CN117858720A

CN117858720A - Combination of CAR T cell therapy and immunomodulatory compounds for treatment of lymphomas

Info

Publication number: CN117858720A
Application number: CN202280037122.0A
Authority: CN
Inventors: N·S·特雷德; T·帕尔汉姆; F·吴; M·普德耐德; J·A·迪斯泰法诺; M·S·卡兰西奥安东; T·J·巴克霍尔兹; M·宝姿; J·秦; R·杜博威; N·布兰德
Original assignee: Juno Therapeutics Inc
Current assignee: Juno Therapeutics Inc
Priority date: 2021-03-29
Filing date: 2022-03-29
Publication date: 2024-04-09

Abstract

Methods, compositions, uses, and articles of manufacture are provided for combination therapies involving immunotherapy, such as adoptive cell therapy, e.g., T-cell therapy, and 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 mixture of enantiomers thereof, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, for treating subjects suffering from diseases and disorders such as certain B-cell malignancies, as well as related methods, compositions, uses, and articles of manufacture. The cells typically express a recombinant receptor, such as a Chimeric Antigen Receptor (CAR). In some embodiments, the disease or disorder is non-hodgkin lymphoma (NHL), such as recurrent or refractory NHL or a particular NHL subtype.

Description

Combination of CAR T cell therapy and immunomodulatory compounds for treatment of lymphomas

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application number 63/167,599 filed on day 29 of 3 months 2021 and U.S. provisional application number 63/277,134 filed on day 8 of 11 of 2021, the contents of each of which are hereby incorporated by reference in their entirety for all purposes.

Incorporated by reference into the sequence listing

The present application is filed with a sequence listing in electronic format. The sequence listing is provided as a file titled 735042024540seqlist. Txt, which was created at 2022, 3, 27, and 36 kilobytes in size. The information in the sequence listing in electronic format is incorporated in its entirety by reference.

Technical Field

The present disclosure relates in some aspects to methods, compositions, uses, and articles of manufacture of combination therapies involving immunotherapy, such as adoptive cell therapy, e.g., T-cell therapy, and 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 mixture of enantiomers thereof, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, for treating subjects suffering from diseases and conditions such as certain B-cell malignancies, and related methods, compositions, uses, and articles of manufacture. The T cell therapies include cells that express recombinant receptors, such as Chimeric Antigen Receptors (CARs). In some embodiments, the disease or disorder is non-hodgkin lymphoma (NHL), such as recurrent or refractory NHL or a particular NHL subtype.

Background

Various strategies are available for immunotherapy, such as the administration of engineered T cells for adoptive therapy. For example, strategies can be used to engineer T cells expressing genetically engineered antigen receptors (e.g., CARs), and to administer compositions containing such cells to a subject. Improved strategies are needed to improve the efficacy of cells, e.g., to improve persistence, activity, and/or proliferation of cells after administration to a subject. Methods, compositions, kits, and systems are provided that meet such needs.

Disclosure of Invention

In some embodiments, provided herein is a method of treating a CD19 expressing cancer, the method comprising administering to a subject having a CD19 expressing cancer a combination therapy comprising: (i) A T cell therapy comprising a dose of engineered cells comprising T cells expressing a Chimeric Antigen Receptor (CAR) for the cancer; and (ii) a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof.

In some of any of the provided methods, the CD19 expressing cancer is a lymphoma.

In some embodiments, provided herein is also a method of treating lymphoma, comprising administering to a subject having cancer that is lymphoma a combination therapy comprising: (i) A T cell therapy comprising a dose of engineered cells comprising T cells expressing a Chimeric Antigen Receptor (CAR) that binds cluster of differentiation 19 (CD 19); and (ii) a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof.

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

In some embodiments, provided herein is also a method of treating a CD19 expressing cancer, the method comprising administering to a subject having a CD19 expressing cancer a T cell therapy comprising a dose of an engineered cell comprising a T cell expressing a Chimeric Antigen Receptor (CAR) targeted to the cancer, wherein the T cell therapy is administered in combination therapy with a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein administration of the compound is initiated prior to administration of the T cell therapy.

In some embodiments, provided herein is also a method of treating lymphoma comprising administering to a subject having a cancer that is lymphoma a T cell therapy comprising a dose of engineered cells comprising T cells expressing a Chimeric Antigen Receptor (CAR) that binds cluster of differentiation 19 (CD 19), wherein the T cell therapy is administered in combination therapy with a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein administration of the compound is initiated prior to administration of the T cell therapy.

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

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

In some embodiments, provided herein is a method of treating a lymphoma comprising administering to a subject having a cancer that is lymphoma a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein the compound is administered in combination therapy with a T cell therapy comprising a dose of engineered cells comprising T cells that express a Chimeric Antigen Receptor (CAR) that binds cluster of differentiation 19 (CD 19), wherein administration of the compound is initiated after administration of the T cell therapy.

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

In some of any of the provided methods, the administration of the compound is initiated between (including the endpoints of) day 1 and day 29 of the combination therapy.

In some of any of the provided methods, the compound is administered in a plurality of doses, wherein each dose is between or about 0.1mg and or about 0.6mg, inclusive.

In some of any of the provided methods, the compound is administered in a plurality of intermittent doses that do not exceed once a week administration.

In some embodiments, provided herein is also a method of treating lymphoma, comprising administering to a subject having lymphoma a combination therapy comprising: (i) A T cell therapy comprising a dose of engineered cells comprising T cells expressing a Chimeric Antigen Receptor (CAR) that binds cluster of differentiation 19 (CD 19), wherein the T cell therapy is administered on day 1 of the combination therapy; and (ii) a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione having the structure:

or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein the compounds are administered in intermittent (i.e., non-daily) dosing regimens.

In some embodiments, provided herein is also a method of treating lymphoma comprising administering to a subject suffering from lymphoma a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione having the structure:

Or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein the compound is administered in combination therapy with a T cell therapy comprising a dose of engineered cells comprising T cells expressing a Chimeric Antigen Receptor (CAR) that binds cluster of differentiation 19 (CD 19), wherein the T cell therapy is administered on day 1 of the combination therapy and the compound is administered on an intermittent (i.e., non-daily) dosing regimen.

In some of any of the provided methods, the compound is administered in a plurality of intermittent doses. In some of any of the provided methods, the compound is administered no more than once a week.

In some of any of the provided methods, each dose of the compound is between or about 0.1mg and or about 0.6mg, inclusive.

Or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein the compound is administered at a plurality of intermittent doses not exceeding once weekly administration, wherein each dose is between or about 0.1mg and or about 0.6mg (inclusive), wherein administration of the compound is initiated between (inclusive of) day 1 and day 29 of the combination therapy.

or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein: administering the compound in combination therapy with a T cell therapy comprising a dose of engineered cells comprising T cells expressing a Chimeric Antigen Receptor (CAR) that binds cluster of differentiation 19 (CD 19), wherein the T cell therapy is administered on day 1 of the combination therapy; and administering the compound in a plurality of intermittent doses not exceeding once weekly administration, wherein each dose is between or about 0.1mg and or about 0.6mg (inclusive), and wherein administration of the compound is initiated between days 1 and 29 of the combination therapy (inclusive).

In some of any of the provided methods, the administration of the compound is started between day 1 and day 22 (inclusive). In some of any of the provided methods, the administration of the compound is started between day 1 and day 15 (inclusive). In some of any of the provided methods, the administration of the compound begins between day 8 and day 15 (inclusive).

In some of any of the provided methods, the administration of the compound is started at or about day 1. In some of any of the provided methods, the administration of the compound is started at or about day 8. In some of any of the provided methods, the administration of the compound is started at or about day 15.

In some of any of the provided methods, each of the plurality of intermittent doses is the same.

In some of any of the provided methods, the compound is administered once per week. In some of any of the provided methods, the compound is administered once every 7 days (Q7D). In some of any of the provided methods, the compound is administered once every two weeks. In some of any of the provided methods, the compound is administered once every 14 days (Q14D).

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

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

In some of any of the provided methods, the dose of the compound is between or about 0.3mg and or about 0.6mg, inclusive. In some of any of the provided methods, the dose of the compound is at or about 0.6mg.

In some of any of the provided methods, the dose of the compound is between or about 0.2mg and or about 0.4mg, inclusive. In some of any of the provided methods, the dose of the compound is at or about 0.4mg. In some of any of the provided methods, the dose of the compound is less than 0.4mg. In some of any of the provided methods, the dose of the compound is at or about 0.3mg. In some of any of the provided methods, the dose of the compound is at or about 0.2mg.

or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein: administering the compound in combination therapy with a T cell therapy comprising a dose of engineered cells comprising T cells expressing a Chimeric Antigen Receptor (CAR) that binds cluster of differentiation 19 (CD 19), wherein the T cell therapy is administered on day 1 of the combination therapy; and the compound was administered in a plurality of intermittent doses administered once every seven days (Q7D) and on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85, wherein each dose is 0.3mg.

Or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein: administering the compound in combination therapy with a T cell therapy comprising a dose of engineered cells comprising T cells expressing a Chimeric Antigen Receptor (CAR) that binds cluster of differentiation 19 (CD 19), wherein the T cell therapy is administered on day 1 of the combination therapy; and the compound was administered in a plurality of intermittent doses administered once every seven days (Q7D) and on days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85, wherein each dose is 0.3mg.

or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein: administering the compound in combination therapy with a T cell therapy comprising a dose of engineered cells comprising T cells expressing a Chimeric Antigen Receptor (CAR) that binds cluster of differentiation 19 (CD 19), wherein the T cell therapy is administered on day 1 of the combination therapy; and the compound was administered in a plurality of intermittent doses administered once every seven days (Q7D) and on days 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85, wherein each dose is 0.3mg.

or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein: administering the compound in combination therapy with a T cell therapy comprising a dose of engineered cells comprising T cells expressing a Chimeric Antigen Receptor (CAR) that binds cluster of differentiation 19 (CD 19), wherein the T cell therapy is administered on day 1 of the combination therapy; and the compound was administered in a plurality of intermittent doses administered once every seven days (Q7D) and on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85, wherein each dose is 0.4mg.

Or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein: administering the compound in combination therapy with a T cell therapy comprising a dose of engineered cells comprising T cells expressing a Chimeric Antigen Receptor (CAR) that binds cluster of differentiation 19 (CD 19), wherein the T cell therapy is administered on day 1 of the combination therapy; and the compound was administered in multiple intermittent doses administered once every 14 days (Q14D) and on days 8, 22, 36, 50, 64 and 78, with each dose being 0.3mg.

or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein: administering the compound in combination therapy with a T cell therapy comprising a dose of engineered cells comprising T cells expressing a Chimeric Antigen Receptor (CAR) that binds cluster of differentiation 19 (CD 19), wherein the T cell therapy is administered on day 1 of the combination therapy; and the compound was administered in a plurality of intermittent doses administered once every seven days (Q7D) and on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85, wherein each dose is 0.2mg.

or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein: administering the compound in combination therapy with a T cell therapy comprising a dose of engineered cells comprising T cells expressing a Chimeric Antigen Receptor (CAR) that binds cluster of differentiation 19 (CD 19), wherein the T cell therapy is administered on day 1 of the combination therapy; and the compound was administered in a plurality of intermittent doses administered once every seven days (Q7D) and on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85, wherein each dose is 0.6mg.

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

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

In some of any of the provided methods, the subject does not exhibit severe toxicity after administration of the T cell therapy at the beginning of administration of the compound. In some of any of the provided methods, the severe toxicity is severe Cytokine Release Syndrome (CRS), optionally a grade 3 or higher CRS, an extended grade 3 or higher CRS, a grade 4 CRS, or a grade 5 CRS; and/or the severe toxicity is severe neurotoxicity, optionally grade 3 or higher, prolonged grade 3 or higher, grade 4 or grade 5 neurotoxicity. In some embodiments, the severe toxicity is severe CRS. In some embodiments, the severe toxicity is severe neurotoxicity.

In some of any of the provided methods, if the subject exhibits toxicity, optionally hematological toxicity, after administration of the compound, administration of the compound is paused and/or the dosage of the compound is modified, optionally reduced. In some of any of the provided methods, administration of the compound is suspended if the subject exhibits toxicity after administration of the compound. In some of any of the provided methods, the dosage of the compound is reduced if the subject exhibits toxicity after administration of the compound. In some of any of the provided methods, the toxicity is hematologic toxicity. In some of any of the provided methods, the toxicity is severe thrombocytopenia, optionally grade 4 thrombocytopenia or prolonged grade 4 thrombocytopenia. In some of any of the provided methods, the toxicity is severe neutropenia, optionally grade 4 neutropenia, prolonged grade 4 neutropenia, or febrile neutropenia, optionally grade 3 or higher grade febrile neutropenia, or prolonged grade 3 or higher grade febrile neutropenia.

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

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

In some of any of the provided methods, the lymphoma is a B cell malignancy. In some of any of the provided methods, the lymphoma is relapsed/refractory lymphoma. In some of any of the provided methods, the lymphoma is invasive lymphoma. In some of any of the provided methods, the lymphoma is non-hodgkin's lymphoma (NHL), optionally wherein the NHL comprises invasive NHL; diffuse large B-cell lymphoma (DLBCL); DLBCL-NOS, optionally transformed inert; EBV positive DLBCL-NOS; t cell/histiocyte enriched large B cell lymphomas; primary mediastinum large B-cell lymphoma (PMBCL); follicular Lymphoma (FL), optionally grade 3B follicular lymphoma (FL 3B); and/or high grade B cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements with DLBCL histology (double/triple hit).

In some of any of the provided methods, the CD19 is human CD19.

In some of any of the provided methods, the Chimeric Antigen Receptor (CAR) comprises an extracellular antigen recognition domain that specifically binds to the CD19 and an intracellular signaling domain comprising ITAM. In some of any of the provided methods, the intracellular signaling domain comprises a CD 3-zeta (CD 3 zeta) chain, optionally a signaling domain of a human CD 3-zeta chain. In some of any of the provided methods, the intracellular signaling domain comprises a signaling domain of a human CD 3-zeta (CD 3 zeta) chain. In some any of the provided methods, the Chimeric Antigen Receptor (CAR) further comprises a costimulatory signaling region. In some of any of the provided methods, the costimulatory signaling region comprises the signaling domain of CD28 or 4-1BB, optionally human CD28 or human 4-1 BB. In some of any of the provided methods, the co-stimulatory signaling region comprises a signaling domain of human CD 28. In some of any of the provided methods, the costimulatory signaling region comprises the signaling domain of human 4-1 BB.

In some of any of the provided methods, the CAR comprises an scFv specific for the CD 19; a transmembrane domain; a cytoplasmic signaling domain derived from a costimulatory molecule, optionally being or comprising 4-1BB, optionally human 4-1BB; and a cytoplasmic signaling domain derived from an ITAM-containing primary signaling molecule, the cytoplasmic signaling domain optionally being or comprising a CD3 zeta signaling domain, optionally a human CD3 zeta signaling domain; and optionally wherein the CAR further comprises a spacer between the transmembrane domain and the scFv. In some of any of the provided methods, the CAR comprises an scFv specific for the CD 19; a transmembrane domain; cytoplasmic signaling domain derived from human 4-1BB; a cytoplasmic signaling domain that is or comprises a human CD3 zeta signaling domain; and a spacer between the transmembrane domain and the scFv.

In some of any of the provided methods, the CAR comprises, in order, an scFv specific for the CD 19; a transmembrane domain; a cytoplasmic signaling domain derived from a costimulatory molecule, said cytoplasmic signaling domain optionally being or comprising a 4-1BB signaling domain, optionally a human 4-1BB signaling domain; and a cytoplasmic signaling domain derived from an ITAM-containing primary signaling molecule, the cytoplasmic signaling domain optionally being a CD3 zeta signaling domain, optionally a human CD3 zeta signaling domain. In some of any of the provided methods, the CAR comprises, in order, an scFv specific for the CD 19; a transmembrane domain; a cytoplasmic signaling domain that is or comprises a human 4-1BB signaling domain; and a cytoplasmic signaling domain that is a human CD3 zeta signaling domain.

In some of any of the provided methods, the CAR comprises, in order, an scFv specific for the CD 19; a spacer; a transmembrane domain; a cytoplasmic signaling domain derived from a costimulatory molecule, said cytoplasmic signaling domain optionally being a 4-1BB signaling domain; and a cytoplasmic signaling domain derived from an ITAM-containing primary signaling molecule, the cytoplasmic signaling domain optionally being or comprising a CD3 zeta signaling domain. In some of any of the provided methods, the CAR comprises, in order, an scFv specific for the CD 19; a spacer; a transmembrane domain; a cytoplasmic signaling domain that is a 4-1BB signaling domain; and a cytoplasmic signaling domain that is or includes a CD3 zeta signaling domain.

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

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

In some of any of the provided methods, the scFv comprises a CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 35), a CDRL2 sequence of SRLHSGV (SEQ ID NO: 36), and/or a CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 37); and/or the CDRH1 sequence of DYGVs (SEQ ID NO: 38), the CDRH2 sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39) and/or the CDRH3 sequence of YAMDYWG (SEQ ID NO: 40). In some of any of the provided methods, the scFv comprises a CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 35), a CDRL2 sequence of SRLHSGV (SEQ ID NO: 36), and a CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 37); and/or the CDRH1 sequence of DYGVs (SEQ ID NO: 38), the CDRH2 sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39) and the CDRH3 sequence of YAMDYWG (SEQ ID NO: 40). In some of any of the provided methods, the scFv comprises a CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 35), a CDRL2 sequence of SRLHSGV (SEQ ID NO: 36), and a CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 37); and the CDRH1 sequence of DYGVs (SEQ ID NO: 38), the CDRH2 sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39) and the CDRH3 sequence of YAMDYWG (SEQ ID NO: 40). In some of any of the provided methods, the scFv comprises a variable light chain region comprising the CDRL1 sequence of FMC63, the CDRL2 sequence of FMC63, the CDRL3 sequence of FMC63, and a variable heavy chain region comprising the CDRH1 sequence of FMC63, the CDRH2 sequence of FMC63, and the CDRH3 sequence of FMC 63. In some of any of the provided methods, the scFv comprises a variable heavy chain region of FMC63 and a variable light chain region of FMC 63. In some embodiments, the scFv comprises a VH comprising the amino acid sequence set forth in SEQ ID NO. 41 and a VL comprising the amino acid sequence set forth in SEQ ID NO. 42. In some of any of the provided methods, the scFv comprises a CDRL1 sequence of FMC63, a CDRL2 sequence of FMC63, a CDRL3 sequence of FMC63, a CDRH1 sequence of FMC63, a CDRH2 sequence of FMC63, and a CDRH3 sequence of FMC 63. In some of any of the provided methods, the scFv comprises a variable heavy chain region of FMC63 and a variable light chain region of FMC 63. In some 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 in SEQ ID NO. 42. In some of any of the provided methods, the scFv has the amino acid sequence set forth in SEQ ID NO. 43.

In some of any of the provided methods, the dose of engineered cells comprises at or from about 1x10 ⁵ To 5x10 ⁸ Total CAR expressing T cells, 1x10 ⁶ To 2.5x10 ⁸ Total CAR expressing T cells, 5x10 ⁶ Up to 1x10 ⁸ Total CAR expressing T cells, 1x10 ⁷ To 2.5x10 ⁸ Individual total CAR expressing T cells or 5x10 ⁷ Up to 1x10 ⁸ Each total CAR expressing T cell contains an endpoint. In some of any of the provided methods, the dose of engineered cells comprises at or from about 1x10 ⁵ To 5x10 ⁸ Total CAR expressing T cells, inclusive. In some of any of the provided methods, the dose of engineered cells comprises at or from about 1x10 ⁶ To 2.5x10 ⁸ Total CAR expressing T cells, inclusive. In some of any of the provided methods, the dose of engineered cells comprises at or from about 5x10 ⁶ Up to 1x10 ⁸ Total CAR expressing T cells, inclusive. In some of any of the provided methods, the dose of engineered cells comprises at or from about 1x10 ⁷ To 2.5x10 ⁸ Total CAR expressing T cells, inclusive. In some of any of the provided methods, the dose of engineered cells comprises at or from about 5x10 ⁷ Up to 1x10 ⁸ Total CAR expressing T cells, inclusive.

