-25 Edited by: SA Brooks and U. Schumacher (copyright) Humana Press Inc., Totowa, New Jersey).

[0362] In some embodiments, a sample or composition of cells to be separated is incubated with a small magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., Dynalbeads or MACS beads). The magnetically responsive material, such as particles, is generally bound directly or indirectly to molecules present on the cells or cell populations to be separated, such as negatively or positively selected cells, such as surface markers, or to binding partners such as antibodies that specifically bind to these molecules, such as antibodies.

[0363] In some embodiments, the magnetic particles or beads include a magnetically responsive material bound to a specific binding partner, such as an antibody or other binding partner. Many well-known magnetically responsive materials exist that are used in magnetic separation methods. Suitable magnetic particles include those described in Molday's U.S. Patent No. 4,452,773 and European Patent Specification EP452342B, which are thus incorporated herein by reference. Other examples include colloidal-sized particles, such as those described in Owen's U.S. Patent No. 4,795,698 and Liberti et al.'s U.S. Patent No. 5,200,084.

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

[0365] In some embodiments, the sample is placed in a magnetic field, and cells having bound magnetically responsive or magnetizable particles are bound to a magnet and separated from unlabeled cells. With respect to positive selection, cells bound to the magnet are retained, and with respect to negative selection, unbound cells (unlabeled cells) are retained. In some embodiments, a combination of positive and negative selection is carried out in the same selection step, where the positive and negative fractions are retained, further processed, and subjected to further 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 to 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 attached. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with a biotinylated primary or secondary antibody.

[0367] In some embodiments, the magnetically responsive particles remain bound to cells that are subsequently incubated, cultured, and / or manipulated, and in some embodiments, 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 competing magnetizable particles or antibodies conjugated to a cleavable linker. In some embodiments, the magnetizable particles are biodegradable.

[0368] In some embodiments, affinity-based selection is performed 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 such a manner that non-target and target species are sequentially eluted after the application of an external magnetic field. That is, cells bound to magnetized particles are maintained in place, while unbound species are eluted. Next, after this first elution step is complete, species that are trapped by the magnetic field and prevented from eluting are released in some manner so that they can be eluted and recovered. In certain embodiments, non-target cells are labeled and depleted from the heterogeneous population of cells.

[0369] In some embodiments, 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 embodiments, the system is used to perform each of these steps in a closed or sterile environment, for example, to minimize errors, user handling, and / or contamination. One example of such a system is the one described in International Patent Application Publication No. WO2009 / 072003 or US20110003380A1.

[0370] In some embodiments, the system or apparatus performs one or more, for example, all, isolation, processing, manipulation, and formulation steps in an integrated or self-contained system and / or in an automated or programmable manner. In some embodiments, the system or apparatus includes a computer and / or computer program that communicates with the system or apparatus, thereby enabling the user to program, control, evaluate, and / or adjust various aspects thereof, the outcomes of the processing, isolation, manipulation, and formulation steps.

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

[0372] In some embodiments, the CliniMACS system uses magnetizable particles coupled with antibodies supplied in a sterile, non-pyrogenic solution. In some embodiments, after labeling cells with magnetic particles, the cells are washed to remove excess particles. The cell preparation bag is then connected to a tube set, which is subsequently connected to a buffer-containing bag and a cell collection bag. The tube set consists of assembled sterile tubes, including a pre-column and a separation column, and is for single use only. After the separation program is started, 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, the cell population for use using the method described herein is unlabeled and is not retained in the column. In some embodiments, the cell population for use using the method described herein is labeled and is retained in the column. In some embodiments, the cell population for use using the method described herein is eluted from the column after the magnetic field is removed and collected in the cell collection bag.

[0373] In certain embodiments, separation and / or other steps are performed using the CliniMACS Prodigy system (Miltenyi Biotec). In some embodiments, the CliniMACS Prodigy system is equipped with a cell processing unit that enables automated cell washing and fractionation by centrifugation. The CliniMACS Prodigy system may also include a built-in camera and image recognition software that determines the optimal cell fractionation endpoint by identifying macroscopic layers of source cell products. For example, peripheral blood is automatically separated into erythrocyte, leukocyte, 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 growth, antigen loading, and long-term cell culture. Input ports may allow for sterile removal and replenishment of culture medium, and cells can be monitored using an integrated microscope. For example, see 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, the cells can be labeled with multiple markers, enabling the isolation of a clearly defined T cell subset with high purity.

[0375] In some embodiments, the antibody or binding partner is labeled with one or more detectable markers to facilitate separation with respect to positive and / or negative selection. For example, separation may be based on binding to a fluorescently labeled antibody. In some examples, cell separation based on the binding of an antibody or other binding partner specific to one or more cell surface markers is carried in a flow stream, for example, by fluorescence-activated cell sorting (FACS) including a preparative scale (FACS) and / or 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 relating to freezing, for example, cryopreserving, cells either before or after isolation, incubation, and / or manipulation. In some embodiments, the freezing, followed by a thawing step, removes granule cells and, to some extent, monocytes from the cell population. In some embodiments, the cells are suspended in a freezing solution after a washing step to remove, for example, plasma and platelets. Any of the various known freezing solutions and parameters may be used in some embodiments. One example involves PBS or other suitable cell freezing medium containing 20% ​​DMSO and 8% human serum albumin (HSA). This is then diluted 1:1 with the medium, resulting in final concentrations of DMSO and HSA of 10% and 4%, respectively. The cells are then typically frozen to -80°C at a rate of 1°C per minute and stored in the gas phase of a liquid nitrogen storage tank.

[0377] In some embodiments, cells are incubated and / or cultured before or in connection with genetic manipulation. The incubation step may include culture, cultivation, stimulation, activation, and / or propagation. Incubation and / or manipulation may be carried out in a culture vessel such as a unit, chamber, well, column, tube, tube set, valve, vial, culture dish, bag, or other container for culturing or growing cells. In some embodiments, the composition or cells are incubated in the presence of stimulating conditions or stimulants. Such conditions include those designed to induce cell proliferation, growth, activation, and / or survival in a population, to mimic antigen exposure, and / or to prime cells for genetic manipulation, for example, for the introduction of recombinant antigen receptors.

[0378] The conditions may include one or more of the following: specific culture medium, temperature, oxygen content, carbon dioxide content, time, drugs such as nutrients, amino acids, antibiotics, ions, and / or stimulants such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other drugs designed to activate cells.

[0379] In some embodiments, the stimulating conditions or agents include one or more agents, e.g., ligands, that can activate or stimulate the intracellular signaling domain of the TCR complex. In some embodiments, the agents activate or initiate the TCR / CD3 intracellular signaling cascade in T cells. Such agents may include antibodies, e.g., TCR-specific ones, e.g., anti-CD3. In some embodiments, the stimulating conditions include one or more agents, e.g., ligands, e.g., anti-CD28, that can stimulate costimulatory receptors. In some embodiments, such agents and / or ligands may be conjugated to a solid support such as beads and / or one or more cytokines. Optionally, the amplification method may further include the step of 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, stimulants include IL-2, IL-15, and / or IL-7. In some embodiments, the IL-2 concentration is at least about 10 units / mL.

[0380] In some embodiments, incubation is carried out in accordance with techniques such as those described in U.S. Patent No. 6,040,177 by 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 increased by adding feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs), to the culture initiation composition (e.g., so that the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells with respect to each T lymphocyte in the initial population to be increased); and by incubating the culture (e.g., for a time sufficient to increase the number of T cells). In some embodiments, the non-dividing feeder cells may 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 embodiments, the feeder cells are added to the culture medium before the addition of the T cell population.

[0382] In some embodiments, the stimulation conditions include temperatures suitable for human T lymphocyte growth, e.g., at least about 25°C, generally at least about 30°C, and generally 37°C or about 37°C. Optionally, the incubation may further include the addition of lymphoblast-like cells (LCLs) transformed with non-mitotic EBV as feeder cells. The LCLs may be irradiated with gamma rays in the range of about 6,000 to 10,000 rads. In some embodiments, the LCL feeder cells are provided in any suitable amount, for example, with a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.