In some of any of the provided methods, the dose of engineered cells comprises at least or at least about 1x10 ⁵ Individual CAR expressing cells, at least, or at least about 2.5x10 ⁵ Individual CAR-expressing cells, at least or at least about 5x10 ⁵ Individual CAR expressing cells, at least or at least about 1x10 ⁶ Individual CAR expressing cells, at least, or at least about 2.5x10 ⁶ Fine individual CAR expressionCells, at least or at least about 5x10 ⁶ Individual CAR expressing cells, at least or at least about 1x10 ⁷ Individual CAR expressing cells, at least, or at least about 2.5x10 ⁷ Individual CAR expressing cells, at least, or at least about 5x10 ⁷ Individual CAR-expressing cells, at least, or at least about 1x10 ⁸ Individual CAR expressing cells, at least, or at least about 2.5x10 ⁸ Individual CAR-expressing cells, or at least about 5x10 ⁸ And (3) CAR expressing cells.

In some of any of the provided methods, the dose of engineered cells comprises at least or at least about 1x10 ⁵ And (3) CAR expressing cells. In some of any of the provided methods, the dose of engineered cells comprises at least or at least about 2.5x10 ⁵ And (3) CAR expressing cells. In some any of the provided methods, the dose of engineered cells comprises at least or at least about 5x10 ⁵ And (3) CAR expressing cells. In some of any of the provided methods, the dose of engineered cells comprises at least or at least about 1x10 ⁶ And (3) CAR expressing cells. In some of any of the provided methods, the dose of engineered cells comprises at least or at least about 2.5x10 ⁶ And (3) CAR expressing cells. In some any of the provided methods, the dose of engineered cells comprises at least or at least about 5x10 ⁶ And (3) CAR expressing cells. In some of any of the provided methods, the dose of engineered cells comprises at least or at least about 1x 10 ⁷ And (3) CAR expressing cells. In some of any of the provided methods, the dose of engineered cells comprises at least or at least about 2.5x10 ⁷ And (3) CAR expressing cells. In some any of the provided methods, the dose of engineered cells comprises at least or at least about 5x10 ⁷ And (3) CAR expressing cells. In some of any of the provided methods, the dose of engineered cells comprises at least or at least about 1x 10 ⁸ And (3) CAR expressing cells. In some of any of the provided methods, the dose of engineered cells comprises at least or at least about 2.5x10 ⁸ And (3) CAR expressing cells. In some any of the provided methods, the dose of engineered cells comprises at least or at least about 5x10 ⁸ And (3) CAR expressing cells.

In some of any of the provided methods, the dose of engineered cells comprises at or about 1x 10 ⁸ And (3) CAR expressing cells.

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

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

In some of any of the provided methods, the dose of engineered cells comprises cd4+ T cells expressing the CAR and cd8+ T cells expressing the CAR and the administration of the dose comprises administering a plurality of separate compositions comprising a first composition comprising one of the cd4+ T cells and the cd8+ T cells and a second composition comprising the other of the cd4+ T cells or the cd8+ T cells.

In some of any of the provided methods, the first composition and the second composition are administered 0 to 12 hours apart, 0 to 6 hours apart, or 0 to 2 hours apart, or wherein the administration of the first composition and the administration of the second composition are performed on the same day, between about 0 and about 12 hours apart, between about 0 and about 6 hours apart, or between about 0 and 2 hours apart; and/or the initiation of the administration of the first composition and the initiation of the administration of the second composition are between about 1 minute and about 1 hour apart or between about 5 minutes and about 30 minutes apart. In some any of the provided methods, the first composition and the second composition are administered no more than 2 hours apart, no more than 1 hour apart, no more than 30 minutes apart, no more than 15 minutes apart, no more than 10 minutes apart, or no more than 5 minutes apart.

In some any of the provided methods, the first composition comprises the cd4+ T cells. In some any of the provided methods, the first composition comprises the cd8+ T cells.

In some of any of the provided methods, the first composition is administered prior to the second composition.

In some of any of the provided methods, the subject has been preconditioned with a lymphocyte removal therapy prior to administration of the T cell therapy, the lymphocyte removal therapy comprising administration of fludarabine and/or cyclophosphamide. In some any of the provided methods, the method further comprises administering to the subject, immediately prior to administering the T cell therapy, a lymphocyte removal therapy comprising administering fludarabine and/or cyclophosphamide.

In some of any of the provided methods, the lymphocyte removal therapy comprises daily administration of about 200-400mg/m ² Comprising the endpoints, optionally at or about 300mg/m ² Cyclophosphamide of (C), and/or about 20-40mg/m ² Optionally 30mg/m ² Is administered for 2-4 days, optionally for 3 days, or wherein the lymphocyte removal therapy comprises administration of about 500mg/m ² Cyclophosphamide of (c). In some of any of the provided methods, the lymphocyte removal therapy comprises daily administration at or about 300mg/m ² Cyclophosphamide sum of about 30mg/m ² Fludarabine of (c) for 3 days; and/or the lymphocyte removal therapy comprises daily administration of at or about 500mg/m ² Cyclophosphamide sum of about 30mg/m ² Is continued for 3 days.

In some of any of the provided methods, the subject is a human.

In some of any of the provided methods, at least 35%, at least 40%, or at least 50% of the subjects treated according to the methods achieve a Complete Response (CR) that may last or last for at least 60%, 70%, 80%, 90%, or 95% of the subjects who achieved the CR for or greater than 6 months or for or greater than 9 months; and/or at least 60%, 70%, 80%, 90% or 95% of subjects who achieved CR by six months remain responsive, maintain CR and/or survive without progression for 3 months or more and/or for 6 months or more and/or for nine months or more; and/OR at least 50%, at least 60%, OR at least 70% of the subjects treated according to the method achieve an Objective Response (OR), optionally wherein the OR may last OR last for 6 months OR more OR for 9 months OR more in at least 60%, 70%, 80%, 90%, OR 95% of the subjects who achieved the OR; and/OR at least 60%, 70%, 80%, 90% OR 95% of subjects who achieved OR by six months remain responsive OR alive for 3 months OR more and/OR for 6 months OR more.

In some of any of the provided methods, at or immediately prior to administration of the dose of engineered cells, the subject has relapsed after remission after treatment with or becomes refractory to: one, two or three previous therapies for one or more of the NHLs, optionally in addition to another dose of engineered cells expressing the CAR. In some of any of the provided methods, at or immediately prior to administration of the dose of engineered cells, the subject relapses after remission or becomes refractory to the following therapy: one prior therapy for the NHL. In some of any of the provided methods, at or immediately prior to administration of the dose of engineered cells, the subject relapses after remission or becomes refractory to the following therapy: two previous therapies for the NHL. In some of any of the provided methods, at or immediately prior to administration of the dose of engineered cells, the subject relapses after remission or becomes refractory to the following therapy: three previous therapies for the NHL. In some of any of the provided methods, the one or more previous therapies do not include another dose of engineered cells expressing the CAR.

In some of any of the provided methods, the dose of engineered cells is administered at or prior to administration of the dose of engineered cells: the subject is identified as or has been identified as having a double/triple hit lymphoma; the subject is identified as having or has been identified as having a chemotherapeutic refractory lymphoma, optionally a chemotherapeutic refractory DLBCL; and/or the subject has not achieved Complete Remission (CR) in response to a previous therapy. In some of any of the provided embodiments, the subject has not achieved Complete Remission (CR) in response to a previous therapy.

In some of any of the provided methods, administration of the compound: reversing the depletion phenotype in CAR-expressing T cells in the subject; preventing, inhibiting, or delaying the onset of a depletion phenotype in CAR-expressing T cells in the subject; reducing the level or extent of a depletion phenotype in CAR-expressing T cells in the subject; or reducing the percentage or total number of CAR-expressing T cells having a depletion phenotype in the subject.

In some of any of the provided methods, the initiation of administration of the compound is performed after administration of the T cell therapy, and after administration of the compound or initiation thereof, the subject exhibits a restoration or rescue of antigen or tumor specific activity or function of the CAR-expressing T cells in the subject, optionally wherein the initiation of the restoration, rescue, and/or administration of the compound is a point in time after CAR-expressing T cells in the subject or in the blood of the subject have exhibited a depletion phenotype.

In some of any of the provided methods, administering the compound comprises administering in an amount, frequency, and/or duration effective to: (a) Achieving an increase in antigen-specific or antigen receptor driving activity of naive or non-depleting T cells in the subject, said naive or non-depleting T cells optionally comprising T cells expressing the CAR, after exposure of said naive or non-depleting T cells to a CD19 antigen or antigen receptor specific agent, as compared to the case of said administration in the absence of said compound; or (b) preventing, inhibiting or delaying the onset of a depletion phenotype in naive or non-depleted T cells in the subject, optionally comprising T cells expressing the CAR, after exposure of the naive or non-depleted T cells to a CD19 antigen or antigen receptor specific agent, as compared to the administration in the absence of the compound; or (c) reversing the depletion phenotype in depleted T cells in the subject, optionally comprising T cells expressing the CAR, as compared to the administration in the absence of the compound.

In some of any of the provided methods, administering the compound comprises administering in an amount, frequency, and/or duration effective to: (i) Achieving the increased activity, and (ii) preventing, inhibiting or delaying the onset of the depletion phenotype and/or reversing the depletion phenotype.

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

In some of any of the provided methods, the depletion phenotype associated with the T cell or T cell population comprises: an increase in the level or extent of surface expression of one or more depletion markers, optionally 2, 3, 4, 5 or 6 depletion markers, or an increase in the percentage of the population of T cells exhibiting said surface expression, over a reference population of T cells under the same conditions; or a decrease in the level or extent of activity exhibited by a T cell or population of T cells when exposed to a CD19 antigen or antigen receptor specific agent as compared to a reference population of T cells under the same conditions.

In some of any of the methods provided, the increase in level, degree, or percentage is greater than or about 1.2-fold, greater than or about 1.5-fold, greater than or about 2.0-fold, greater than or about 3-fold, greater than or about 4-fold, greater than or about 5-fold, greater than or about 6-fold, greater than or about 7-fold, greater than or about 8-fold, greater than or about 9-fold, greater than or about 10-fold, or more. In some of any of the provided methods, the reduction in level, degree, or percentage is greater than or about 1.2-fold, greater than or about 1.5-fold, greater than or about 2.0-fold, greater than or about 3-fold, greater than or about 4-fold, greater than or about 5-fold, greater than or about 6-fold, greater than or about 7-fold, greater than or about 8-fold, greater than or about 9-fold, greater than or about 10-fold, or more.

In some of any of the provided methods, the reference T cell population is a T cell population known to have a non-depleting phenotype, is a naive T cell population, is a central memory T cell population, or is a stem cell central memory T cell (stem centralmemory T cell) population, optionally from the same subject or the same species as the subject from which the one or more T cells having the depleting phenotype were derived. In some of any of the provided methods, the reference T cell population (a) is a subject-matched population comprising a plurality of T cells isolated from blood of a subject from which the one or more T cells having the depletion phenotype are derived, optionally wherein the plurality of T cells do not express the CAR, and/or (b) is obtained from a subject from which the one or more T cells having the depletion phenotype are derived prior to receiving administration of a dose of T cells expressing the CAR. In some of any of the provided methods, the reference T cell population is a composition comprising a sample of the T cell therapy (e.g., a sample of T cells expressing the CAR) or a pharmaceutical composition comprising T cells expressing the CAR prior to its administration to the subject, optionally wherein the composition is a cryopreserved sample.

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

In some of any of the provided methods, the activity is one or more of proliferation, cytotoxicity, or production of one or a combination of inflammatory cytokines, optionally wherein the one or combination of cytokines is selected from IL-2, IFN- γ, and TNF- α.

In some of any of the provided methods, exposing to the CD19 antigen or antigen receptor specific agent comprises incubating with the CD19 antigen or antigen receptor specific agent, optionally an agent that binds to an antigen binding domain of the CAR. In some of any of the provided methods, exposing the CD19 antigen or antigen receptor specific agent comprises exposing the T cell to a target cell expressing CD19 antigen, optionally a cell of the B cell malignancy.

Also provided are combination therapies for use in a method of treating a CD19 expressing cancer according to any of the provided methods, the combination therapies comprising a T cell therapy comprising a dose of an engineered cell comprising a T cell expressing a Chimeric Antigen Receptor (CAR) targeted to the cancer and a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof.

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

Also provided are T cell therapies comprising a dose of engineered cells comprising T cells expressing a Chimeric Antigen Receptor (CAR) targeted to a CD19 expressing cancer for use in a method of treating the cancer according to any of the provided methods.

Also provided is a use of a combination therapy comprising a T cell therapy comprising a dose of an engineered cell comprising T cells expressing a Chimeric Antigen Receptor (CAR) targeted to the cancer and a compound which is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, for treating a CD19 expressing cancer according to any of the provided methods.

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

Also provided is the use of a T cell therapy comprising a dose of engineered cells comprising T cells expressing a Chimeric Antigen Receptor (CAR) that targets a CD19 expressing cancer according to any of the provided methods.

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

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

Also provided is the use of a T cell therapy comprising a dose of engineered cells comprising T cells expressing a Chimeric Antigen Receptor (CAR) that targets a CD19 expressing cancer for the manufacture of a medicament for treating cancer according to any of the provided methods.

In some any of the provided embodiments, the CAR binds to cluster of differentiation 19 (CD 19). In some embodiments, the CD19 expressing cancer is a lymphoma.

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

Drawings

FIG. 1 shows the expression levels of Aiolos and Ikaros transcription factors in anti-CD 19 CAR T cells following stimulation with CAR specific anti-idiotype antibodies in the presence of various immunomodulatory compounds.

Figure 2A shows the percentage of live anti-CD 19 CAR T cells after stimulation with CAR-specific anti-idiotype antibodies in the presence of various immunomodulatory compounds. Figures 2B and 2C show the cell doubling numbers of anti-CD 19 CAR T cells after stimulation with CAR-specific anti-idiotype antibodies in the presence of various immunomodulatory compounds. Figures 2D and 2E show the percentage of anti-CD 19 CAR T cells at G1 phase of the cell cycle after stimulation with CAR-specific anti-idiotype antibodies in the presence of various immunomodulatory compounds. Figure 2F shows cytokine levels of anti-CD 19 CAR T cells after stimulation with CAR-specific anti-idiotype antibodies in the presence of various immunomodulatory compounds. Log greater than zero ₂ The change times value is indicated with "+".

Figure 2G shows the cell number of RL tumor cells treated with anti-CD 19 CAR T cells and compound C. Figure 2H shows the cell number of RL tumor cells treated with anti-CD 19 CAR T cells and compound 2.

Figure 3A shows cytokine production by anti-CD 19 CAR T cells following chronic stimulation. Figure 3B shows the cytolytic function of anti-CD 19 CAR T cells following chronic stimulation.

Figure 3C shows a representative image of CD19 expressing tumor spheres on day 9 co-cultured with anti-CD 19 CAR T cells stimulated in the presence of compound C (concurrent treatment).

Figures 3D and 3E show the volume of CD19 expressing tumor spheres co-cultured with anti-CD 19 CAR T cells stimulated in the presence of compound C after chronic stimulation (concurrent treatment). Fig. 3F shows ifnγ concentrations in co-cultured cell supernatants.

Figure 3G shows Ikaros expression in anti-CD 19 CART cells six days after chronic stimulation in the presence of compound C or compound 2.

Figure 3H depicts a volcanic plot showing genes differentially expressed in chronically stimulated anti-CD 19 CAR T cells treated in parallel with compound C. FIG. 3I shows the effect on gene expression profile induced by compound C parallel treatment of anti-CD 19 CAR T cells that have been chronically stimulated (log ₂ Fold change). Figure 3J shows KEGG pathway enrichment analysis of differentially expressed genes in chronically stimulated anti-CD 19 CAR T cells after concurrent treatment with compound C.

Figure 4A shows a representative image of CD19 expressing tumor spheres at day 9 in the presence of compound C (rescue treatment) co-cultured with anti-CD 19 CAR T cells.

Fig. 4B and 4C show the volume of CD19 expressing tumor spheres co-cultured with anti-CD 19 CAR T cells in the presence of compound C (rescue treatment). Fig. 4D shows ifnγ concentrations in co-cultured cell supernatants.

Figure 4E depicts a volcanic plot showing genes differentially expressed in chronically stimulated anti-CD 19CAR T cells following rescue treatment with compound C. FIG. 4F shows the effect on gene expression profile induced by compound C rescue treatment of anti-CD 19CAR T cells that have been chronically stimulated (log ₂ Fold change). Figure 4G shows KEGG pathway enrichment analysis of differentially expressed genes in chronically stimulated anti-CD 19CAR T cells following rescue treatment with compound C.

Figure 5A shows the number of tumor cells during re-excitation of chronically stimulated CAR T cells in the presence of compound C (concurrent treatment). Figure 5B shows the number of tumor cells during re-priming of CAR T cells in the presence of compound C (rescue treatment).

Fig. 6A shows the expected plasma concentration levels of compound C based on a once every seven day (Q7D) dosing regimen.

Fig. 6B and 6C show Ikaros levels in anti-CD 19CAR T cells after 24 hours (fig. 6B) or six days (fig. 6C) exposure to compound C.

Fig. 6D shows Absolute Neutrophil Count (ANC) spectra predicted based on the Q7D dosing regimen of compound C, as generated using four different pharmacodynamic models.

Figure 7A shows the number of tumor cells during re-stimulation of chronically stimulated CAR T cells in the presence of compound C (including chronically stimulated CAR T cells in the presence of compound C, wherein compound C at a high concentration continues for one day, then compound C at a low concentration continues for five days). Figures 7B and 7C show the cytolytic activity and CD27/CCR7 expression, respectively, of chronically stimulated CAR T cells in the presence of compound C (including chronically stimulated CAR T cells in the presence of compound C, wherein compound C is present at a high concentration for one day and then compound C is present at a low concentration for six days).

Detailed Description

Provided herein are methods and uses of cell therapies, such as cell therapies containing engineered T cells (e.g., CAR-T cells), and compounds that are (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione having the structure:

or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer or racemic mixtures (compound C) thereof, and compositions thereof, for use in the treatment of subjects suffering from cancer or proliferative diseases. In some aspects, the cell therapy is T cell therapy. In some aspects, the cell therapy is adoptive T cell therapy comprising T cells that specifically recognize and/or target an antigen associated with a cancer or proliferative disease, such as an antigen associated with a B cell malignancy (e.g., a lymphoma, such as non-hodgkin's lymphoma (NHL) or subtype thereof). In some aspects, the cell therapy comprises T cells engineered with a Chimeric Antigen Receptor (CAR) comprising an antigen binding domain that binds (e.g., specifically binds) to an antigen. In some cases, the antigen targeted by the cell therapy is CD19. In some aspects, the cancer (e.g., lymphoma) expresses CD19. Also provided herein are combinations and articles of manufacture, such as kits, containing a composition comprising a cell therapy and/or a composition comprising compound C, as well as the use of such compositions and combinations for the treatment or prevention of diseases, conditions, and disorders, including cancers, such as lymphomas.

(S) -2- (2, 6-Dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione (compound C) is an oral cereblon modulator (CELMOD). The cereblon acts as a substrate receptor for CRL4 ubiquitin E3 ligase and binding of the cereblon modulating compounds induces recruitment, ubiquitination and disruption of key target substrates such as Ikaros family zinc finger proteins 1 and 3 (IKZF 1 and IKZF3, also known as Ikaros and Aiolos, respectively) to mediate cellular effects. As shown herein, administration of compound C resulted in deep degradation of Ikaros and Aiolos, deeper than other CELMoD in its class (e.g., lenalidomide, atorvastatin, and Ai Baidu amine (iberdomide)). However, a potential side effect of Ikaros and Aiolos degradation is the development of neutropenia secondary to neutrophil maturation arrest. Neutropenia may develop as a result of depletion of the key transcription factor Ikaros that controls hematopoiesis and is due to late maturation arrest of neutrophil progenitors.