[0383] In one embodiment, antigen-specific T cells, such as antigen-specific CD4 + and / or CD8 + T cells are 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 cytomegalovirus antigens by isolating T cells from an infection target and stimulating the cells in vitro with the same antigen.

[0384] III. Outcomes of exemplary treatments and methods for evaluating them In some embodiments of the methods, compositions, combinations, uses, kits, and products provided herein, the combination therapies provided result in one or more therapeutic outcomes, characteristics associated with one or more of the therapy or parameters associated with the therapy, such as those described below. In some embodiments, the method is one of those described in Section I. In some embodiments, the method further includes evaluation of the exposure, persistence, and proliferation of T cells, e.g., T cells administered for T cell-based therapy. In some embodiments, the cell exposure, or long-term growth and / or persistence, and / or changes in cellular phenotype or functional activity of cells in the methods provided herein, e.g., cells administered for immunotherapy, e.g., T cell therapy, can be measured by evaluating the properties of T cells in vivo or ex vivo. In some embodiments, such assays can be used to determine or confirm the function of T cells, e.g., T cell therapy, before, during, or after administration of the combination therapy provided herein.

[0385] In some embodiments, the combination therapy may further include one or more screening steps for identifying subjects with respect to treatment using the combination therapy and / or for continuing the combination therapy, and / or for evaluating treatment outcomes and / or for monitoring treatment outcomes. In some embodiments, the steps for evaluating treatment may include steps for evaluating and / or monitoring treatment and / or for identifying subjects with respect to the administration of further or remaining steps of the therapy and / or for repeated therapy. In some embodiments, the screening steps for treatment outcomes and / or evaluation can be used to determine the dose, frequency, duration, timing and / or sequence of the combination therapies provided herein.

[0386] In some embodiments, any of the treatment outcome screening steps and / or evaluations described herein can be used before, during, or after the administration of one or more steps of the combination therapy provided, e.g., administration of T-cell therapy (e.g., CAR-expressing T cells) and / or compound C. In some embodiments, the evaluation is performed before, during, or after the implementation of any of the methods provided herein. In some embodiments, the evaluation is performed before the implementation of any of the methods provided herein. In some embodiments, the evaluation is performed after the implementation of one or more steps of the methods provided herein. In some embodiments, the evaluation is performed before the administration of one or more steps of the combination therapy provided, e.g., to screen and identify patients who are suitable and / or likely to receive the combination therapy. In some embodiments, the evaluation is performed during, or after the administration of one or more steps of the combination therapy provided, e.g., to evaluate intermediate or final treatment outcomes, e.g., to determine the effectiveness of the therapy and / or to determine whether to continue or repeat the therapy and / or to determine whether to administer the remaining steps of the combination therapy.

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

[0388] In some embodiments, the screening and / or evaluation of treatment outcomes includes assessing the survival and / or function of T cells administered for cell-based therapy. In some embodiments, the screening and / or evaluation of treatment outcomes includes assessing the levels of cytokines or growth factors. In some embodiments, the screening and / or evaluation of treatment outcomes includes assessing disease burden and / or improvement, e.g., tumor burden and / or clinical outcomes. In some embodiments, any of ...

Claims

1. A therapeutic agent for cancer expressing differentiation antigen group 19 (CD19), characterized in that the therapeutic agent is used in combination therapy to subjects suffering from cancer expressing CD19. (i) A T-cell therapy agent comprising a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds to differentiation antigen group 19 (CD19), which is administered on day 1 of combination therapy; and (ii)(S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or its enantiomer or mixture of enantiomers, or pharmaceutically acceptable salts, solvates, hydrates, cocrystals, clathrates or polymorphs A therapeutic agent, including combination therapy drugs.