Cell therapies, such as T cell-based therapies, e.g., adoptive T cell therapies, including those involving administration of cells expressing chimeric receptors (e.g., chimeric Antigen Receptors (CARs) and/or other recombinant antigen receptors) specific for a disease or disorder of interest, as well as other adoptive immune cell and adoptive T cell therapies, may be effective in the treatment of diseases and disorders such as B cell malignancies. Engineered expression of recombinant receptors, such as Chimeric Antigen Receptors (CARs), on the surface of T cells enables specific redirection of T cells. In clinical studies, CAR-T cells (e.g., anti-CD 19CAR-T cells) have produced a durable, complete response in 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).

In some situations, the viable approach to adoptive cell therapy may not always be entirely satisfactory. For example, although CAR T cell persistence can be detected in many subjects with lymphoma, fewer Complete Responses (CRs) are observed in subjects with NHL compared to subjects with ALL. More specifically, although higher overall response rates of up to 80% (CR rate 47% to 60%) after CAR T cell infusion have been reported, in some subjects the response was transient, and subjects have been shown to relapse in the presence of durable CAR T cells (neelpau, 58th Annual Meeting of the American Society of Hematology (ASH): 2016; san diego, ca, usa. Abstract number LBA-6.2016; abramson, blood.2016, 12 month 1 day; 128 (22): 4192). Another study reported a long term CR of 40% (Schuster, ann Hematol.2016, month 10; 95 (11): 1805-10).

In some aspects, the explanation for this is immune depletion of circulating CAR-expressing T cells and/or a change in T lymphocyte populations. This is because, in some circumstances, optimal efficacy may depend on the ability of the administered cells to have the following properties: is activated, expanded, exerts various effector functions (including cytotoxic killing and secretion of various factors such as cytokines), persists (including long-term presence), differentiates, converts or participates in reprogramming to certain phenotypic states (such as long-term memory, low differentiation and effector states), avoids or reduces immunosuppressive conditions in the local microenvironment of the disease, provides an effective and robust recall response upon clearance and re-exposure to target ligands or antigens, and avoids or reduces depletion, anergy, peripheral tolerance, terminal differentiation and/or differentiation to an inhibited state.

In some aspects, the engineered cells are reduced or decreased in exposure, persistence, and function after administration to a subject. In some aspects, following prolonged stimulation or exposure to an antigen and/or exposure to conditions in the tumor microenvironment, T cells may become hypofunctional and/or exhibit characteristics associated with a depleted state over time. In some aspects, this reduces the persistence and efficacy of T cells against antigens and limits their ability to take effect. However, observations suggest that in some cases, the administered cells expressing the recombinant receptor may be re-expanded and/or re-activated in vivo (e.g., exhibiting an increased number or duration of cells over time) to improve the efficacy and therapeutic outcome of adoptive cell therapy. There is a need for methods of improving the efficacy and function of CAR T cells, particularly minimizing, reducing, preventing or reversing the reduced or depleted state.

The method provided is based on the following observations: treatment with compound C may improve T cell function, including functions related to the ability of T cells to produce one or more cytokines, cytotoxicity, expansion, proliferation, and persistence. In some aspects, provided methods enhance or modulate proliferation and/or activity of T cell activity associated with administration of a cell therapy (e.g., CAR expressing T cells). Such methods and uses have been found to provide or achieve improved or greater T cell function and thereby improved anti-tumor efficacy.

It has also been found herein that treatment with compound C can reverse, delay or prevent T cell depletion, including by increasing T cell signaling and/or altering one or more genes that are differentially regulated following chronic stimulation, in addition to enhancing T cell function. Although in some cases agents that increase or potentiate T cell activity may drive the cells to a depleted state, it is found herein that the activity of compound C that exerts a potentiating effect on T cell activity is not associated with T cell depletion. Furthermore, observations herein show that compound C exhibits activity to rescue T cells from T cell depletion, such as by restoring or partially restoring one or more T cell activities after the cells have been characterized as having been depleted. Notably, the results herein show that T cells that have been chronically stimulated and that exhibit characteristics of depleting T cells are able to restore or partially restore their activity upon exposure to compound C. Observations herein support that the provided methods can also achieve improved or more durable responses as compared to certain alternative methods, as in a particular subject group being treated.

It is also shown herein that low dose compound C treatment (e.g., 1 nM) is capable of deeply degrading both Ikaros and Aiolos expression in chronically stimulated CART cells. However, it is also shown herein that higher doses of compound C (e.g., 10 and 100 nM) can lead to sustained target degradation of Ikaros and Aiolos and to reduced cytolytic function. These results indicate that excessive sustained target degradation by Ikaros and Aiolos may be counterproductive and result in CAR T cell dysfunction. Based on these results, and because compound C can degrade Ikaros and Aiolos more deeply than other CELMoD compounds targeting Ikaros and Aiolos, it is assumed herein that a single weekly dose will be sufficient to achieve deep but transient degradation of Ikaros and Aiolos, thus potentially allowing Ikaros and/or Aiolos expression to resume between doses. As shown herein, pharmacokinetic modeling of compound C at a weekly dose (Q7D) of 0.3mg demonstrated fast C _max (about 20 nM) and biphasic elimination of Compound C, C on day 7 _min Is about 1nM. Modeling also showed that the weekly dose of 0.3mg did not substantially affect neutrophil counts. As also shown in the preclinical model herein, anti-CD 19 CAR T cell lytic activity against cd19+ target cells was most evident after a brief (one day) exposure to high concentration of compound C (20-100 nM), followed by rinsing and a chronic low concentration of compound C (for five days at 1 nM), which was selected to model the pharmacokinetic profile of 0.3mg of compound C once every seven days (Q7D). In contrast, higher chronic concentrations of compound C have deleterious effects on cell lysis function.

Thus, based on the results shown herein, the combination of cell therapy (e.g., CAR T cell therapy) with intermittent (e.g., once every seven days) dosing of compound C can provide a useful therapeutic approach for preventing premature CAR T cell depletion, enhancing and prolonging T cell function, and leading to a more pronounced and sustained response in cancers such as lymphomas, while also reducing the risk of the patient suffering from neutropenia secondary to neutrophil maturation arrest.

In some embodiments, T cell function (including functions related to expansion, proliferation, and persistence of T cells) of an engineered T cell therapy administered according to the provided methods is improved by compound C. In some embodiments, the method is advantageous as a result of administering a T cell therapy in combination with compound C, such as a composition comprising cells for adoptive cell therapy (e.g., like T cell therapy, e.g., CAR expressing T cells). In some aspects, the provided methods and uses provide or achieve improved or more durable reactions or efficacy, as compared to certain alternative methods. In some aspects, provided methods enhance or modulate proliferation and/or activity of T cells associated with administration of the T cell therapy (e.g., CAR expressing T cells). In particular embodiments, combination therapies using compound C can provide useful therapeutic methods for enhancing and prolonging the activity of CAR T cells in B cell malignancies by modulating tumor microenvironment by improving the durable anti-tumor function of the CAR T cells.

In some embodiments, compound C is administered to the subject for a time sufficient after receiving the lymphocyte removal therapy such that the myelosuppressive effects of compound C and the lymphocyte removal therapy are minimized.

In some embodiments, the provided methods are used when T cell therapies (e.g., CAR T cells) can exhibit or may exhibit depletion characteristics. In some embodiments, the depletion phenotype is apparent after the number of T cells in the subject's blood that have reached peak expansion begins to decline. In some embodiments, the method of exposing or contacting a T cell of a T cell therapy (CAR T cell) to compound C is performed when the T cell exhibits an increase in a reduced or depleted state compared to immediately prior to exposure of the T cell to the antigen (baseline) or to a point in time at which the cell has been exposed to the antigen but continues to proliferate and has not reached peak proliferation. In some embodiments, an increase in the reduced function or depletion state can be determined by increased expression of the depletion marker as compared to a previous earlier time point. In some embodiments, an increase in the reduced function or depleted state (e.g., an increase in expression of a depletion marker) occurs at a time after administration of a T cell therapy (e.g., CAR T cells) to a subject suffering from a disease or disorder associated with an antigen to which the T cell therapy is targeted. T cells, such as T cells in peripheral blood, may be monitored for markers of T cell activation or depletion (e.g., PD-1, TIM-3, and LAG-3) after administration to a subject.

In some embodiments, the provided methods call for administration of a T cell therapy, e.g., a CAR T cell, and administration of compound C is initiated at a time prior to the CAR T cell exhibiting or likely exhibiting a depletion phenotype. In some embodiments, administration of compound C is initiated at a point in time when the CAR T cells are still expanding or are still able to expand. In some embodiments, administration of compound C is initiated prior to or suspected of being at a point in time prior to the presence of a peak CAR T cell number in the blood of the subject. In some aspects, starting administration of compound C at this point in time enhances CAR T cell function. In some aspects, starting administration of compound C at this point in time also delays or prevents CAR T cell depletion.

In some embodiments, administration of compound C is initiated before or at about the time at which the peak CAR-T cells are or are suspected of being present in the blood of the subject (e.g., within 21 days after administration of the T cells is initiated). In some cases, the peak CAR-T cells are present within 11-15 days after administration of the CAR T cells. In some embodiments, compound C is administered beginning at a time of 1 to 15 days (e.g., at or about 1 day or 8 days or 15 days) after the initiation of administration of the cell therapy. In some embodiments, compound C is administered at a time when the subject does not exhibit severe toxicity following administration of the cell therapy.

In some embodiments, the provided methods do not result in, or reduce, a high rate or likelihood of toxicity or toxic outcome, such as Neurotoxicity (NT), cytokine Release Syndrome (CRS), or hematologic toxicity, such as neutropenia, as compared to certain other cell therapies or immunomodulatory drug regimens.

In some embodiments, the methods do not result in, or increase the risk of, certain hematological toxicities (such as neutropenia or thrombocytopenia). In some embodiments, no more than 50% of the subjects exhibit a level 3 neutropenia, such as prolonged level 3 neutropenia or level 4 neutropenia, and/or a level 3 thrombocytopenia, such as level 3 or level 4 thrombocytopenia. In some embodiments, at least 50% of subjects treated according to the methods (e.g., at least 60%, at least 70%, at least 80%, at least 90% or more of the treated subjects) do not exhibit grade 3 or higher severe neutropenia or severe thrombocytopenia.

In some embodiments, the method does not result in or increase the risk of: severe NT (sNT), severe CRS (sccrs), macrophage activation syndrome, tumor lysis syndrome, fever at or about 38 degrees celsius for three or more days, and plasma CRP levels of at least or about 20 mg/dL. In some embodiments, greater than or greater than about 30%, 35%, 40%, 50%, 55%, 60% or more of the subjects treated according to the provided methods do not exhibit any fractionated CRS or any fractionated neurotoxicity. In some embodiments, no more than 50% of the treated subjects (e.g., at least 60%, at least 70%, at least 80%, at least 90% or more of the treated subjects) exhibit a Cytokine Release Syndrome (CRS) higher than grade 2 and/or a neurotoxicity higher than grade 2. In some embodiments, at least 50% of the subjects treated according to the methods (e.g., at least 60%, at least 70%, at least 80%, at least 90% or more of the treated subjects) do not exhibit severe toxicity consequences (e.g., severe CRS or severe neurotoxicity), such as do not exhibit grade 3 or higher graded neurotoxicity and/or do not exhibit severe CRS, or do not exhibit such for some period of time after the treatment (e.g., within one week, two weeks, or one month of administration of the cells).

In some aspects, the dose, timing of the dose, or number of doses is not expected to cause severe toxicity, such as grade 4 neutropenia or grade 4 thrombocytopenia. In some aspects, the provided methods minimize or avoid toxicity after administration of T cell therapy and/or compound C to a subject (e.g., during the entire intermittent dosing of compound C). In some aspects, the methods provided herein involve administering a dose that is significantly lower than the dose that may be used for compound C in a monotherapy method and administered at a reduced frequency. In some aspects, the methods provided herein involve administering a dose that is less than the dose that may be used for compound C in a monotherapy method. In some aspects, the methods provided herein involve administering intermittent doses of compound C, and the period of time between doses of compound C is longer than the period of time that compound C might be used in a monotherapy method.

In some cases, compound C is administered when it can efficiently/effectively strengthen or prime the cells. In some embodiments, administration of compound C begins when or before a peak or maximum level of cells of the cell therapy can be detected in the blood of the subject. In some embodiments, the provided methods can augment T cell therapies, such as CAR-T cell therapies, which can improve treatment outcome in some aspects. In some embodiments, the methods are particularly advantageous in subjects in which cells of a T cell therapy exhibit weak expansion in the subject, have become depleted, exhibit reduced or reduced persistence, and/or have cancers that are resistant to or refractory to other therapies and/or are invasive or high risk cancers.

In some embodiments, a subject who has received administration of a T cell therapy (e.g., CAR-T cells) is monitored for the presence, absence, or level of T cells of the therapy in the subject (e.g., in a biological sample of the subject, e.g., in the blood of the subject). In some embodiments, the provided methods result in genetically engineered cells having increased persistence and/or better efficacy in the subject to which they are administered. In some embodiments, the persistence of genetically engineered cells, such as CAR-expressing T cells, in a subject is higher as compared to persistence that can be achieved by alternative methods (such as those involving administration of T cell therapy but in the absence of administration of compound C). In some embodiments, the persistence is increased by at least or about at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more.

In some embodiments, the extent or range of persistence of the administered cells can be detected or quantified after administration to a subject. For example, in some aspects, quantitative PCR (qPCR) is used to assess the amount of cells (e.g., CAR expressing cells) expressing a recombinant receptor in the blood or serum or organ or tissue (e.g., disease site) of a subject. In some aspects, persistence is quantified as a copy of the DNA or plasmid encoding a receptor (e.g., CAR) per microgram of DNA, or as the number of receptor expressing (e.g., CAR expressing) cells per microliter of sample (e.g., blood or serum) or per microliter of total number of Peripheral Blood Mononuclear Cells (PBMCs) or leukocytes or T cells in the sample. In some embodiments, flow cytometry assays may also be performed, which typically detect cells expressing a receptor using antibodies specific for the receptor. Cell-based assays can also be used to detect the number or percentage of functional cells, such as cells that can bind to and/or neutralize a disease or disorder or cells that express an antigen recognized by a receptor and/or cells that can induce a response (e.g., a cytotoxic response) against the cells. In any such embodiment, the degree or level of expression of another marker associated with the recombinant receptor (e.g., CAR expressing cells) can be used to distinguish the administered cells from endogenous cells of the subject.

In some embodiments, administration of compound C is continued for a period of time to enhance, increase, or optimize the persistence of the reaction. In some aspects, the provided methods are based on the following observations: subjects who achieve or are in Complete Remission (CR) at 3 months are more likely to sustain a response longer, such as survival or progression free survival for greater than or greater than about three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or twelve months after treatment is completed or after first achieving a Complete Response (CR) after administration of the combination therapy. In some aspects, the method is performed to administer compound C after initiation of administration of T cell therapy, such as in a specific intermittent dosing regimen as described, for a period of at least 3 months or about three months.

In some aspects, the provided methods and uses provide or achieve an improved or more durable response or efficacy as compared to certain alternative methods, e.g., comprising administering T cell therapy or compound C as monotherapy or not as a method of combination therapy as described herein, such as in a particular group of treated subjects. In some embodiments, the method is advantageous due to administration of a T cell therapy, such as a composition comprising cells for adoptive cell therapy (e.g., like T cell therapy, e.g., CAR expressing T cells), and compound C. In some embodiments, such a response is observed in high risk patients with poor prognosis (such as those with high risk diseases, e.g., high risk NHL). In some aspects, the methods treat subjects with some form of invasive and/or poorly prognosis B cell non-hodgkin lymphoma (NHL), such as NHL that is recurrent or refractory (R/R) to standard therapy or has a poor prognosis. In some embodiments, a subject treated according to the provided methods suffers from diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma.

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 the subjects treated according to the provided methods and/or with the provided articles, kits, or compositions achieve a Complete Response (CR). In some embodiments, the subject is in CR and exhibits Minimal Residual Disease (MRD). In some embodiments, the subject is in CR and is MRD-. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the subjects treated according to the provided methods and/or with the provided articles, kits, or compositions achieve an objective response of Partial Response (PR). In some embodiments, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more of the subjects according to the provided methods and/or treated with the provided articles, kits or compositions achieve CR or PR six months, seven months, eight months, nine months, ten months, eleven months or one year after starting administration of the cell therapy.

In some embodiments, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, OR twelve months OR longer after initiation of administration of the cell therapy, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% OR more of the subjects according to the provided methods and/OR treated with the provided articles, kits, OR compositions remain responsive, such as retaining CR OR Objective Response (OR). In some embodiments, such a response, such as CR OR, may last for at least three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, OR longer, such as in at least OR about at least 60%, at least 70%, at least 80%, at least 90%, at least 95% OR more of the subjects treated according to the provided methods OR in such subjects who achieve CR by three months, four months, five months, OR six months. In some embodiments, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more of the subjects according to the provided methods and/or treated with the provided articles, kits or compositions, or such subjects achieving CR for up to three, four, five or six months survive or survive without progression for greater than or greater than about six, seven, eight, nine, ten, eleven, twelve or more months.

All publications (including patent documents, scientific articles, and databases) mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. If the definition set forth herein is contrary to or otherwise inconsistent with the definition set forth in the patents, applications, published applications and other publications incorporated by reference, the definition set forth herein takes precedence over the definition incorporated by reference.

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

I. Combination therapy

Provided herein are engineered cells such as T cells (e.g., CAR-T cells) and (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione or a compound of formula I

Or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, or racemic mixture (compound C), including compositions thereof, for use in treating a subject having cancer. In some embodiments, the methods are for treating a subject having lymphoma. In some embodiments, the lymphoma is a B cell malignancy. In some aspects, the lymphoma is non-hodgkin's lymphoma (NHL). In some embodiments, the cancer (e.g., lymphoma) expresses CD19. In some aspects, the methods and uses provide or achieve improved response and/or more durable response or efficacy, e.g., in a particular group of treated subjects, as compared to certain alternative methods.

Methods of preparing compound C are described in US 2019/032647, which is incorporated herein by reference in its entirety.

In some embodiments, the methods and uses comprise 1) administering to a subject a cell therapy comprising T cells expressing a genetically engineered cell surface receptor (e.g., a recombinant antigen receptor, e.g., a chimeric receptor such as a Chimeric Antigen Receptor (CAR)), which recognizes an antigen expressed by, associated with, and/or characteristic of a lymphoma (e.g., a B cell malignancy such as NHL) and/or a cell type from which the lymphoma is derived, and 2) administering to a subject compound C. In some embodiments, administration of compound C is initiated after (subsequent to) administration of the cell therapy or after (subsequent to) initiation of administration of the cell therapy. In some cases, compound C is administered to a subject who has received administration of a cell therapy. The method may involve administering one or more doses of the engineered cells and more than one dose of compound C to the subject.

The combination therapies described herein (e.g., comprising an engineered cell expressing a recombinant receptor such as a Chimeric Antigen Receptor (CAR) and compound C) or compositions comprising the engineered cell and/or compound C) can be used in a variety of therapeutic, diagnostic, and prophylactic indications. For example, the combination may be used to treat a variety of diseases and disorders in a subject. Such methods and uses include therapeutic methods and uses, for example, which involve administering engineered cells, compound C, and/or compositions containing one or both to a subject suffering from a disease, condition, or disorder (e.g., a tumor or cancer). In some embodiments, the engineered cells, compound C, and/or a composition containing one or both are administered in an amount effective to effect treatment of the disease or disorder. Uses include the use of engineered cells, compound C, and/or compositions containing one or both in such methods and treatments, and in the manufacture of medicaments for performing such therapeutic methods. In some embodiments, the methods are performed by administering the engineered cells, compound C, and/or a composition containing one or both to a subject having or suspected of having a disease or disorder. In some embodiments, the method thereby treats a disease or condition or disorder in a subject. In some embodiments, the engineered cell is any one as described in section II.

In some embodiments, the combination therapy is administered to a subject having lymphoma. In some embodiments, the combination therapy is administered to a subject having a particular B cell malignancy. The B cell malignancy being treated can be any one in which expression of an antigen is associated with and/or involved in the etiology of the B cell malignancy (e.g., causes, exacerbates, or otherwise participates in the B cell malignancy). Exemplary B cell malignancies may include diseases or disorders (e.g., cancer) associated with the transformation of the malignancy or cells. Exemplary antigens are described herein, including antigens associated with various B cell malignancies that can be treated. In certain embodiments, the chimeric antigen receptor specifically binds to an antigen associated with a disease or disorder. In some embodiments, the receptor-targeted antigen comprises an antigen associated with a B cell malignancy, such as any of a number of known B cell markers. In some embodiments, the antigen is expressed by or on B cells (including human B cells). In some embodiments, the receptor-targeted antigen is CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, igκ, igλ, CD79a, CD79b, or CD30. In some embodiments, the antigen is CD19 and the chimeric antigen receptor specifically binds CD19. In some embodiments, the CD19 antigen is human CD19. In some embodiments, the lymphoma comprises B cells expressing CD19. It will be appreciated that the description of any of the methods provided herein in which the CAR expresses a T cell specific for CD19 can also be made by targeting another B cell antigen or an antigen associated with or expressed on a cell of a T cell malignancy (such as any of the above).

In some embodiments, B-cell malignancies to be treated include leukemias and lymphomas, e.g., acute myeloid (or myelogenous) leukemia (AML), chronic myeloid (or myelogenous) 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's Lymphoma (HL), non-hodgkin's 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 disorder is a B cell malignancy selected from the group consisting of: acute Lymphoblastic Leukemia (ALL), adult ALL, chronic Lymphoblastic Leukemia (CLL), non-hodgkin lymphoma (NHL), and diffuse large B-cell lymphoma (DLBCL). In some embodiments, the disease or disorder is NHL, and the NHL is selected from invasive NHL, diffuse large B-cell lymphoma (DLBCL) NOS type (de novo and indolent), primary mediastinal large B-cell lymphoma (PMBCL), T-cell/tissue cell enriched large B-cell lymphoma (TCHRBCL), burkitt lymphoma, mantle Cell Lymphoma (MCL), and/or Follicular Lymphoma (FL) (optionally, grade 3B follicular lymphoma (FL 3B)).

In some embodiments, the methods involve treating a subject with a lymphoma such as non-hodgkin's 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 after administration of the recombinant receptor-expressing cell (e.g., CAR-expressing cell), such as after or after starting administration of the recombinant receptor-expressing cell (e.g., CAR-expressing cell).

In some embodiments, NHL can be staged based on the Lugano (Lugano) classification (see, e.g., cheson et al, (2014) JCO 32 (27): 3059-3067; cheson, B.D. (2015) Chin Clin Oncol 4 (1): 5). In some cases, the stages are described in terms of roman numerals I through IV (1-4), and localized (I or II) lymphomas affecting extra-lymphatic organs (extra-nodal organs) are indicated by E. Phase I represents a single extranodular lesion (IE) affected in one node or a group of adjacent nodes, or without a node affected. Phase 2 represents two or more groups of nodules involving the ipsilateral side of the diaphragm, or phase I or phase II (IIE) with limited continuous extra-nodular involvement depending on the extent of the nodule. Stage III represents the involvement of nodules on both sides of the diaphragm or nodules above the diaphragm and spleen involvement. Stage IV represents additional discontinuous extra-lymphatic involvement. In addition, "macromass disease" can be used to describe large tumors in the chest, especially for stage II tumors. The extent of the disease is determined by Positron Emission Tomography (PET) -computed tomography (CT, for avid lymphomas) and CT (for non-avid lymphomas).

In some embodiments, an eastern tumor collaboration group (Eastern Cooperative Oncology Group, ECOG) physical stamina indicator may be used to evaluate or select subjects for treatment, e.g., subjects with poor physical performance due to previous therapies (see, e.g., oken et al (1982) Am J Clin oncol.5:649-655). In some embodiments, the ECOG status of the subject is less than or equal to 1.ECOG physical energy state meters describe the level of functioning of a patient in terms of his own ability, daily activities, and physical abilities (e.g., walking, working, etc.). In some embodiments, an ECOG physical state of 0 indicates that the subject may be performing normal activities. In some aspects, a subject with ECOG physical performance status of 1 exhibits some limitation in physical activity, but the subject is fully ambulatory. In some aspects, a patient with ECOG physical ability status 2 can walk greater than 50%. In some cases, a subject with ECOG physical ability status 2 may also be able to self-care; see for example,et al, (1993) Br J Cancer 67 (4) 773-775. Criteria reflecting ECOG physical performance status are described in table 1 below:

in some embodiments, the subject has or has been identified as having a double/triple hit lymphoma or a double/triple hit subtype of lymphoma. In some embodiments, the lymphoma is a double-hit lymphoma characterized by the presence of MYC (myelomatosis oncogene), BCL2 (B-cell lymphoma 2), and/or BCL6 (B-cell lymphoma 6) gene rearrangements (e.g., translocations). In some embodiments, the lymphoma is a triple-hit lymphoma characterized by the presence of MYC, BCL2, and BCL6 gene rearrangements; see, for example, aukema et al, (2011) Blood 117:2319-2331. In some aspects of such embodiments, the subject is ECOG 0-1. In aspects, the therapy is indicated for such subjects, and/or the instructions indicate administration to the subjects within this population. In some embodiments, a double/triple-hit lymphoma may be considered as a high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangement with DLBCL histology (double/triple hit) based on the 2016WHO standard (Swerdlow et al, (2016) Blood 127 (20): 2375-2390).

In some embodiments, the combination therapy is administered to a subject who is or is likely to be or is predicted to be a poor responder to a treatment with a cell therapy (e.g., car+ T cells), and/or who is likely to be and/or is predicted to be and/or is not to be, to some extent, responsive to the treatment. In some embodiments, the combination therapy is administered to a subject who does not exhibit or is unlikely to exhibit or is predicted to not exhibit a complete or overall response, such as within 1 month, within two months, or within three months after initiation of administration of the cell therapy. In some embodiments, the combination therapy is administered to a subject who exhibits, or is likely to exhibit, or is predicted to exhibit disease Progression (PD) within 1 month, two months, or three months after administration of the cell therapy. In some embodiments, based on subjects so treated or having similar circumstances previously treated with the cell therapy, the subject may or is not predicted to exhibit a response or a certain response.

In some embodiments, provided methods include treating a particular group or subset of subjects, e.g., subjects identified as having a high risk disease (e.g., high risk NHL). In some aspects, the methods treat subjects with some form of invasive and/or poorly prognosis B-cell non-hodgkin lymphoma (NHL), such as NHL with a poor prognosis for recurrent or refractory (R/R) to standard therapy. In some cases, the total response rate (ORR) to an available therapy, to a standard of care, or to a reference therapy is less than 40% and/or the Complete Response (CR) is less than 20% for the disease and/or patient population for which the therapy is indicated. In some embodiments, in a chemotherapeutic refractory DLBCL, the ORR with reference or available treatment or standard of care therapy is about 26% and the CR is about 8% (Crump et al Outomesin refractory aggressive diffuse large B-cell lymphoma (DLBCL): results from theinternational SCHOLAR study. ASCO 2016[ Abstract 7516 ]). In some aspects, the provided methods, compositions, uses, and articles of manufacture achieve improved and superior responses to available therapies.

In some embodiments, methods and uses for treating a subject described herein involve selecting or identifying a particular group or subset of subjects, e.g., based on a particular disease type, diagnostic criteria, previous treatments, and/or responses to previous treatments. In some embodiments, the methods involve treating a subject who relapses or becomes refractory to one or more previous therapies after remission following treatment with the one or more previous therapies; or a subject who is relapsed or refractory (R/R) to one or more previous therapies (e.g., one or more standard therapies normals, including those as described herein).

In some embodiments, the subject has undergone more than one, two, three, four, five, or six prior therapies. In some embodiments, the subject has undergone a prior therapy. In some embodiments, the subject has undergone about two to four prior therapies. In some embodiments, the subject has undergone about five to six prior therapies. In some embodiments, the subject has undergone more than six prior therapies.

In some embodiments, the subject has been previously treated with a therapy or therapeutic agent that targets lymphoma (e.g., NHL) prior to administration of cells expressing the recombinant receptor. In some embodiments, the subject has been previously treated with a 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 a poor prognosis and/or one or more previous treatment lines have failed following treatment with standard therapies. In some embodiments, the subject has been treated or has previously received at least or about 1, 2, 3, 4, 5, 6, or 7 other therapies for treating NHL in addition to lymphocyte removal therapy. In some embodiments, the subject has been previously treated with chemotherapy or radiation therapy. In some aspects, the subject is refractory or non-responsive to other therapies or therapeutic agents. In some embodiments, the subject suffers from a persistent or recurrent disease, e.g., after treatment with another therapy or therapeutic intervention (including chemotherapy or radiation).

In some embodiments, the combination therapy is administered to a subject who has progressed on prior therapy. In some embodiments, the combination therapy is administered to a subject who has stopped responding to a previous therapy. In some embodiments, the combination therapy is administered to a subject who has relapsed after remission after a prior treatment. In some embodiments, the combination therapy is administered to a subject who is refractory to prior treatment. In some embodiments, the combination therapy is administered to a subject having less than optimal response (e.g., complete response, partial response, or disease stabilization) to a prior therapy.

In some embodiments, the subject is refractory to the last prior therapy. In some embodiments, the subject has relapsed for the last previous therapy. The status is refractory if the subject achieves less than a partial response to the last previous therapy. In some embodiments, the subject is undergoing prior chemotherapy. In some embodiments, the subject is refractory to chemotherapy for prior chemotherapy. In some embodiments, the subject is chemosensitive to prior therapy. A condition is refractory to chemotherapy if the subject achieves disease Stabilization (SD) or disease Progression (PD) for the last regimen containing chemotherapy or relapses less than 12 months after autologous stem cell transplantation. Otherwise, the state is chemically sensitive.

In some embodiments, the prior treatment or therapy comprises an agent that targets CD 20. In some embodiments, the prior treatment or therapy comprises an anthracycline. In some embodiments, the prior treatment or therapy comprises a cell therapy (e.g., a T cell therapy, such as a CAR T cell therapy).

In some embodiments, the methods, uses, and articles of manufacture relate to or are used to treat a subject, the methods, uses, and articles of manufacture relate to selecting or identifying a particular group or subset of subjects, e.g., based on a particular disease type, diagnostic criteria, previous treatments, and/or responses to previous treatments, such as any subject group as described. In some embodiments, the methods involve treating a subject who relapses or becomes refractory to one or more previous therapies after remission following treatment with the one or more previous therapies; or a subject that is relapsed or refractory (R/R) to one or more previous therapies (e.g., one or more standard treatment lines, such as cell therapies (e.g., car+t cells)). In some embodiments, the method involves treating a subject suffering from: diffuse large B-cell lymphoma (DLBCL) is not specific (NOS: de novo and from indolent transformation), primary mediastinal (thymic) large B-cell lymphoma (PMBCL) or grade 3B follicular lymphoma (FL 3B), EBV positive DLBCL or EBV positive NOS. In some embodiments, the methods relate to treating a subject having an eastern tumor cooperative group energy status (ECOG) of less than 1 (e.g., 0-1). In some embodiments, the methods treat a poor prognosis population or a poor prognosis population of a DLBCL patient or subject thereof that is generally poorly responsive to therapy or a particular reference therapy, such as a poor prognosis population with one or more (e.g., two or three) chromosomal translocations (e.g., so-called "double-hit" or "triple-hit" lymphomas, which are highly graded B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements with DLBCL histology, a poor prognosis population with translocations MYC/8q24 loci, typically in combination with t (14; 18) (q 32; q 21) BCL-2 gene or/and BCL6/3q27 chromosomal translocations, see, e.g., xu et al (2013) Int J Clin Exp Pathol 6 (4): 788-794), and/or a poor prognosis population that has relapsed, optionally relapsed within 12 months, and/or has been considered as a chemotherapeutic refractory.

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

In some embodiments, an antigen receptor (e.g., CAR) specifically binds to a target antigen associated with a disease or disorder (e.g., associated with NHL). In some embodiments, the antigen associated with the disease or disorder is selected from CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, igκ, igλ, CD79a, CD79b, or CD30. In some embodiments, the antigen is CD19. In some embodiments, the CD19 antigen is human CD19.

In some embodiments, the method comprises administering a cell therapy and compound C to a subject at risk of suffering from or suspected of suffering from a B-cell malignancy.

In some embodiments, the method comprises administering the cells to a subject selected or identified as having a certain prognosis or risk of NHL. Non-hodgkin lymphomas (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 may be classified into groups that may inform disease prognosis and/or recommended treatment strategies. In some cases, these groups may be "low risk", "moderate risk", "high risk", and/or "extremely high risk", and the patient may be so classified according to a variety of factors including, but not limited to, genetic abnormalities and/or morphological or physical characteristics. In some embodiments, subjects treated according to the methods and/or with the articles or compositions are classified or identified based on risk of NHL. In some embodiments, the subject is a subject with a high risk of NHL.

In some embodiments, the subject to be treated comprises a group of subjects suffering from: invasive NHL, in particular large B-cell lymphoma (NOS: de novo and from indolent), T-cell/tissue cell enriched large B-cell lymphoma (PMBCL), primary mediastinal (thymic) large B-cell lymphoma (PMBCL), grade 3B follicular lymphoma (FL 3B), EBV positive DLBCL, EBV positive NOS, or high grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangement and DLBCL histology ("double-hit" or "triple-hit" lymphoma). In some embodiments, the disease of the subject has relapsed or is refractory to at least two previous treatment lines. In some embodiments, the prior therapy comprises an agent that targets CD20 and/or an anthracycline. In some embodiments, the subject has an ECOG score of 0-1 at the time of screening. In some embodiments, the subject suffers from a Positron Emission Tomography (PET) positive disease according to the rukino classification (Cheson, 2014). In some embodiments, the subject may optionally have been previously treated with allogeneic Stem Cell Transplantation (SCT).

In some embodiments, the subject is an adult. In some embodiments, the subject is a male. In some embodiments, the subject is a female. In some embodiments, the subject is at least 40 years old when administered the combination therapy (e.g., when they are administered the cell therapy). In some embodiments, the subject is less than 40 years old when administered the combination therapy (e.g., when they are administered the cell therapy). In some embodiments, the subject is about 40-65 years old when administered the combination therapy (e.g., when they are administered the cell therapy). In some embodiments, the subject is at least 65 years old when administered the combination therapy (e.g., when they are administered the cell therapy).

A. Administration of cell therapies

Methods for cell administration for adoptive cell therapy are known and may be used in combination with the provided methods, compositions and articles of manufacture, and kits. Adoptive T cell therapy methods are described, for example, in the following documents: U.S. patent application publication No. 2003/0170238 to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; rosenberg (2011) Nat Rev Clin Oncol.8 (10): 577-85). See, e.g., themeli et al (2013) NatBiotechnol.31 (10): 928-933; tsukahara et al (2013) Biochem Biophys Res Commun 438 (1): 84-9; davila et al (2013) PLoS ONE 8 (4): e61338.

In some embodiments, the cells used in or administered in conjunction with the provided methods contain or are engineered to contain an engineered receptor, such as an engineered antigen receptor (e.g., chimeric Antigen Receptor (CAR)) or a T Cell Receptor (TCR). Compositions include pharmaceutical compositions and formulations for administration (e.g., for adoptive cell therapy). Also provided are therapeutic methods for administering the cells and compositions to a subject (e.g., patient) according to the provided methods and/or with the provided articles or compositions.

Cells typically express recombinant receptors such as antigen receptors (including functional non-TCR antigen receptors, e.g., chimeric Antigen Receptors (CARs)) and other antigen binding receptors such as transgenic T Cell Receptors (TCRs). Receptors also include other chimeric receptors. Exemplary engineered cells administered as cell therapies in the provided methods are described in section II.

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

In some embodiments, cell therapy (e.g., adoptive T cell therapy) is performed by allogeneic transfer, wherein the cells are isolated and/or otherwise prepared from a subject other than the subject (e.g., the first subject) that is about to receive or ultimately receive the cell therapy. 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.

The cells of the T cell therapy may be administered in a composition formulated for administration, or alternatively in more than one composition (e.g., two compositions) formulated for separate administration. The one or more doses of cells may include a specific number or relative number of cells or engineered cells, and/or a defined ratio or composition of two or more subtypes (e.g., CD4 to CD8T cells) within the composition.

Cells may be administered by any suitable means, such as by bolus infusion, by injection such as intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intracoronary injection, anterior chamber injection, subconjunctival (subconjunctival) injection, sub-Tenon injection, retrobulbar injection, periocular injection, or posterior juxtascleral (postjuxta-clip) delivery. In some embodiments, they are administered by parenteral, intrapulmonary and intranasal, and if desired for topical treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In some embodiments, the given dose is administered by a single bolus administration of the cells. In some embodiments, a given dose is administered by multiple bolus injections of cells, for example, over a period of no more than 3 days, or by continuous infusion of cells. In some embodiments, administration of the cell dose or any other therapy (e.g., lymphocyte removal therapy, intervention therapy, and/or combination therapy) is via an outpatient delivery.

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 clinical history of the subject and the response to the cell, and the discretion of the attending physician. In some embodiments, the composition and cells are suitable for administration to a subject at one time or over a series of treatments.

In some aspects, preconditioning a subject with an immune clearance (e.g., lymphocyte clearance) therapy can improve the efficacy of Adoptive Cell Therapy (ACT).

Thus, in some embodiments, the method comprises administering to the subject a preconditioning agent, such as a lymphocyte scavenger or a chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof, prior to initiating cell therapy. For example, the preconditioning agent may be administered to the subject at least 2 days (e.g., at least 3, 4, 5, 6, or 7 days before) prior to initiation of the cell therapy. In some embodiments, the preconditioning agent is administered to the subject no more than 7 days (e.g., no more than 6, 5, 4, 3, or 2 days before) prior to initiation of the cell therapy.

In some embodiments, the subject is preconditioned with cyclophosphamide at a dose of between or about 20mg/kg and 100mg/kg, such as between or about 40mg/kg and 80 mg/kg. In some aspects, the subject is preconditioned with or with about 60mg/kg cyclophosphamide. In some embodiments, cyclophosphamide may be administered in a single dose or may be administered in multiple doses, such as daily, every other day, or every third day. In some embodiments, cyclophosphamide is administered once daily for one or two days. In some embodiments, where the lymphocyte scavenger comprises cyclophosphamide, the subject is administered cyclophosphamide at the following doses: at or about 100mg/m ² With 500mg/m ² Between, e.g. at or about 200mg/m ² With 400mg/m ² Between or 250mg/m ² And 350mg/m ² And the end value is included. In some cases, about 300mg/m is administered to the subject ² Cyclophosphamide of (c). In some cases, about 500mg/m is administered to the subject ² Cyclophosphamide of (c). In some embodiments, cyclophosphamide may be administered in a single dose or may be administered in multiple doses, such as daily, every other day, or every third day. In some embodiments, cyclophosphamide is administered daily, such as for 1-5 days, for example for 3 to 5 days. In some cases, about 300mg/m is administered to the subject daily prior to initiation of the cell therapy ² Cyclophosphamide of (c) for 3 days. In some cases, about 500mg/m is administered to the subject daily prior to initiation of the cell therapy ² Is characterized in that the cyclophosphamide of (C) is a cyclic phosphoramide,for 3 days.

In some embodiments, when the lymphocyte scavenger comprises fludarabine, the subject is administered fludarabine at the following doses: at or about 1mg/m ² With 100mg/m ² Between, e.g. at or about 10mg/m ² And 75mg/m ² Between 15mg/m ² With 50mg/m ² Between 20mg/m ² And 40mg/m ² Between or 24mg/m ² And 35mg/m ² And the end value is included. In some cases, about 30mg/m is administered to the subject ² Fludarabine of (c). In some embodiments, fludarabine may be administered in a single dose or may be administered in multiple doses, such as daily, every other day, or every third day. In some embodiments, fludarabine is administered daily, such as for 1-5 days, for example for 3 to 5 days. In some cases, about 30mg/m is administered to the subject daily prior to initiation of the cell therapy ² Is continued for 3 days.