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

3. A therapeutic agent for cancer expressing CD19, wherein the therapeutic agent comprises a compound which is (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or an enantiomer or mixture of enantiomers thereof, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate or polymorph thereof, and the compound or so A therapeutic agent characterized in that an enantiomer or mixture of enantiomers, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate, or polymorph thereof, is used in combination therapy with a T-cell therapy agent containing a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds to differentiation antigen group 19 (CD19), wherein the T-cell therapy agent is administered on day 1 of the combination therapy.

4. A therapeutic agent for lymphoma, characterized in that the therapeutic agent is used in combination therapy to a subject suffering from the said cancer. (i) A T-cell therapy agent comprising a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds to differentiation antigen group 19 (CD19), which is administered on day 1 of combination therapy; and (ii)(S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or its enantiomer or mixture of enantiomers, or pharmaceutically acceptable salts, solvates, hydrates, cocrystals, clathrates or polymorphs A therapeutic agent, including combination therapy drugs.

5. A therapeutic agent for lymphoma, wherein the therapeutic agent comprises a T-cell therapy agent comprising a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds to differentiation antigen group 19 (CD19), wherein the T-cell therapy agent is used to be administered to a subject with the cancer in combination therapy with a compound that is (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or an enantiomer or mixture of enantiomers thereof, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate or polymorph thereof, wherein the T-cell therapy agent is administered on day 1 of the combination therapy.

6. A therapeutic agent for lymphoma, wherein the therapeutic agent comprises a compound that is (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or an enantiomer or mixture of enantiomers thereof, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate or polymorph thereof, wherein the compound is used in combination therapy with a T-cell therapy agent comprising a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds to differentiation antigen group 19 (CD19), the T-cell therapy agent being administered on day 1 of the combination therapy.

7. (i) The administration of the compound is initiated after the administration of the T-cell therapy agent, (ii) The compound is administered in multiple doses, each dose being 0.1 mg or about 0.1 mg to 0.6 mg or about 0.6 mg (including upper and lower limits), or (iii) The therapeutic agent according to any one of claims 1 to 6, wherein the compound is administered as a plurality of intermittent doses administered once a week or less.

8. The therapeutic agent according to any one of claims 1 to 7, wherein the administration of the compound is started between day 1 and day 22 (including the upper and lower limits).

9. The therapeutic agent according to claim 7 or 8, wherein each of the multiple intermittent doses is the same.

10. (i) The compound is administered (a) once a week 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), A therapeutic agent according to any one of claims 1 to 9.

11. (i) The compound is administered for at least 12 weeks after the administration of the T-cell therapy agent, or (ii) The therapeutic agent according to any one of claims 1 to 10, wherein the compound is administered for up to 12 weeks after the administration of the T-cell therapeutic agent.

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 therapeutic agent according to any one of claims 1 to 11, wherein the compound is administered on days 8, 22, 36, 50, 64 and 78.

13. The therapeutic agent according to any one of claims 1 to 12, wherein the dose of the compound is 0.3 mg or about 0.3 mg to 0.6 mg or about 0.6 mg (including upper and lower limits).

14. The therapeutic agent according to any one of claims 1 to 13, wherein the compound is a pharmaceutically acceptable salt or polymorph of (S)-2-(2,6-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-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-dioxopiperidine-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidine-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 after administration of the T-cell therapy drug, and the severe toxicity is severe cytokine release syndrome (CRS) or severe neurotoxicity, or (ii) If the subject exhibits toxicity after administration of the compound, administration of the compound is withheld or the dose of the compound is modified, and the toxicity is severe thrombocytopenia or severe neutropenia. A therapeutic agent according to any one of claims 1 to 15.

17. The therapeutic agent according to claim 16, wherein administration of the compound is resumed after the subject no longer exhibits toxicity.

18. The therapeutic agent according to any one of claims 1 to 3 and 7 to 17, wherein the cancer is a B-cell malignant lesion.