In some embodiments, the lymphocyte scavenger comprises a combination of agents, such as cyclophosphamide and fludarabine. Thus, a combination of agents may include cyclophosphamide at any dose or administration regimen (such as those described above) and fludarabine at any dose or administration regimen (such as those described above). For example, in some aspects, 60mg/kg (about 2 g/m) is administered to the subject prior to the first dose or subsequent doses ² ) 25mg/m of cyclophosphamide and 3 to 5 doses ² Fludarabine. In some embodiments, about 300mg/m is administered daily to the subject prior to initiation of the cell therapy ² Cyclophosphamide and 30mg/m ² Is continued for 3 days. In some embodiments, about 500mg/m is administered daily to the subject prior to initiation of the cell therapy ² Cyclophosphamide and 30mg/m ² Is continued for 3 days.

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

1. Compositions and formulations

In some embodiments, the dose of cells of a cell therapy (e.g., T cell therapy, which comprises cells engineered with a recombinant antigen receptor (e.g., CAR or TCR)) is provided as a composition or formulation (e.g., a pharmaceutical composition or formulation). Such compositions may be used according to the provided methods and/or with the provided articles or compositions, such as in the treatment of B cell malignancies.

The term "pharmaceutical formulation" refers to a formulation which is in a form such that the biological activity of the active ingredient contained therein is effective, and which is free of additional components having unacceptable toxicity to the subject to whom the formulation is administered.

By "pharmaceutically acceptable carrier" is meant an ingredient of the pharmaceutical formulation that is non-toxic to the subject in addition to the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

In some embodiments, the cell therapy (e.g., engineered T cells (e.g., CAR T cells)) is formulated with a pharmaceutically acceptable carrier. In some aspects, the choice of carrier is determined in part by the particular cell or agent and/or by the method of administration. Thus, there are a variety of suitable formulations. For example, the pharmaceutical composition may contain a preservative. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixture thereof is typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Vectors are described, for example, in the following documents: remington' sPharmaceutical Sciences, 16 th edition, osol, editions a. (1980). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethylbenzyl ammonium chloride, hexa methyl ammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol, or benzyl alcohol, alkyl parabens such as methyl or propyl parabens, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); a low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG).

In some aspects, a buffer is included in the composition. Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffers is used. The buffer or mixture thereof is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, remington, theScience and Practice of Pharmacy, lippincott Williams & Wilkins; 21 st edition (month 1 of 2005 5).

The formulation may comprise an aqueous solution. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease or condition being treated with the cell or agent, wherein the respective activities do not adversely affect each other. Such active ingredients are suitably present in combination in an amount effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and the like.

In some embodiments, the pharmaceutical composition contains an amount (e.g., a therapeutically effective amount or a prophylactically effective amount) of the cells effective to treat the disease or disorder. In some embodiments, the efficacy of the treatment is monitored by periodic assessment of the subject being treated. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until the desired inhibition of disease symptoms occurs. However, other administration regimens may be useful and may be determined. The desired dose may be delivered by administering the composition by a single bolus, by administering the composition by multiple bolus injections, or by administering the composition by continuous infusion.

The cells may be administered using standard administration techniques, formulations and/or devices. Formulations and devices (e.g., syringes and vials) for storing and administering the compositions are provided. With respect to cells, administration may be autologous or heterologous. For example, an immune response cell or progenitor cell can be obtained from one subject and administered to the same subject or a different compatible subject. The peripheral blood-derived immune response cells or their progeny (e.g., in vivo, ex vivo, or derived in vitro) may be administered via local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When a therapeutic composition (e.g., a pharmaceutical composition containing genetically modified immune response cells) is administered, it is typically formulated in unit dose injectable form (solution, suspension, emulsion).

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

In some embodiments, the compositions are provided as sterile liquid formulations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH in some aspects. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, the liquid composition is somewhat more convenient to administer, particularly by injection. On the other hand, the adhesive composition may be formulated within an appropriate viscosity range to provide longer contact times with specific tissues. The liquid or viscous composition may comprise a carrier, which may be a solvent or dispersion medium, containing, for example, water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

Sterile injectable solutions may be prepared by incorporating the cells in a solvent, for example, with suitable carriers, diluents or excipients such as sterile water, physiological saline, dextrose and the like.

Formulations for in vivo administration are typically sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

2. Administration of drugs

In some embodiments, a dose of cells is administered to a subject according to the provided methods and/or with the provided articles or compositions. In some embodiments, the size or timing of the dose is determined according to the particular disease or disorder (e.g., cancer, such as a B-cell malignancy) of the subject. In some cases, the size or timing of the dose for a particular disease may be determined empirically from the description provided.

In some embodiments, the dose of cells is contained at or about 2x 10 ⁵ Individual cells/kg and is at or about 2x 10 ⁶ Between individual cells/kg, e.g. at or about 4X10 ⁵ Individual cells/kg and is at or about 1x 10 ⁶ Between individual cells/kg, or at or about 6x10 ⁵ Individual cells/kg and are at or about 8x 10 ⁵ Between individual cells/kg. In some embodiments, the dose of cells comprises no more than 2x 10 ⁵ Individual cells (e.g., antigen-expressing cells, such as CAR-expressing cells) per kilogram body weight (cells/kg) of the subject, such as no more than or about 3x 10 ⁵ Individual cells/kg, no more than or about 4x10 ⁵ Individual cells/kg, no more than or about 5x 10 ⁵ Individual cells/kg, no more than or about 6x10 ⁵ Individual cells/kg, no more than or about 7x 10 ⁵ Individual cells/kg, no more than or about 8x 10 ⁵ Individual cells/kg, no more than about 9x 10 ⁵ Individual cells/kg, no more than or about 1x 10 ⁶ Individual cells/kg, or no more than or about 2x 10 ⁶ Individual cells/kg. In some embodiments, the dose of cells comprises at least or at least about or at or about 2x 10 ⁵ Individual cells (e.g., antigen-expressing cells, such as CAR-expressing cells) per kilogram body weight (cells/kg) of the subject, such as at least or at least about or at or about 3x 10 ⁵ Individual cells/kg, at least or at least about or about 4x 10 ⁵ Individual cells/kg, at least or at least about or about 5x 10 ⁵ Individual cells/kg, at least or at least about or about 6x 10 ⁵ Individual cells/kg, at least or at least about or about 7x 10 ⁵ Individual cells/kg, at least or at least about or about 8x 10 ⁵ Individual cells/kg, at least or at least about or about 9x 10 ⁵ Individual cells/kg, at least or at least about or about 1x 10 ⁶ Individual cells/kg, or at least about or about 2x 10 ⁶ Individual cells/kg.

In certain embodiments, the individual population of cells or subtypes of cells is administered to the subject in a range of about 100 to about 1000 million cells and/or in such an amount of cells per kilogram of body weight, such as, for example, 100 to about 500 million cells (e.g., about 500 million cells, about 5 million cells, about 10 million cells, about 50 million cells, about 200 million cells, about 300 million cells, about 400 million cells, or a range defined by any two of the foregoing values), such as about 1000 to about 1000 million cells (e.g., about 2000 million cells, about 3000 million cells, about 4000 million cells, about 6000 million cells, about 7000 million cells, about 8000 million cells, about 9000 million cells, about 100 million cells, about 250 million cells, about 500 million cells, about 750 cells, about 900 million cells, or a range defined by any two of the foregoing values), and in some cases about 1 million cells to about 500 cells (e.g., about 1.2, about 2.5 million cells, about 5 million cells, about 4.5 million cells, about 5 million cells, about 5.5 million cells, about 9, or a range between about 5.5 million cells, about 3.5 million cells, about 5 million cells, about 3.5 million cells, about 9, or any of these cells. The dosage may vary depending on the disease or disorder and/or the patient and/or other treatment-specific attributes.

In some embodiments, the dose of cells is a flat dose of cells or a fixed dose of cells such that the cell dose is independent of or based on the body surface area or body weight of the subject.

In some embodiments, for example, where the subject is a human, the dosage comprises less than about 5x 10 ⁸ Total recombinant receptor (e.g., CAR) expressing cells, T cells, or Peripheral Blood Mononuclear Cells (PBMCs), e.g., at about 1x 10 ⁶ To 5x 10 ⁸ Within the scope of such cells, e.g. 2X 10 ⁶ 、5x 10 ⁶ 、1x 10 ⁷ 、5x 10 ⁷ 、1x 10 ⁸ 、2x 10 ⁸ 、3x 10 ⁸ Or 4x 10 ⁸ Total such cells, or a range between any two of the foregoing values. In some embodiments, where the subject is a human, the dose is included at about 1x 10 ⁶ And 3x 10 ⁸ Total recombinant receptor (e.g., CAR) expressing cells between individuals, e.g., at about 1x 10 ⁷ Up to 2x 10 ⁸ Within the scope of such cells, e.g. 1X 10 ⁷ 、5x 10 ⁷ 、1x 10 ⁸ Or 1.5X10 ⁸ Total such cells, or a range between any two of the foregoing values. In some embodiments, multiple doses are administered to the patient, and each dose or total dose may be within any of the foregoing values. In some implementationsIn embodiments, the dose of cells comprises administration of at or from about 1x 10 ⁵ To 5x 10 ⁸ Total recombinant receptor (e.g., CAR) expressing T cells or total T cells, 1x 10 ⁵ To 1x 10 ⁸ Total recombinant receptor (e.g., CAR) expressing T cells or total T cells, from or about 5x 10 ⁵ To 1x 10 ⁷ Total recombinant receptor (e.g., CAR) expressing T cells or total T cells or from or about 1x 10 ⁶ To 1x 10 ⁷ Total recombinant receptor (e.g., CAR) expressing T cells or total T cells, each comprising an endpoint.

In some embodiments, the dose of T cells comprises cd4+ T cells, cd8+ T cells, or cd4+ and cd8+ T cells.

In some embodiments, for example, where the subject is a human, the dose (including in the dose comprising cd4+ and cd8+ T cells) of cd8+ T cells comprises about 1x 10 ⁶ And 1x 10 ⁸ Total recombinant receptor (e.g., CAR) expressing cd8+ cells between the individuals, e.g., at about 5x 10 ⁶ To 1x 10 ⁸ Within the scope of such cells, e.g. 1X 10 ⁷ 、2.5x 10 ⁷ 、5x 10 ⁷ 、7.5x 10 ⁷ Or 1x 10 ⁸ Total such cells, or a range between any two of the foregoing values. In some embodiments, multiple doses are administered to the patient, and each dose or total dose may be within any of the foregoing values. In some embodiments, the dose of cells comprises administration of at or from about 1x 10 ⁷ To 0.75x 10 ⁸ Total recombinant receptor expressing cd8+ T cells, 1x 10 ⁷ To 2.5x10 ⁷ Total recombinant receptor expressing cd8+ T cells, from or about 1x 10 ⁷ To 0.75x10 ⁸ Total recombinant receptor expressing cd8+ T cells each contain an endpoint. In some embodiments, the dosage of cells comprises administration at or about 1x 10 ⁷ 、2.5x 10 ⁷ 、5x 10 ⁷ 、7.5x 10 ⁷ Or 1x 10 ⁸ The total recombinant receptor expressed cd8+ T cells.

In some embodiments, for example, where the subject is a human, the dose (including in the dose comprising cd4+ and cd8+ T cells) of cd4+ T cells comprises about 1x 10 ⁶ And 1x 10 ⁸ Total weight between eachGroup receptor (e.g., CAR) expressing cd4+ cells, e.g., at about 5x10 ⁶ To 1x 10 ⁸ Within the scope of such cells, e.g. 1X 10 ⁷ 、2.5x 10 ⁷ 、5x 10 ⁷ 、7.5x 10 ⁷ Or 1x 10 ⁸ Total such cells, or a range between any two of the foregoing values. In some embodiments, multiple doses are administered to the patient, and each dose or total dose may be within any of the foregoing values. In some embodiments, the dose of cells comprises administration of at or from about 1x 10 ⁷ To 0.75x10 ⁸ Total recombinant receptor expressing CD4+ T cells, 1x 10 ⁷ To 2.5x10 ⁷ Total recombinant receptor expressing CD4+ T cells, from or about 1x 10 ⁷ To 0.75x10 ⁸ Total recombinant receptor expressing cd4+ T cells each contain an endpoint. In some embodiments, the dosage of cells comprises administration at or about 1x 10 ⁷ 、2.5x 10 ⁷ 、5x 10 ⁷ 、7.5x 10 ⁷ Or 1x 10 ⁸ The total recombinant receptor expressed cd4+ T cells.

In some embodiments, the dose of cells (e.g., recombinant receptor expressing T cells) is administered to the subject as a single dose, or is administered only once over a period of two weeks, one month, three months, six months, 1 year, or more.

In the case of adoptive cell therapy, administration of a given "dose" encompasses administration of a given amount or number of cells as a single composition and/or as a single uninterrupted administration (e.g., as a single injection or continuous infusion), and also encompasses administration of a given amount or number of cells provided in multiple separate compositions or infusions as divided doses or as multiple compositions over a specified period of time (e.g., no more than 3 days). Thus, in some instances, a dose is a single or continuous administration of a specified number of cells, administered or initiated at a single point in time. However, in some circumstances, the dose is administered as multiple injections or infusions over a period of no more than three days, such as once a day for three or two days or by multiple infusions over a period of one day.

Thus, in some aspects, the dose of cells is administered as a single pharmaceutical composition. In some embodiments, the dose of cells is administered in a plurality of compositions that collectively contain the dose of cells.

In some embodiments, the term "split dose" refers to a dose that is split such that it is administered over a period of more than one day. This type of administration is encompassed in the methods of the invention and is considered a single dose.

Thus, the dose of cells may be administered as a split dose, e.g., a split dose administered over time. For example, in some embodiments, the dose may be administered to the subject within 2 days or 3 days. An exemplary method for split dosing includes administering 25% of the dose on the first day and administering the remaining 75% of the dose on the second day. In other embodiments, 33% of the dose may be administered on the first day and the remaining 67% on the second day. In some aspects, 10% of the dose is administered on the first day, 30% of the dose is administered on the second day, and 60% of the dose is administered on the third day. In some embodiments, the split dose lasts no more than 3 days.

In some embodiments, the dose of cells may be administered by administering a plurality of compositions or solutions (e.g., first and second, optionally more), each composition or solution containing the dose of some cells. In some aspects, multiple compositions are administered, either separately or independently, optionally over a period of time, each composition containing a different cell population and/or cell subtype. For example, the cell population or cell subtype may each include CD8 ⁺ And CD4 ⁺ T cells, and/or populations enriched for cd8+ and cd4+, respectively, such as cd4+ and/or cd8+ T cells, each individually comprising cells genetically engineered to express recombinant receptors. In some embodiments, the administering of the dose comprises administering a first composition comprising a dose of cd8+ T cells or a dose of cd4+ T cells; and administering a second composition comprising another dose of cd4+ T cells and cd8+ T cells.

In some embodiments, administration of a composition or dose (e.g., administration of multiple cell compositions) involves separate administration of the cell compositions. In some aspects, separate administrations are performed simultaneously or sequentially in any order. In some embodiments, the dosage comprises a first composition and a second composition, and the administration of the first composition and the second composition is separated by 0 to 12 hours, by 0 to 6 hours, or by 0 to 2 hours. In some embodiments, the beginning of the administration of the first composition and the beginning of the administration of the second composition are separated by no more than 2 hours, no more than 1 hour, or no more than 30 minutes, by no more than 15 minutes, no more than 10 minutes, or no more than 5 minutes. In some embodiments, the beginning and/or completion of the administration of the first composition and the completion and/or beginning of the administration of the second composition are separated by no more than 2 hours, no more than 1 hour, or no more than 30 minutes, by no more than 15 minutes, no more than 10 minutes, or no more than 5 minutes.

In some embodiments, the first composition is mixed with the second composition prior to administration to the subject. In some embodiments, the first composition is mixed with the second composition shortly 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 composition is mixed with the second composition immediately prior to administration.

In some compositions, the first composition (e.g., a dose of the first composition) comprises cd4+ T cells. In some compositions, the first composition (e.g., a dose of the first composition) comprises cd8+ T cells. In some embodiments, the first composition is applied before the second composition.

In some embodiments, the dose or composition of cells includes a defined or target ratio of cd4+ cells expressing recombinant receptors to cd8+ cells expressing recombinant receptors and/or cd4+ cells to cd8+ cells, optionally at a ratio of about 1:1, or between about 1:3 and about 3:1, such as about 1:1. In some aspects, administration of a composition or dose of a different cell population having a target or desired ratio (e.g., cd4: cd8+ ratio or CAR + cd4: CAR + cd8+ ratio, e.g., 1:1) involves administration of a cell composition containing one of the populations, followed by administration of a separate cell composition comprising the other of the populations, wherein the administration is at or substantially at the target or desired ratio. In some aspects, administering a dose or composition of cells at a defined ratio results in improved expansion, persistence, and/or anti-tumor activity of the T cell therapy.

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 continuous dose, e.g., a second dose, about 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, a plurality of consecutive doses is administered after the first dose, such that another one or more doses are administered after the consecutive doses are administered. In some aspects, the number of cells administered to the subject at the additional dose is the same or similar to the first dose and/or the consecutive doses. In some embodiments, the additional dose or doses are greater than the previous dose.

In some aspects, the size of the first and/or consecutive doses is determined based on one or more criteria, such as the likelihood or incidence of a subject's response to a previous treatment (e.g., chemotherapy), a subject's disease burden (e.g., tumor burden, volume, size, or extent), the extent or type of metastasis, staging, and/or the subject's occurrence of toxic outcome (e.g., CRS, macrophage activation syndrome, oncolytic syndrome, neurotoxicity, and/or host immune response to the administered cells and/or recombinant receptors).

In some aspects, the time between administration of the first dose and administration of the successive doses is from about 9 to about 35 days, from about 14 to about 28 days, or from 15 to 27 days. In some embodiments, administration of successive doses is at a time point exceeding about 14 days and less than about 28 days after administration of the first dose. In some aspects, the time between the first dose and the consecutive dose is about 21 days. In some embodiments, additional one or more doses are administered after administration of the consecutive doses (e.g., consecutive doses). In some aspects, the additional one or more consecutive doses are administered at least about 14 days and less than about 28 days after the administration of the previous dose. In some embodiments, an additional dose is administered less than about 14 days after the previous dose (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 days after the previous dose). In some embodiments, no dose is administered less than about 14 days after the previous dose, and/or no dose is administered more than about 28 days after the previous dose.

In some embodiments, the dose of cells (e.g., recombinant receptor expressing cells) comprises two doses (e.g., a double dose), a first dose comprising T cells and a continuous dose of T cells, wherein one or both of the first dose and the second dose comprises administering a split dose of T cells.

In some embodiments, the dose of cells is generally large enough to effectively reduce disease burden.

In some embodiments, the cells are administered at a desired dose, which in some aspects includes a desired dose or number of cells or one or more cell types and/or a desired ratio of cell types. Thus, in some embodiments, the dose of cells is based on the total number of cells (or number per kg body weight) and the desired ratio of individual populations or subtypes, such as the ratio of cd4+ to cd8+. In some embodiments, the dose of cells is based on the desired total number of cells in the individual population or individual cell types (or number per kg body weight). In some embodiments, the dose is based on a combination of such features as the total number of cells desired, the ratio desired, and the total number of cells desired in the individual populations.

In some embodiments, the population or subtype of cells, such as CD8, is administered at or within a tolerance difference in the desired dose of total cells (e.g., the desired dose of T cells) ⁺ And CD4 ⁺ T cells. In some aspects, the desired dose is a desired number of cells or a desired number of cells per unit body weight of a subject to which the cells are administered, e.g., cells/kg. In some aspects, the required dose is equal to or higher than a minimum cell number or minimum cell number per unit body weight. In some aspects, individual populations or subtypes are administered in total cells at a desired dose at or near a desired output ratio (e.g., CD4 ⁺ With CD8 ⁺ Ratio), for example, within a certain tolerance difference or error of such ratio.

In some embodiments, the cells are administered at or within the tolerance of the desired dose of one or more individual cell populations or subtypes, such as the desired dose of cd4+ cells and/or the desired dose of cd8+ cells. In some aspects, the desired dose is the desired subtype or population of cells or the desired number of such cells per unit weight (e.g., cells/kg) of the subject to whom the cells are administered. In some aspects, the required dose is equal to or higher than the minimum population or subtype cell number or minimum population or subtype cell number per unit body weight.

Thus, in some embodiments, the dose is based on a fixed dose of the total cells desired and the ratio desired, and/or based on a fixed dose of one or more individual subtypes or sub-populations (e.g., each) desired. Thus, in some embodiments, the dose is based on a fixed or minimum dose of T cells desired and CD4 desired ⁺ With CD8 ⁺ Cell ratio, and/or based on desired CD4 ⁺ And/or CD8 ⁺ Fixed or minimal dose of cells.

In some embodiments, the cells are administered at or within the tolerance range of a desired output ratio of a plurality of cell populations or subtypes (e.g., cd4+ and cd8+ cells or subtypes). In some aspects, the desired ratio may be a particular ratio or may be a series of ratios. For example, in some embodiments, the desired ratio (e.g., CD4 ⁺ With CD8 ⁺ The ratio of cells) is between or about 1:5 and or about 5:1 (or greater than about 1:5 and less than about 5:1), or between or about 1:3 and or about 3:1 (or greater than about 1:3 and less than about 3:1), such as between or about 2:1 and or about 1:5 (or greater than about 1:5 and less than about 2:1), such as is or about 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, 1:1.2, 1:3, 1:4, 1.5, 1:6, 1.7, 1:1, 1:1.8, 1:1.5, 3:1.5, 4:1.5, 1.1:1, 4:1.5, 1.5:1, 3:1.5, 4:1.5, 4:1.5:1). In some aspects, the tolerance difference is about 1%, about 2%, about 3% of the desired ratioWithin about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, including any values between these ranges.

In particular embodiments, the number and/or concentration of cells refers to the number of cells expressing a recombinant receptor (e.g., CAR). In other embodiments, the number and/or concentration of cells refers to the number or concentration of all cells, T cells, or Peripheral Blood Mononuclear Cells (PBMCs) administered.

In some aspects, the size of the dose is determined based on one or more criteria, such as the likelihood or incidence of the subject's response to a previous treatment (e.g., chemotherapy), the subject's disease burden (e.g., tumor burden, volume, size, or extent), the extent or type of metastasis, staging, and/or the subject's toxic outcome (e.g., CRS, macrophage activation syndrome, oncolytic syndrome, neurotoxicity, and/or host immune response to the administered cells and/or recombinant receptors).

In some embodiments, the method further comprises administering one or more additional doses of Chimeric Antigen Receptor (CAR) -expressing cell and/or lymphocyte removal therapy, and/or repeating one or more steps of the method. In some embodiments, the one or more additional doses are the same as the initial dose. In some embodiments, the one or more additional doses are different from the initial dose, e.g., higher, such as 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more higher than the initial dose, or lower, such as 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more lower than the initial dose. In some embodiments, the administration of one or more additional doses is determined based on: the likelihood or incidence of a subject's response to an initial treatment or any previous treatment, the subject's disease burden (e.g., tumor burden, volume, size, or extent), the extent or type of metastasis, staging, and/or the subject's occurrence of toxic outcome (e.g., CRS, macrophage activation syndrome, oncolytic syndrome, neurotoxicity, and/or host immune response to the administered cells and/or recombinant receptors).

B. Administration of Compound C

In some embodiments of the methods, compositions, combinations, kits, or articles of manufacture provided herein, the combination therapy comprises administering a compound C having the chemical name (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione and/or having the structure of formula I:

or an enantiomer or mixture of enantiomers thereof, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof.

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

In certain embodiments, compound C is a solid. In certain embodiments, compound C is hydrated. In certain embodiments, compound C is solvated. In certain embodiments, compound C is anhydrous. In certain embodiments, compound C is non-hygroscopic.

In certain embodiments, compound C is amorphous. In certain embodiments, compound C is crystalline.

In some embodiments, compound C is a pharmaceutically acceptable salt of (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione. As used herein, the term "pharmaceutically acceptable salt" refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases (including inorganic acids and bases and 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' -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, furancarboxylic acid, galacturonic acid, gluconic acid, glucuronic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, propionic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, sulfuric acid, tartaric acid, and p-toluenesulfonic acid. Other are well known in the art, see, for example, remington's PharmaceuticalSciences, 18 th edition, mack Publishing, easton PA (1990) or Remington: the Science andPractice of Pharmacy, 19 th edition, mack Publishing, easton PA (1995).

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

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

In some embodiments, compound C provided herein contains one chiral center and may exist as a mixture of enantiomers (e.g., a racemic mixture). The present disclosure encompasses the use of stereoisomerically pure forms of such compounds, as well as the use of mixtures of those forms. For example, mixtures comprising equal or unequal amounts of enantiomers of compound C provided herein may be used in the methods and compositions disclosed herein. These isomers may be asymmetrically synthesized or resolved using standard techniques such as chiral columns or chiral resolving agents. See, e.g., jacques, j. Et al, enantomers, racemates and Resolutions (Wiley-Interscience, new York, 1981); wilen, S.H. et al Tetrahedron 33:2725 (1977); eliel, e.l., stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S.H., tables of Resolving Agents and OpticalResolutions, page 268 (edited by E L.Eliel, univ. Of Notre Dame Press, notre Dame, IN, 1972).

It is understood that the chiral center of compound C may undergo epimerization in vivo. Thus, one skilled in the art will recognize that in the case of in vivo epimerization, administration of compound C in (R) form may be equivalent to administration of compound C in (S) form.

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

In some embodiments, compound C is a solvate of (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione. The term "solvate" means a physical association of a compound with one or more solvent molecules (whether organic or inorganic). Such physical associations contain hydrogen bonds. In some cases, the solvate will be able to separate, for example when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid. "solvate" encompasses both solution phases and isolatable solvates. Exemplary solvates include hydrates, ethanolates, methanolates, isopropanolates, acetonitrile solvates, and ethyl acetate solvates. In some embodiments, compound C is a hydrate of (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione. Methods of solvation are known in the art.

"tautomer" refers to the isomeric forms of a compound that are balanced with each other. The concentration of the isomeric forms will depend on the environment in which the compound is located and may vary depending on, for example, whether the compound is in a solid or in an organic or aqueous solution. For example, in aqueous solutions, pyrazoles can exhibit the following isomeric forms, which are referred to as tautomers of each other:

as one of ordinary skill in the art readily appreciates, a wide variety of functional groups and other structures may exhibit tautomerism, and the administration of any tautomer of compound C is within the scope of the methods provided herein.

It should also be noted that compound C as used in the provided methods may contain an unnatural proportion of atomic isotopes at one or more atoms. For example, compound C may be administered with a radioisotope (e.g., such as tritium @ ³ H) Iodine-125% ¹²⁵ I) Sulfur-35% ³⁵ S) or C-14% ¹⁴ C) Radiolabelling, or isotopic enrichment, e.g. deuterium enrichment ² H) Carbon-13% ¹³ C) Or nitrogen-15% ¹⁵ N). As used herein, an "isotopologue" is an isotopically enriched compound. The term "isotopically enriched" refers to an atom having an isotopic composition different from the natural isotopic composition of the atom. "isotopically enriched" may also refer to a compound having at least one atom whose isotopic composition differs from the natural isotopic composition of the atom. The term "isotopic composition" refers to the amount of each isotope present for a given atom. Radiolabeled and isotopically enriched compounds are useful as therapeutic agents, for example, cancer therapeutic agents, research reagents (e.g., binding assay reagents), and diagnostic agents (e.g., in vivo imaging agents). Methods involving administration of any isotopic variant of compound C (whether radioactive or not) are intended to be encompassed within the scope of the methods provided herein. In some embodiments, provided herein are isotopologues (e.g., deuterium-enriched, carbon-13 # ¹³ C) And/or nitrogen-15% ¹⁵ N) compound. As used herein, "deuterated" means a compound in which at least one hydrogen (H) has been deuterated (by D or by ² H indicates) substitution, i.e., the compound is deuterium-enriched at least one position.

It will be appreciated that, regardless of the stereoisomers or isotopic composition, compound C can be administered in the form of any of the pharmaceutically acceptable salts described herein. Likewise, it is understood that the isotopic composition can vary independently of the stereoisomeric composition of compound C. Furthermore, the isotopic composition, although limited to those elements present in compound C or a salt thereof, can additionally vary independently of the selection of a pharmaceutically acceptable salt of compound C.

It should be noted that if there is a difference between the depicted structure and the name given to the structure, the depicted structure is the dominant. In addition, a structure or a part of a structure should be interpreted as including all stereoisomers of the structure if the stereochemistry of the structure or the part of the structure is not indicated with, for example, bold or dashed lines.

1. Compositions and formulations

In some embodiments of the combination therapy methods, compositions, combinations, kits, and uses provided herein, the combination therapy can be administered in one or more compositions (e.g., a pharmaceutical composition containing compound C).

In some embodiments, the composition (e.g., a pharmaceutical composition containing compound C) may include a carrier, such as a diluent, adjuvant, excipient, or vehicle, for administration with compound C and/or the cells. Examples of suitable drug carriers are described by e.w. martin in "Remington's Pharmaceutical Sciences". Such a composition will contain a therapeutically effective amount of compound C (typically in purified form) and a suitable amount of carrier to provide a form for appropriate administration to a patient. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil and sesame oil. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. The pharmaceutical composition may contain any one or more of one or more diluents, one or more adjuvants, one or more anti-adherent agents, one or more binders, one or more coatings, one or more fillers, one or more flavoring agents, one or more pigments, one or more lubricants, one or more glidants, one or more preservatives, one or more detergents, one or more adsorbents, one or more emulsifiers, one or more pharmaceutical excipients, one or more pH buffers, or one or more sweeteners, and combinations thereof. In some embodiments, the pharmaceutical composition may be a liquid, a solid, a lyophilized powder, in gel form, and/or combinations thereof. In some aspects, the choice of carrier depends in part on the particular inhibitor and/or method of administration.

Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethylbenzyl ammonium chloride, hexa methyl ammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol, or benzyl alcohol, alkyl parabens such as methyl or propyl parabens, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); a low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG); stabilizers and/or preservatives. The composition containing compound C may also be lyophilized.

In some embodiments, the pharmaceutical composition may be formulated for administration by any route known to those of skill in the art, including intramuscular, intravenous, intradermal, intralesional, intraperitoneal injection, subcutaneous, intratumoral, epidural, nasal, oral, vaginal, rectal, topical (topical), topical (local), aural, inhalation, buccal (e.g., sublingual), and transdermal administration or any route. In some embodiments, other modes of administration are also contemplated. In some embodiments, administration is by bolus infusion, by injection such as intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intracoronary injection, anterior chamber injection, subconjunctival (subconjunctival) injection, sub-Tenon) injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral (posterior juxtascleral) delivery. In some embodiments, administration is by parenteral, intrapulmonary, and intranasal administration, and if topical treatment is desired, by intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus administration. In some embodiments, a given dose is administered by multiple bolus administration, e.g., over a period of no more than 3 days, or by continuous infusion administration.

In some embodiments, administration may be topical, or systemic, depending on the treatment site. In some embodiments, topical application to the area in need of treatment may be accomplished by, for example, but not limited to, local infusion during surgery, topical application (e.g., in conjunction with a post-surgical wound dressing), by injection, by catheter, by suppository, or by implant. In some embodiments, the composition may also be administered with other bioactive agents, sequentially, intermittently, or in the same composition. In some embodiments, administration may also include a controlled release system, including controlled release formulations and devices for controlled release, such as by means of a pump. In some embodiments, the administration is oral.

In some embodiments, compound C is typically formulated and administered in a unit dosage form or multiple dosage forms. Each unit dose contains a predetermined amount of therapeutically active compound C sufficient to produce the desired therapeutic effect in combination with a desired 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 containing suitable amounts of compound C, as well as oil-water emulsions. The unit dosage form may be contained in ampoules and syringes or in individually packaged tablets or capsules. The unit dosage form may be administered in fractions or multiples thereof. In some embodiments, the multiple dosage form is a plurality of identical unit dosage forms packaged in a single container to be administered in separate unit dosage forms. Examples of multi-dose forms include vials, tablet or capsule bottles or pints or gallon bottles.

2. Administration of drugs

In some embodiments, the provided combination therapy methods involve beginning administration of compound C before, after, during, simultaneously with, nearly simultaneously with, sequentially, concurrently with, and/or intermittently with the initiation of a cell therapy, such as a T cell therapy (e.g., CAR expressing T cells). In some embodiments, administration of compound C in the provided combination therapy methods begins after or subsequent to the initiation of administration of T cell therapy.

In some embodiments, administration of compound C is initiated after (later) initiation of a cell therapy, such as a T cell therapy (e.g., CAR expressing T cells). In some embodiments, administration of compound C begins at or before the peak or maximum level of cells of the T cell therapy can be detected in the blood of the subject.

In some cases, the onset of administration of compound C is performed one week or within one week (e.g., within 1, 2, or 3 days) prior to the following time: (i) A time at which a peak or maximum level of cells of the T cell therapy is detectable in the blood of the subject; (ii) The number of cells of the detectable T cell therapy in the blood is undetectable or reduced after having been detectable in the blood, optionally reduced compared to a prior time point after administration of the T cell therapy; (iii) The number of detectable T cell therapy cells in the blood is reduced or reduced by more than a factor of 1.5, 2.0, 3.0, 4.0, 5.0, 10 or more compared to the peak or maximum number of detectable T cell therapy cells in the blood of the subject after initiation of administration of the T cell therapy; (iv) After a peak or maximum level of cells of the T cell therapy is detectable in the subject's blood, the number of cells of the cells detectable in the subject's blood or the number of cells derived from the cells 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 after treatment with T cell therapy and/or has relapsed after remission; and/or (iv) the subject exhibits an increased tumor burden as compared to the tumor burden at a time before or after administration of the cells and before starting administration of compound C. In certain aspects, the provided methods are performed to enhance, augment, or boost T cell therapy in a subject to improve response to T cell therapy, e.g., the presence of T cells and/or a decrease in tumor burden.

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

In some embodiments, administration of compound C is initiated after (later) initiation of cell therapy. In some embodiments, administration of compound C begins after (after) the initiation of T cell therapy and as early as the same day that T cell therapy was initiated (i.e., as early as day 1 of combination therapy). In some embodiments, compound C is administered beginning between day 1 and day 29 of the combination therapy (inclusive). In some embodiments, compound C is administered beginning between day 1 and day 22 of the combination therapy (inclusive). In some embodiments, compound C is administered beginning between day 1 and day 15 of the combination therapy (inclusive). In some embodiments, compound C is administered beginning between day 2 and day 15 of the combination therapy (inclusive). In some embodiments, compound C is administered beginning between (including the endpoints of) day 3 and day 15 of the combination therapy. In some embodiments, compound C is administered beginning between day 4 and day 15 of the combination therapy (inclusive). In some embodiments, compound C is administered beginning between (including the endpoints of) day 5 and day 15 of the combination therapy. In some embodiments, compound C is administered beginning between (including the endpoints of) day 6 and day 15 of the combination therapy. In some embodiments, compound C is administered beginning between (including the endpoints of) day 7 and day 15 of the combination therapy. In some embodiments, compound C is administered beginning between (including the endpoints of) day 8 and day 15 of the combination therapy.

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

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

In some embodiments, administration of compound C begins one month prior to administration of the cell therapy. In some embodiments, administration of compound C is initiated four weeks prior to administration of the cell therapy. In some embodiments, administration of compound C begins three weeks prior to administration of the cell therapy. In some embodiments, administration of compound C begins two weeks prior to administration of the cell therapy. In some embodiments, administration of compound C begins one week prior to administration of the cell therapy. In some embodiments, administration of compound C begins six days prior to administration of the cell therapy. In some embodiments, administration of compound C begins five days prior to administration of the cell therapy. In some embodiments, administration of compound C begins four days prior to administration of the cell therapy. In some embodiments, administration of compound C begins three days prior to administration of the cell therapy. In some embodiments, administration of compound C begins two days prior to administration of the cell therapy. In some embodiments, administration of compound C begins one day prior to administration of the cell therapy.

In some embodiments, administration of compound C begins after administration of lymphocyte removal therapy and prior to administration of cell therapy. In some embodiments, the cell therapy is administered two to seven days after the end of administration of the lymphocyte removal therapy. In some embodiments, administration of compound C begins the last day of lymphocyte removal therapy. In some embodiments, administration of compound C begins seven days after the end of administration of the lymphocyte removal therapy. In some embodiments, administration of compound C begins within six days after the end of administration of the lymphocyte removal therapy. In some embodiments, administration of compound C begins within five days after the end of administration of the lymphocyte removal therapy. In some embodiments, administration of compound C begins within four days after the end of administration of the lymphocyte removal therapy. In some embodiments, administration of compound C begins within three days after the end of administration of the lymphocyte removal therapy. In some embodiments, administration of compound C begins within two days after the end of administration of the lymphocyte removal therapy. In some embodiments, administration of compound C begins within one day after administration of the lymphocyte removal therapy is complete.

In some embodiments, administration of compound C begins seven days after the end of administration of lymphocyte removal therapy. In some embodiments, administration of compound C begins six days after the end of administration of lymphocyte removal therapy. In some embodiments, administration of compound C begins five days after the end of administration of lymphocyte removal therapy. In some embodiments, administration of compound C begins four days after the end of administration of lymphocyte removal therapy. In some embodiments, administration of compound C begins three days after the end of administration of lymphocyte removal therapy. In some embodiments, administration of compound C begins two days after the end of administration of lymphocyte removal therapy. In some embodiments, administration of compound C begins one day after the end of administration of lymphocyte removal therapy.

In some embodiments, the subject does not exhibit signs or symptoms of severe toxicity, such as severe Cytokine Release Syndrome (CRS) or severe toxicity, at the time of first administration of compound C to the subject, and/or at any subsequent time after the start of said administration. In some embodiments, administration of compound C is performed 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 (e.g., prolonged grade 3 CRS or grade 4 or grade 5 CRS). In some embodiments, administration of compound C is performed 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 (e.g., prolonged grade 3 neurotoxicity or grade 4 or 5 neurotoxicity). In some aspects, between the time of starting administration of T cell therapy and the time of administration of compound C, the subject does not exhibit severe CRS and/or does not exhibit 3-grade or higher-grade CRS, such as prolonged 3-grade CRS or 4-grade or 5-grade CRS. In some cases, the subject does not exhibit severe neurotoxicity and/or does not exhibit grade 3 or higher grade neurotoxicity, such as prolonged grade 3 neurotoxicity or grade 4 or 5 neurotoxicity, between the time of initiation of administration of the T cell therapy and the time of administration of compound C.

In some embodiments, compound C is administered on an intermittent (i.e., non-daily) dosing regimen. In some embodiments, compound C is administered in multiple intermittent doses. In some embodiments, each of the plurality of intermittent doses of compound C is the same. In other embodiments, the multiple intermittent doses of compound C may be different amounts.

In some embodiments, the dose of compound C is between at or about 0.1mg and at or about 1.0mg, such as between or about 0.1mg and 0.9mg, between or about 0.1mg and 0.8mg, between or about 0.1mg and 0.7mg, between or about 0.1mg and 0.6mg, between or about 0.1mg and 0.5mg, between or about 0.1mg and 0.4mg, between or about 0.1mg and 0.3mg, between or about 0.1mg and 0.2mg, between or about 0.2mg and 1.0mg, between or about 0.2mg and 0.9mg, between or about 0.2mg and 0.8mg, between or about 0.2mg and 0.7mg, between or about 0.2mg and 0.6mg, between or about 0.2mg and 0.5mg, between or about 0.2mg and 0.4mg, between or about 0.2mg and 0.3mg, between or about 0.3mg and 0.3mg, between or about 0.2mg and 0.3mg, between or about 0.2mg and 0.9mg, between or about 0.2mg and 0.7mg between or about 0.3mg and 0.4mg, between or about 0.4mg and 1.0mg, between or about 0.4mg and 0.9mg, between or about 0.4mg and 0.8mg, between or about 0.4mg and 0.7mg, between or about 0.4mg and 0.6mg, between or about 0.4mg and 0.5mg, between or about 0.5mg and 1.0mg, between or about 0.5mg and 0.9mg, between or about 0.5mg and 0.8mg, between or about 0.5mg and 0.7mg, between or about 0.5mg and 0.6mg, between or about 0.6mg and 1.0mg, between or about 0.6mg and 0.8mg, between or about 0.6mg and 0.7mg, between or about 0.7mg and 0.8mg, each containing an end value. In some embodiments, the dose of compound C is between at or about 0.1mg and at or about 0.6mg. In some embodiments, the dose of compound C is between at or about 0.1mg and at or about 0.5mg. In some embodiments, the dose of compound C is between at or about 0.1mg and at or about 0.4mg. In some embodiments, the dose of compound C is between or about 0.1mg and or about 0.3mg. In some embodiments, the dose of compound C is at or about 0.1mg. In some embodiments, the dose of compound C is at or about 0.2mg. In some embodiments, the dose of compound C is at or about 0.3mg. In some embodiments, the dose of compound C is at or about 0.4mg. In some embodiments, the dose of compound C is at or about 0.5mg. In some embodiments, the dose of compound C is at or about 0.6mg. In any of the preceding embodiments, each of the plurality of intermittent doses of compound C is the same.