19. The therapeutic agent according to any one of claims 4 to 17, wherein the cancer is non-Hodgkin lymphoma (NHL).

20. The therapeutic agent according to claim 19, wherein the NHL includes invasive NHL; diffuse large B-cell lymphoma (DLBCL); DLBCL-NOS; EBV-positive DLBCL-NOS; T-cell / histiocytocyte-rich large B-cell lymphoma; primary mediastinal large B-cell lymphoma (PMBCL); follicular lymphoma (FL); or MYC having a DLBCL histological appearance and high-grade B-cell lymphoma (double / triple hit) having BCL2 and / or BCL6 rearrangement.

21. The therapeutic agent according to claim 20, wherein the DLBCL-NOS is transformed from low-grade malignancy.

22. The therapeutic agent according to claim 20, wherein the follicular lymphoma (FL) is follicular lymphoma grade 3B (FL3B).

23. The therapeutic agent according to any one of claims 1 to 22, wherein the CD19 is human CD19.

24. (i) The chimeric antigen receptor (CAR) comprises an intracellular signaling domain including ITAM, and the intracellular signaling domain comprises a CD3-zeta (CD3ζ) chain signaling domain, (ii) The chimeric antigen receptor (CAR) further comprises a co-stimulatory signaling region, A therapeutic agent according to any one of claims 1 to 23.

25. The therapeutic agent according to claim 24, wherein the co-stimulatory signaling region comprises a signaling domain of CD28 or 4-1BB.

26. The therapeutic agent according to claim 25, wherein the CD3-zeta (CD3ζ) chain is a human CD3-zeta chain, and the CD28 or 4-1BB is human CD28 or human 4-1BB.

27. (i) The CAR comprises a CD19-specific scFv; a transmembrane domain; a cytoplasmic signaling domain derived from a co-stimulatory molecule; and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, (ii) The CAR comprises, in order, a CD19-specific scFv; a transmembrane domain; a cytoplasmic signaling domain derived from a co-stimulatory molecule; and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, or (iii) The CAR comprises, in order, a CD19-specific scFv; a spacer; a transmembrane domain; a cytoplasmic signaling domain derived from a co-stimulatory molecule; and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule. A therapeutic agent according to any one of claims 1 to 26.

28. (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 or a modified version thereof, and / or comprises about 15 amino acids or less. (iii) The spacer is 12 amino acids long or about 12 amino acids long and / or comprises or consists of all or part of an immunoglobulin hinge or a modified version thereof, (iv) The spacer has or consists of any of the above variants having at least 85% sequence identity thereto; sequence code for sequence number 1; sequence code for sequence number 2, sequence number 30, sequence number 31, sequence number 32, sequence number 33, sequence number 34; or sequence identity thereto (v) The cytoplasmic signaling domain derived from the costimulatory molecule comprises the sequence described in Sequence ID No. 12, or a variant thereof having at least 85% sequence identity thereto. (vi) The cytoplasmic signaling domain derived from the primary signaling ITAM-containing molecule comprises any sequence of SEQ ID NOs: 13 to 15, or a variant thereof having at least 85% sequence identity thereto. (vii) The scFv includes 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), or (viiii) The therapeutic agent according to claim 27, wherein the scFv comprises 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).

29. The aforementioned scFv is, Variable heavy chain region of FMC63 and 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 A therapeutic agent according to claim 27 or 28, comprising:

30. The therapeutic agent according to any one of claims 27 to 29, wherein the scFv has the amino acid sequence described in Sequence ID No.

43.

31. The dose of the manipulated cells is approximately 1 × 10 ５ pieces ~ 5×10 ８ It includes a total of 100 CAR-expressing T cells (including upper and lower limits), and The dose of the manipulated cells is administered parenterally. A therapeutic agent according to any one of claims 1 to 30.

32. (i) whether the T cells are (a) primary T cells obtained from the subject, and / or (b) autologous to the subject, (ii) The T cells are allogeneic to the subject, A therapeutic agent according to any one of claims 1 to 31.

33. The therapeutic agent according to any one of claims 1 to 32, wherein the dose of the manipulated cells comprises CAR-expressing CD4+ T cells and CAR-expressing CD8+ T cells, and the administration of the dose comprises the step of administering a plurality of separate compositions, wherein the plurality of separate compositions comprises a first composition comprising one of CD4+ T cells and CD8+ T cells and a second composition comprising the other of CD4+ T cells and CD8+ T cells.

34. (i) The first composition and the second composition are administered at intervals of 0 to 12 hours, (ii) The administration of the first composition and the administration of the second composition are performed at intervals of approximately 1 minute to approximately 1 hour, and / or (iii) The therapeutic agent according to claim 33, wherein the first composition and the second composition are administered at intervals of two hours or less.

35. (i) The first composition comprises (a) CD4+ T cells or (b) CD8+ T cells, (ii) The therapeutic agent according to claim 33 or 34, wherein the first composition is administered before the second composition.

36. The therapeutic agent according to any one of claims 1 to 35, wherein the subject is preconditioned with lymphocyte depletion therapy including the administration of fludarabine and / or cyclophosphamide prior to the administration of the T-cell therapeutic agent.

37. The therapeutic agent according to any one of claims 1 to 35, further comprising the step of administering a lymphocyte depletion therapy to the subject, including the administration of fludarabine and / or cyclophosphamide, immediately before the administration of the T-cell therapeutic agent.

38. The aforementioned lymphocyte depletion therapy involves administering approximately 200-400 mg / m² daily for 2-4 days. ２ Cyclophosphamide (including upper and lower limits) and / or approximately 20-40 mg / m² ２ The administration of fludarabine, or the lymphocyte depletion therapy, is approximately 500 mg / m². ２ A therapeutic agent according to claim 36 or 37, comprising the administration of cyclophosphamide.

39. The therapeutic agent according to any one of claims 1 to 38, wherein the subject is a human.

40. At least 35% of the treated subjects achieve a sustained complete response (CR) for six months or longer, or at least 60% of the subjects who achieve a CR achieve a sustained CR; and / or At least 60% of patients who achieve complete response (CR) by the sixth month maintain a response, maintain CR, survive, or survive without progression for three months or longer; or The therapeutic agent according to any one of claims 1 to 39, wherein at least 50% of subjects treated according to the method achieve an objective response (OR).

41. The therapeutic agent according to any one of claims 1 to 40, wherein at the time of or immediately before administration of the dose of the manipulated cells, the subject relapses or becomes refractory to one or more prior therapies for lymphoma other than another dose of manipulated cells expressing CAR.

42. At or before administering the dose of the manipulated cells, The subject is identified as having or suffering from double / triple hit lymphoma; The subject is identified as having or suffering from chemotherapy-resistant lymphoma; and / or The therapeutic agent according to any one of claims 1 to 41, wherein the subject has not achieved complete remission (CR) in response to previous therapy.

43. The administration of the aforementioned compound The exhausted phenotype in CAR-expressing T cells in the aforementioned subjects was reversed; To prevent, inhibit, or delay the development of exhaustion phenotype in CAR-expressing T cells in the aforementioned subjects; To reduce the level or degree of exhaustion phenotype in CAR-expressing T cells in the subject; or A therapeutic agent according to any one of claims 1 to 42, which reduces the percentage or total number of CAR-expressing T cells in a subject having an exhausted phenotype.

44. The therapeutic agent according to any one of claims 1 to 43, wherein the administration of the compound is initiated after the administration of the T-cell therapeutic agent, and following the 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.

45. The administration of the aforementioned compound (a) Compared to the absence of the administration of the compound, exposure of T cells to the CD19 antigen or antigen receptor-specific agent results in an increase in the antigen-specific or antigen receptor-driven activity of naive or unexhausted T cells in the subject; (b) Compared to the absence of the administration of the compound, preventing, inhibiting, or delaying the development of exhaustion phenotypes in naive or non-exhausted T cells in a subject after exposure to CD19 antigen or antigen receptor-specific agents; or (c) The therapeutic agent according to any one of claims 1 to 44, comprising administration in an amount, frequency and / or duration effective to reverse the exhaustion phenotype in exhausted T cells in a subject compared to the absence of the administration of the compound.

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