In some embodiments, compound C is administered no more than once every five days as part of an intermittent dosing regimen. In some embodiments, compound C is administered no more than once every six days. In some embodiments, compound C is administered no more than once every seven days. In some embodiments, compound C is administered no more than once every eight days. In some embodiments, compound C is administered no more than once every nine days. In some embodiments, compound C is administered no more than once every 10 days. In some embodiments, compound C is administered no more than once every 11 days. In some embodiments, compound C is administered no more than once every 12 days. In some embodiments, compound C is administered no more than once every 13 days. In some embodiments, compound C is administered no more than once every 14 days.

In some embodiments, compound C is administered once every five days as part of an intermittent dosing regimen. In some embodiments, compound C is administered once every six days. In some embodiments, compound C is administered once a week. In some embodiments, compound C is administered once every seven days (Q7D). In some embodiments, compound C is administered once every eight days. In some embodiments, compound C is administered once every nine days. In some embodiments, compound C is administered once every 10 days. In some embodiments, compound C is administered once every 11 days. In some embodiments, compound C is administered once every 12 days. In some embodiments, compound C is administered once every 13 days. In some embodiments, compound C is administered once every two weeks. In some embodiments, compound C is administered once every 14 days (Q14D).

In some embodiments, compound C is administered for a period of time after the initiation of the administration of the cell therapy. In some embodiments, compound C is administered at least one week after the initiation of administration of the cell therapy. In some embodiments, compound C is administered at least two weeks after the initiation of administration of the cell therapy. In some embodiments, compound C is administered at least three weeks after the initiation of administration of the cell therapy. In some embodiments, compound C is administered at least four weeks after the initiation of administration of the cell therapy. In some embodiments, compound C is administered at least five weeks after the initiation of administration of the cell therapy. In some embodiments, compound C is administered at least six weeks after the initiation of administration of the cell therapy. In some embodiments, compound C is administered at least seven weeks after the initiation of administration of the cell therapy. In some embodiments, compound C is administered at least eight weeks after the initiation of administration of the cell therapy. In some embodiments, compound C is administered at least nine weeks after the initiation of administration of the cell therapy. In some embodiments, compound C is administered at least 10 weeks after the initiation of administration of the cell therapy. In some embodiments, compound C is administered at least 11 weeks after the initiation of administration of the cell therapy. In some embodiments, compound C is administered for at least 12 weeks after the initiation of administration of the cell therapy. In some embodiments, compound C is administered up to 12 weeks after the initiation of administration of the cell therapy. In any of the preceding embodiments, compound C is administered up to 12 weeks after the initiation of administration of the cell therapy. Thus, with respect to combination therapies in which the cell therapy is administered on day 1 of the combination therapy, in some embodiments, compound C is not administered after day 85 of the combination therapy.

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.

In some embodiments, each of the plurality of intermittent doses of compound C is 0.3mg, and compound C is administered on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85.

In some embodiments, each of the plurality of intermittent doses of compound C is 0.3mg, and compound C is administered on days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85.

In some embodiments, each of the plurality of intermittent doses of compound C is 0.3mg, and compound C is administered on days 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85.

In some embodiments, each of the plurality of intermittent doses of compound C is 0.4mg, and compound C is administered on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85.

In some embodiments, each of the plurality of intermittent doses of compound C is 0.3mg, and compound C is administered on days 8, 22, 36, 50, 64, and 78.

In some embodiments, each of the plurality of intermittent doses of compound C is 0.2mg, and compound C is administered on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85.

In some embodiments, each of the plurality of intermittent doses of compound C is 0.6mg, and compound C is administered on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85.

In some cases, the cycling regimen may be interrupted at any time and/or one or more times. In some cases, if the subject experiences one or more adverse events, dose Limiting Toxicity (DLT), neutropenia or febrile neutropenia, thrombocytopenia, cytokine Release Syndrome (CRS), and/or Neurotoxicity (NT) (such as those described in section IV), the circulation regimen is interrupted or modified. In some embodiments, the amount of compound C per day in each administration or certain days of the week is altered after the subject has developed one or more adverse events, dose Limiting Toxicity (DLT), neutropenia or febrile neutropenia, thrombocytopenia, cytokine Release Syndrome (CRS), and/or Neurotoxicity (NT) (such as those described in section IV).

Cell therapy and engineering cells

In some embodiments, cell therapies (e.g., T cell therapies) for use in accordance with the provided combination therapy methods include administering engineered cells that express recombinant receptors designed to recognize and/or specifically bind to antigens associated with a disease or disorder (e.g., cancer, e.g., B cell malignancy). In some embodiments, binding to an antigen results in a reaction, such as an immune response against such antigen. In some embodiments, the cell contains or is engineered to contain an engineered receptor or recombinant receptor, e.g., an engineered antigen receptor, such as a Chimeric Antigen Receptor (CAR). Recombinant receptors, such as CARs, typically include an extracellular antigen (or ligand) binding domain linked (in some aspects via a linker and/or one or more transmembrane domains) to one or more intracellular signaling components. In some aspects, the engineered cells are provided as pharmaceutical compositions and formulations suitable for administration to a subject, such as suitable for adoptive cell therapy. Therapeutic methods for administering cells and compositions to a subject (e.g., a patient) are also provided. In some embodiments, the method is any one as described in section I.

In some embodiments, the cells include one or more nucleic acids introduced by genetic engineering, and thereby express recombinant products or genetically engineered products of such nucleic acids. In some embodiments, gene transfer is accomplished by: cells are first stimulated, such as by combining them with a stimulus that induces a response (such as proliferation, survival and/or activation, e.g., as measured by expression of a cytokine or activation marker), and then the activated cells are transduced and expanded in culture to an amount sufficient for clinical use.

A. Chimeric antigen receptor

In some embodiments of the provided methods and uses (e.g., any of those described in section I), an engineered cell, such as a T cell, expresses a chimeric receptor (e.g., chimeric Antigen Receptor (CAR)) comprising one or more domains that in combination provide a ligand binding domain (e.g., an antibody or antibody fragment) specific for a desired antigen (e.g., a tumor antigen) with an intracellular signaling domain. In some embodiments, the intracellular signaling domain is an activating intracellular domain portion, such as a T cell activating domain, thereby providing a primary activation signal. In some embodiments, the intracellular signaling domain contains or otherwise contains a costimulatory signaling domain to facilitate effector function. Upon specific binding to a molecule, e.g., an antigen, the receptor typically delivers an immunostimulatory signal (e.g., an ITAM-transduced signal) into the cell, thereby facilitating an immune response that targets the disease or disorder. In some embodiments, the chimeric receptor, when genetically engineered into immune cells, can modulate T cell activity, and in some cases can modulate T cell differentiation or homeostasis, thereby producing genetically engineered cells with improved longevity, survival, and/or persistence in vivo, such as for adoptive cell therapy methods.

Exemplary antigen receptors (including CARs) and methods of engineering and introducing such receptors into cells include, for example, those described in the following documents: international patent application publication nos. WO 200014257, WO 2013126726, WO 2012/129514, WO 2014031687, WO 2013/166321, WO 2013/071154, WO 2013/123061, U.S. patent application publication nos. US2002131960, US2013287748, US20130149337, U.S. patent nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353 and 8,479,118, and european patent application No. EP 2537416; and/or those described in the following documents: sadelain et al, cancer discover.2013, month 4; 3 (4) 388-398; davila et al (2013) PLoS ONE 8 (4): e61338; turtle et al, curr. Opin. Immunol, 10, 2012; 24 633-39; wu et al, cancer,2012, 3, 18 (2): 160-75. In some aspects, antigen receptors include CARs as described in U.S. Pat. No. 7,446,190, and those described in international patent application publication No. WO/2014055668 A1. Examples of CARs include CARs as disclosed in any of the above publications, such as WO 2014031687, US 8,339,645, US 7,446,179, US2013/0149337, U.S. Pat. nos. 7,446,190, U.S. Pat. No. 8,389,282; kochenderfer et al 2013,Nature Reviews Clinical Oncology,10,267-276 (2013); wang et al (2012) J.Immunother35 (9): 689-701; and Brentjens et al, sciTransl Med.2013 (177). See also WO 2014031687, US 8,339,645, US 7,446,179, US2013/0149337, U.S. Pat. No. 7,446,190 and U.S. Pat. No. 8,389,282.

Exemplary CAR T cell therapies targeting CD19 include those that are or are being studied in clinical trials NCT02644655, NCT03744676, NCT01087294, NCT03366350, NCT03790891, NCT03497533, NCT04007029, NCT03960840, NCT04049383, NCT04094766, NCT03366324, NCT02546739, NCT03448393, NCT03467256, NCT03488160, NCT04012879, NCT03016377, NCT03468153, NCT03483688, NCT03398967, NCT03229876, NCT03455972, NCT03423706, NCT03497533, and NCT04002401, including FDA approved products(Li Jimai Ronchi), TECARTUS ^TM (Bromoxef), kymriah ^TM (Texarensai) and YESCARTA ^TM (Alkylrensai). Exemplary engineered cells include->TECARTUS ^TM 、KYMRIAH ^TM 、YESCARTA ^TM UCART19 and ALLO-501. In some aspects, the engineered cells include any of those described in Marofi et al, front.immunol. (2021) 12:689984, which is incorporated herein by reference in its entirety.

In some embodiments, the engineered cell (e.g., T cell) expresses a recombinant receptor (e.g., chimeric Antigen Receptor (CAR)) that is specific for a particular antigen (or marker or ligand) (e.g., an antigen expressed on the surface of a particular cell type). In some embodiments, the antigen to which the receptor is targeted is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or over-expressed on cells of a disease or disorder (e.g., tumor or pathogenic cells) as compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or on engineered cells.

In some embodiments, the receptor-targeted antigen comprises an antigen associated with a B cell malignancy, such as any of a number of known B cell markers. In some embodiments, the receptor-targeted antigen is CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, igκ, igλ, CD79a, CD79b, or CD30. In a particular aspect, the antigen is CD19. In some embodiments, any such antigen is an antigen expressed on human B cells.

Chimeric receptors (e.g., CARs) typically include an extracellular antigen-binding domain that is one or more antigen-binding portions of an antibody molecule. In some embodiments, the antigen binding domain is part of an antibody molecule, typically a Variable Heavy (VH) chain region and/or a Variable Light (VL) chain region of an antibody, e.g., a scFv antibody fragment. In some embodiments, the antigen binding domain is a single domain antibody (sdAb), such as sdFv, nanobody, V _H H and V _NAR . In some embodiments, the antigen binding fragment comprises antibody variable regions linked by a flexible linker.

In some embodiments, the antibody or antigen binding fragment (e.g., scFv or V _H Domain) specifically recognizes an antigen, such as CD19. In some embodiments, the antibody or antigen binding fragment is derived from an antibody or antigen binding fragment that specifically binds CD19, or is a variant of an antibody or antigen binding fragment that specifically binds CD19.

In some embodiments, the antigen is CD19. In some embodiments, the scFv comprises V derived from an antibody or antibody fragment specific for CD19 _H And V _L . In some embodiments, the antibody or antibody fragment that binds CD19 is a mouse-derived antibody, such as FMC63 and SJ25C1. In some implementationsIn embodiments, the antibody or antibody fragment is a human antibody, e.g., as described in U.S. patent publication No. US 2016/0152723.

In some embodiments, the antigen binding domain comprises V derived from FMC63 _H And/or V _L Which in some aspects may be an scFv. FMC63 is typically a mouse monoclonal IgG1 antibody raised against human-derived Nalm-1 and Nalm-16 expressing CD19 cells (Ling, N.R. et al (1987) Leucocyte typing III.302). In some embodiments, the FMC63 antibody comprises CDR-H1 and CDR-H2 as set forth in SEQ ID NOS: 38 and 39, respectively, and CDR-H3 as set forth in SEQ ID NOS: 40 or 54; and CDR-L1 as shown in SEQ ID NO:35, and CDR-L2 as shown in SEQ ID NO:36 or 55 and CDR-L3 as shown in SEQ ID NO:37 or 56. In some embodiments, the FMC63 antibody comprises a heavy chain variable region (V _H ) And a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO. 42 _L )。

In some embodiments, the scFv comprises a variable light chain comprising 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 comprising the CDR-H1 sequence of SEQ ID NO:38, the CDR-H2 sequence of SEQ ID NO:39 and the CDR-H3 sequence of SEQ ID NO:40, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the scFv comprises the FMC63 variable heavy chain region set forth in SEQ ID NO. 41 and the FMC63 variable light chain region set forth in SEQ ID NO. 42, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the variable heavy chain and the variable light chain are linked by a linker. In some embodiments, the linker is as set forth in SEQ ID NO. 59. In some embodiments, the scFv comprises V in order _H Linker and V _L . In some embodiments, the scFv comprises V in turn _L Linker and V _H . In some embodiments, the scFv consists of SEQ IDThe nucleotide sequence set forth in SEQ ID NO. 57 or a sequence encoding 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 comprises the amino acid sequence shown 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-CD 19 CAR is(Li Jimai rensai) anti-CD 19 CAR. In some embodiments, the T cell therapy is +.>(Li Jimai Lun Site).

In some embodiments, the antigen binding domain comprises V derived from SJ25C1 _H And/or V _L Which in some aspects may be an scFv. SJ25C1 is a mouse monoclonal IgG1 antibody raised against human-derived Nalm-1 and Nalm-16 expressing CD19 (Ling, N.R. et al (1987) Leucocyte typing III.302). In some embodiments, the SJ25C1 antibody comprises the CDR-H1, CDR-H2 and CDR-H3 shown in SEQ ID NOS: 47-49, respectively, and the CDR-L1, CDR-L2 and CDR-L3 sequences shown in SEQ ID NOS: 44-46, respectively. In some embodiments, the SJ25C1 antibody comprises a heavy chain variable region (V _H ) And a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO. 51 _L ). In some embodiments, the scFv comprises a variable light chain comprising 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 comprising the CDR-H1 sequence of SEQ ID NO:47, the CDR-H2 sequence of SEQ ID NO:48 and the CDR-H3 sequence of SEQ ID NO:49, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodimentsThe scFv comprises the SJ25C1 variable heavy chain region set forth in SEQ ID No. 50 and the SJ25C1 variable light chain region set forth in SEQ ID No. 51, or a variant of any one of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the variable heavy chain and the variable light chain are linked by a linker. In some embodiments, the linker is as set forth in SEQ ID NO. 52. In some embodiments, the scFv comprises V in turn _H Linker and V _L . In some embodiments, the scFv comprises V in turn _L Linker and V _H . In some embodiments, the scFv comprises the amino acid sequence shown 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 to SEQ ID NO. 53.

In some embodiments, the anti-CD 19 CAR is TECARTUS ^TM An anti-CD 19 CAR of (buulosa). In some embodiments, the T cell therapy is TECARTUS ^TM (Broadside).

In some embodiments, the anti-CD 19 CAR is kymeriah ^TM anti-CD 19 CAR of (temazei). In some embodiments, the T cell therapy is KYMRIAH ^TM (Tixarensai).

In some embodiments, the anti-CD 19 CAR is YESCARTA ^TM An anti-CD 19 CAR of (alemtuzite). In some embodiments, the T cell therapy is YESCARTA ^TM (Alkylrensai).

The term "antibody" is used herein in its broadest sense and includes polyclonal and monoclonal antibodies, including whole antibodies and functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F (ab') ₂ Fragments, fab' fragments, fv fragments, recombinant IgG (rIgG) fragments, variable heavy chains capable of specifically binding antigen (V _H ) Regions, single chain antibody fragments (including single chain variable fragments (scfvs)), single domain antibodies (e.g., sdabs, sdfvs, nanobodies, V) _H H or V _NAR ) Or fragments. The term encompasses immunoglobulin eggsWhite genetically engineered and/or otherwise modified forms such as intracellular antibodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugate antibodies, multispecific (e.g., bispecific) antibodies, diabodies, triabodies and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise indicated, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses whole or full length antibodies, including antibodies of any class or subclass (including IgG and subclasses thereof, igM, igE, igA and IgD). In some aspects, the CAR is a bispecific CAR, e.g., contains two antigen binding domains with different specificities.

In some embodiments, antigen binding proteins, antibodies, and antigen binding fragments thereof specifically recognize the antigen of a full length antibody. In some embodiments, the heavy and light chains of an antibody may be full length or may be antigen binding portions (Fab, F (ab') 2, fv, or single chain Fv fragments (scFv)). In other embodiments, the antibody heavy chain constant region is selected from, for example, igG1, igG2, igG3, igG4, igM, igA1, igA2, igD, and IgE, particularly selected from, for example, igG1, igG2, igG3, and IgG4, more particularly IgG1 (e.g., human IgG 1). In another embodiment, the antibody light chain constant region is selected from, for example, kappa or lambda, especially kappa.

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

The exact amino acid sequence boundaries for a given CDR or FR can be readily determined using any of a number of well known schemes, including those described in the following documents: kabat et al (1991), "Sequences of Proteins of Immunological Interest," 5 th edition 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), "anti-body-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 variabledomains and Ig superfamily V-like domains," Dev Comp Immunol, month 1 2003; 27 (1) 55-77 ("IMGT" numbering scheme); honegger A and Pl Uckthun A, "Yet another numberingscheme for immunoglobulin variabledomains: an automatic modeling and analysis tool," JMol Biol, no. 6/8 2001; 309 (3) 657-70 ("Aho" numbering scheme); and Martin et al, "Modelingantibody hypervariable loops: a combined algorithm," PNAS,1989,86 (23): 9268-9272 ("AbM" numbering scheme).

The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. Numbering of the Kabat and Chothia protocols is based on the most common antibody region sequence length, with insertions provided by insert letters such as "30a" and deletions in some antibodies. Both of these schemes place certain insertions and deletions ("indels") at different positions, resulting in different numbers. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM protocol is a compromise between Kabat and Chothia definitions and is a protocol based on the use of Oxford Molecular's AbM antibody modeling software.

Table 2 below lists exemplary location boundaries for CDR-L1, CDR-L2, CDR-L3, and CDR-H1, CDR-H2, CDR-H3 identified by the Kabat, chothia, abM and Contact schemes, respectively. For CDR-H1, residue numbers are listed using the two numbering schemes of Kabat and Chothia. FR is located between the CDRs, e.g., FR-L1 is located before CDR-L1, FR-L2 is located between CDR-L1 and CDR-L2, FR-L3 is located between CDR-L2 and CDR-L3, etc. It should be noted that because the Kabat numbering scheme shown places insertions at H35A and H35B, when numbered using the Kabat numbering convention shown, the ends of the Chothia CDR-H1 loop vary between H32 and H34 depending on the length of the loop.

Table 2 CDR boundaries according to various numbering schemes.

1-Kabat et al (1991), "Sequences of Proteins of Immunological Interest," 5 th edition Public Health Service, national Institutes of Health, bethesda, MD

2-Al-Lazikani et Al, (1997) JMB 273,927-948

Thus, unless otherwise specified, it is to be understood that a "CDR" or "complementarity determining region" or a separately specified CDR (e.g., CDR-H1, CDR-H2, CDR-H3) of a given antibody or region thereof (e.g., variable region thereof) encompasses one (or a particular) complementarity determining region as defined by any of the foregoing schemes or other known schemes. For example, in stating that a particular CDR (e.g., CDR-H3) contains a given V _H Or V _L In the case of the amino acid sequence of a corresponding CDR in the region amino acid sequence, it is to be understood that such CDR has the sequence of the corresponding CDR (e.g., CDR-H3) within the variable region, as defined by any of the foregoing schemes or other known schemes. In some embodiments, specific CDR sequences are specified. Exemplary CDR sequences of the provided antibodies are described using various numbering schemes, but it should be understood that the provided antibodies may include CDRs as described according to any other of the above-described numbering schemes or other numbering schemes known to the skilled artisan.

Likewise, unless otherwise specified, the FR of a given antibody or region thereof, such as its variable region, or a separately specified FR(s) (e.g., FR-H1, FR-H2, FR-H3, FR-H4) should be understood to encompass one (or a particular) framework region as defined by any known scheme. In some cases, an identification scheme for identifying a particular CDR, FR, or a plurality of particular FR or CDRs is specified, such as a CDR defined by Kabat, chothia, abM or Contact methods or other known schemes. In other cases, specific amino acid sequences of CDRs or FR are given.

The term "variable region" or "variable domain" refers to a domain of an antibody that is involved in the binding of the antibody to an antigen in the heavy or light chain of the antibody. The variable regions of the heavy and light chains of natural antibodies (V respectively _H And V _L ) Typically have a similar structure, wherein each domain comprises four conserved Framework Regions (FR) and three CDRs. (see, e.g., kit et al KubyImmunology, 6 th edition, w.h. freeman and co., p. 91 (2007)). Single V _H Or V _L The domain may be sufficient to confer antigen binding specificity. In addition, V from antigen-binding antibodies can be used _H Or V _L Domain isolation of antibodies binding to the specific antigen to screen complementary V _L Or V _H Library of domains. See, for example, portolano et al, J.Immunol.150:880-887 (1993); clarkson et al Nature 352:624-628 (1991).

Antibodies provided include antibody fragments. An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ') ₂ The method comprises the steps of carrying out a first treatment on the surface of the A diabody; a linear antibody; variable heavy chain (V) _H ) Regions, single chain antibody molecules (e.g., scFv) and single domain V _H A single antibody; and multispecific antibodies formed from antibody fragments. In particular embodiments, the antibody is a single chain antibody fragment, such as an scFv, comprising a variable heavy chain region and/or a variable light chain region.

The term "variable region" or "variable domain" refers to a domain of an antibody that is involved in the binding of the antibody to an antigen in the heavy or light chain of the antibody. The variable domains of the heavy and light chains of natural antibodies (V respectively _H And V _L ) Typically having a similar structure, each domain comprises four conserved Framework Regions (FR) and three CDRs. (see, e.g., kit et al KubyImmunology, 6 th edition, w.h. freeman and co., p. 91 (2007)). Single V _H Or V _L The domain may be sufficient to confer antigen binding specificity. In addition, canV using antibodies from binding antigens _H Or V _L Domain isolation of antibodies binding to the specific antigen to screen complementary V _L Or V _H Library of domains. See, for example, portolano et al, J.Immunol.150:880-887 (1993); clarkson et al Nature 352:624-628 (1991).

A single domain antibody (sdAb) is an antibody fragment that comprises all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody. In some embodiments, the CAR comprises an antibody heavy chain domain that specifically binds an antigen, such as a cancer marker or a cell surface antigen of a cell or disease to be targeted (e.g., a tumor cell or cancer cell), such as any of the target antigens described or known herein. Exemplary single domain antibodies include sdFv, nanobody, V _H H or V _NAR 。

Antibody fragments may 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 antibodies are recombinantly produced fragments, such as fragments comprising a naturally non-occurring arrangement (e.g., those having two or more antibody regions or chains joined by a synthetic linker (e.g., a peptide linker), and/or fragments that are not produced by enzymatic digestion of a naturally occurring intact antibody. In some embodiments, the antibody fragment is an scFv.

A "humanized" antibody is one 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. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized form" of a non-human antibody refers to a variant of a non-human antibody that has undergone humanization to generally reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody from which CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

In some aspects, a recombinant receptor (e.g., chimeric antigen receptor) includes an extracellular portion that contains one or more ligand (e.g., antigen) binding domains (e.g., antibodies or fragments thereof); and one or more intracellular signaling regions or domains (also interchangeably referred to as cytoplasmic signaling domains or regions). In some aspects, the recombinant receptor (e.g., CAR) further comprises a spacer and/or a transmembrane domain or portion. In some aspects, the spacer and/or transmembrane domain can connect an extracellular portion containing a ligand (e.g., antigen) binding domain and one or more intracellular signaling regions or domains.

In some embodiments, the recombinant receptor (e.g., CAR) further comprises a spacer, which may be or include at least a portion of an immunoglobulin constant region or variant or modified form thereof, such as a hinge region (e.g., an IgG4 hinge region) and/or C _H 1/C _L And/or an Fc region. In some embodiments, the recombinant receptor further comprises a spacer and/or a hinge region. In some embodiments, the constant region or portion is a constant region or portion of a human IgG (e.g., igG4 or IgG 1). In some aspects, the portion of the constant region serves as a spacer region between the antigen recognition component (e.g., scFv) and the transmembrane domain. The length of the spacer may provide enhanced cellular reactivity following antigen binding compared to the absence of the spacer. In some examples, the spacer has a length of or about 12 amino acids or has a length of no more than 12 amino acids. Exemplary spacers include those having the following: at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between the endpoints of any of the listed ranges. In some embodiments, the spacer region has about 12 or fewer amino acids, about 119 or fewer amino acids, or about 229 or fewer amino acids. Example Sex spacers include IgG4 only hinges, igG4 hinges linked to CH2 and CH3 domains, or IgG4 hinges linked to CH3 domains. Exemplary spacers include, but are not limited to, those described in the following documents: hudecek et al (2013) Clin.cancer Res.,19:3153; hudecek et al (2015) cancer Immunol Res.3 (2): 125-135 or International patent application publication No. WO 2014031687.

In some embodiments, the spacer contains only the hinge region of IgG, such as an IgG4 or IgG1 only hinge, such as the hinge-only spacer shown in SEQ ID NO:1 and encoded by the sequence shown in SEQ ID NO: 2. In some embodiments, the spacer is with C _H 2 and/or C _H 3 domain linked Ig hinge, e.g., igG4 hinge. In some embodiments, the spacer is with C _H 2 and C _H 3 domain linked Ig hinge, e.g.IgG 4 hinge, as shown in SEQ ID NO: 3. In some embodiments, the spacer is attached to C only _H 3 domain linked Ig hinge, e.g.IgG 4 hinge, as shown in SEQ ID NO. 4. In some embodiments, the spacer is or includes a glycine-serine rich sequence or other flexible linker, such as known flexible linkers. In some embodiments, the constant region or portion is a constant region or portion of IgD. In some embodiments, the spacer has the sequence set forth in SEQ ID NO. 5. In some embodiments, the spacer has an amino acid sequence that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs 1, 3, 4 and 5.

In some aspects, the spacer is a polypeptide spacer that: (a) Comprising or consisting of all or a portion of an immunoglobulin hinge or modified form thereof, or comprising about 15 or less amino acids and not comprising a CD28 extracellular region or a CD8 extracellular region, (b) comprising or consisting of an immunoglobulin hinge, optionally an IgG4 hinge, or all or a portion of a modified form thereof,and/or comprises about 15 or fewer amino acids and does not comprise a CD28 extracellular region or a CD8 extracellular region, or (c) is or about 12 amino acids in length and/or comprises, or consists of, an immunoglobulin hinge, optionally an IgG4 hinge, or all or a portion of a modified form thereof; or (d) consists of or comprises the following: 1, 3-5, 27-34, or 58, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or (e) comprising formula X ₁ PPX ₂ P or consist of, wherein X ₁ Is glycine, cysteine or arginine and X ₂ Is cysteine or threonine.

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

In one embodiment, a transmembrane domain is used that naturally associates with one of the domains in the receptor (e.g., CAR). In some cases, the transmembrane domains are selected or modified by amino acid substitutions to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

In some embodiments, the transmembrane domain is derived from a natural or synthetic source. When the source is natural, in some aspects, the domain is derived from any membrane-bound protein or transmembrane protein. The transmembrane regions include those derived from (i.e., comprising at least one or more of the transmembrane regions of): the α, β or ζ chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 (4-1 BB) or CD154. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues, such as leucine and valine. In some aspects, triplets of phenylalanine, tryptophan and valine will be found at each end of the synthetic transmembrane domain. In some embodiments, the connection is through a linker, spacer and/or one or more transmembrane domains. In some aspects, the transmembrane domain comprises a transmembrane portion of CD28 or variant thereof. The extracellular domain and the transmembrane domain may be directly or indirectly linked. In some embodiments, the extracellular domain and the transmembrane domain are connected by a spacer (any spacer as described herein).

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

In some embodiments, a recombinant receptor (e.g., CAR) includes at least one or more intracellular signaling components, such as an intracellular signaling region or domain. In some aspects, T cell activation is described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components. Intracellular signaling regions include those that mimic or approximate: signals via natural antigen receptors, signals via a combination of such receptors and co-stimulatory receptors, and/or signals via co-stimulatory receptors alone. In some embodiments, a short oligopeptide or polypeptide linker (e.g., a linker between 2 and 10 amino acids in length, such as a glycine and serine containing linker, e.g., a glycine-serine duplex) is present between the transmembrane domain and cytoplasmic signaling domain of the CAR and forms a linkage.

In some embodiments, upon connection of the CAR, the cytoplasmic domain or intracellular signaling region of the CAR activates at least one of the normal effector functions or responses of an immune cell (e.g., a T cell engineered to express the CAR). For example, in some contexts, the CAR induces a function of a T cell, such as cytolytic activity or T helper cell activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling region of the antigen receptor component or co-stimulatory molecule (e.g., if it transduces an effector function signal) is used in place of the intact immunostimulatory chain. In some embodiments, the intracellular signaling region (e.g., comprising one or more intracellular signaling domains) comprises the cytoplasmic sequence of a T Cell Receptor (TCR), and in some aspects also comprises co-receptors (which act synergistically with such receptors in a natural context to initiate signal transduction upon antigen receptor engagement) and/or those of any derivative or variant of such molecules, and/or any synthetic sequence having the same functional ability. In some embodiments, the intracellular signaling region (e.g., comprising one or more intracellular domains) includes a cytoplasmic sequence of regions or domains that are involved in providing a costimulatory signal.

In some aspects, the CAR comprises a primary cytoplasmic signaling sequence that modulates primary activation of the TCR complex. The primary cytoplasmic signaling sequence that acts in a stimulatory manner may contain a signaling motif (which is referred to as an immunoreceptor tyrosine activation motif or ITAM). Examples of primary cytoplasmic signaling sequences containing ITAM include those derived from the cd3ζ chain, fcrγ, cd3γ, cd3δ, and cd3ε. In some embodiments, one or more cytoplasmic signaling molecules in the CAR contain a cytoplasmic signaling domain derived from cd3ζ, portion or sequence thereof.

In some embodiments, the receptor comprises an intracellular component of the TCR complex, such as a TCR CD3 chain, e.g., a cd3ζ chain, that mediates T cell activation and cytotoxicity. Thus, in some aspects, the antigen binding portion is linked to one or more cell signaling modules. In some embodiments, the cell signaling module comprises a CD3 transmembrane domain, a CD3 intracellular signaling domain, and/or other CD transmembrane domain. In some embodiments, the receptor (e.g., CAR) further comprises a portion of one or more additional molecules (e.g., fc receptor gamma, CD8 a, CD8 beta, CD4, CD25, or CD 16). For example, in some aspects, the CAR or other chimeric receptor comprises a chimeric molecule between CD3- ζ (cd3ζ) or Fc receptor γ and CD8 a, CD8 β, CD4, CD25, or CD 16.

In some embodiments, the intracellular (or cytoplasmic) signaling region comprises a human CD3 chain, optionally a CD3 zeta-stimulating signaling domain, or a functional variant thereof, such as the cytoplasmic domain of 112 AA of dysmorphism 3 of human CD3 zeta (accession number: P20963.2) or a CD3 zeta signaling domain as described in U.S. Pat. No. 7,446,190 or U.S. Pat. No. 8,911,993. In some embodiments, the intracellular signaling region comprises the amino acid sequence shown in SEQ ID No. 13, 14, or 15 or an amino acid sequence exhibiting at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 13, 14, or 15.

In the case of native TCRs, complete activation typically requires not only signaling via the TCR, but also a co-stimulatory signal. Thus, in some embodiments, to facilitate complete activation, components for generating secondary or co-stimulatory signals are also included in the CAR. In other embodiments, the CAR does not include a component for generating a co-stimulatory signal. In some aspects, the additional CAR is expressed in the same cell and provides a component for generating a secondary or co-stimulatory signal.

In some embodiments, the chimeric antigen receptor comprises an intracellular domain of a T cell costimulatory molecule. In some embodiments, the CAR comprises a signaling domain and/or transmembrane portion of a co-stimulatory receptor (e.g., CD28, 4-1BB, OX40 (CD 134), CD27, DAP10, DAP12, ICOS, and/or other co-stimulatory receptor). In some embodiments, the CAR comprises a co-stimulatory region or domain of CD28 or 4-1BB (e.g., human CD28 or human 4-1 BB).

In some embodiments, the intracellular signaling region or domain comprises the intracellular co-stimulatory signaling domain of human CD28 or a functional variant or portion thereof, such as a 41 amino acid domain thereof and/or such a domain having LL to GG substitutions at positions 186-187 of the native CD28 protein. In some embodiments, the intracellular signaling domain may comprise the amino acid sequence shown in SEQ ID No. 10 or 11 or an amino acid sequence exhibiting at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 10 or 11. In some embodiments, the intracellular region comprises the intracellular co-stimulatory signaling domain of 4-1BB or a functional variant or portion thereof, such as the cytoplasmic domain of 42 amino acids of human 4-1BB (accession No. Q07011.1) or a functional variant or portion thereof, such as the amino acid sequence shown in SEQ ID NO:12 or an amino acid sequence exhibiting at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12.

In some aspects, the same CAR includes both a primary (or activating) cytoplasmic signaling region and a costimulatory signaling component.

In some embodiments, the activation domain is included within one CAR, and the co-stimulatory component is provided by another CAR that recognizes another antigen. In some embodiments, the CAR comprises an activated or stimulated CAR, a co-stimulated CAR (see WO 2014/055668), all expressed on the same cell. In some aspects, the cell comprises one or more stimulating or activating CARs and/or co-stimulating CARs. In some embodiments, the cell further comprises an inhibitory CAR (iCAR, see Fedorov et al, sci.tranl.medicine, 5 (215) (month 12 2013), such as a CAR that recognizes an antigen other than an antigen associated with and/or characteristic of a disease or disorder, wherein an activation signal delivered by a disease-targeted CAR is reduced or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.

In some cases, the CAR is referred to as a first, second, and/or third generation CAR. In some aspects, the first generation CAR is a CAR that provides only CD3 chain-induced signaling upon antigen binding; in some aspects, the second generation CAR is a CAR that provides such signals and co-stimulatory signals, such as a CAR comprising an intracellular signaling domain from a co-stimulatory receptor (e.g., CD28 or CD 137); in some aspects, the third generation CAR is a CAR comprising multiple co-stimulatory domains of different co-stimulatory receptors.

In some embodiments, the CAR encompasses one or more (e.g., two or more) co-stimulatory domains and an activation domain (e.g., a primary activation domain) in the cytoplasmic portion. Exemplary CARs include intracellular components of CD 3-zeta, CD28, and 4-1 BB.

In some embodiments, the antigen receptor further comprises a label, and/or the cell expressing the CAR or other antigen receptor further comprises a surrogate marker, such as a cell surface marker, which can be used to confirm that the cell is transduced or engineered to express the receptor. In some aspects, the marker comprises all or part (e.g., a truncated form) of CD34, NGFR, or an epidermal growth factor receptor, such as a truncated form of such a cell surface receptor (e.g., tgfr). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding a linker sequence (e.g., a cleavable linker sequence, such as T2A). For example, the tag and optionally the linker sequence may be any as disclosed in published patent application number WO 2014031687. For example, the marker may be truncated EGFR (tEGFR), which is optionally linked to a linker sequence, such as a T2A cleavable linker sequence.

Exemplary polypeptides of truncated EGFR (e.g., tEGFR) comprise the amino acid sequence set forth in SEQ ID NO:7 or 16 or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:7 or 16. Exemplary T2A linker sequences comprise the amino acid sequences shown in SEQ ID NO. 6 or 17 or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 6 or 17.

In some embodiments, the marker is a molecule (e.g., a cell surface protein) or portion thereof that is not found naturally on a T cell or is not found naturally on a T cell surface. In some embodiments, the molecule is a non-self molecule, e.g., a non-self protein, i.e., a molecule that is not recognized as "self" by the host immune system of the adoptively transferred cell.

In some embodiments, the marker does not play any therapeutic role and/or does not produce a role other than that used as a marker for genetic engineering (e.g., for selection of successfully engineered cells). In other embodiments, the marker may be a therapeutic molecule or a molecule that otherwise exerts some desired effect, such as a ligand of a cell that is encountered in vivo, such as a co-stimulatory or immune checkpoint molecule for enhancing and/or attenuating a cellular response upon adoptive transfer of the cell and encountering the ligand.

In some embodiments, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or fragment described herein. In some aspects, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or fragment described herein and an intracellular signaling domain. In some implementations In embodiments, the antibody or fragment comprises scFv or single domain V _H Antibodies, and intracellular domains contain ITAM. In some aspects, the intracellular signaling domain comprises a signaling domain of the zeta chain of the CD 3-zeta (CD 3 zeta) chain. In some embodiments, the CD 3-zeta chain is a human CD 3-zeta chain. In some embodiments, the intracellular signaling region further comprises a CD28 and CD137 (4-1 bb, tnfrsf 9) costimulatory domain linked to the cd3ζ 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 comprises a transmembrane domain disposed between an extracellular domain and an intracellular signaling region. In some aspects, the transmembrane domain comprises a transmembrane portion of CD28. The extracellular domain and the transmembrane domain may be directly or indirectly linked. In some embodiments, the extracellular domain and the transmembrane domain are connected by a spacer (any spacer as described herein).

In some embodiments, the CAR contains 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 containing 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 (e.g., an antibody fragment, including scFv, e.g., specific for CD19, as any of the above), a spacer (e.g., a spacer comprising a portion of an immunoglobulin molecule (e.g., a hinge region and/or one or more constant regions of a heavy chain molecule), such as a spacer comprising an Ig hinge, a transmembrane domain comprising all or a portion of a CD 28-derived transmembrane domain, a CD 28-derived intracellular signaling domain, and a CD3 zeta signaling domain.

In some embodiments, the CAR contains an antibody (e.g., an antibody fragment), is or contains a transmembrane domain of a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing 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 comprising a portion of an Ig molecule (e.g., a human Ig molecule), such as an Ig hinge, e.g., an IgG4 hinge, such as a hinge-only spacer. In some embodiments, the CAR comprises, for example, an antibody or fragment specific for CD19 (e.g., scFv, any of the above), a spacer (e.g., any Ig hinge-containing spacer), a CD 28-derived transmembrane domain, a 4-1 BB-derived intracellular signaling domain, and a cd3ζ -derived signaling domain.

B. Nucleic acids, vectors and methods for genetic engineering

In some embodiments, cells (e.g., T cells) are genetically engineered to express recombinant receptors. In some embodiments, engineering is performed by introducing a polynucleotide encoding a recombinant receptor. Polynucleotides encoding recombinant receptors are also provided, as are vectors or constructs comprising such nucleic acids and/or polynucleotides.

In some cases, the nucleic acid sequence encoding the recombinant receptor comprises a signal sequence encoding a signal peptide. In some aspects, the signal sequence may encode a signal peptide derived from a native polypeptide. In other aspects, the signal sequence may encode a heterologous or non-native signal peptide, such as an exemplary signal peptide of the GMCSFR alpha chain shown in SEQ ID NO:25 and encoded by the nucleotide sequence shown in SEQ ID NO: 24. In some cases, the nucleic acid sequence encoding a recombinant receptor (e.g., chimeric Antigen Receptor (CAR)) contains a signal sequence encoding a signal peptide. Non-limiting illustrative examples of signal peptides include, for example, the GMCSFR alpha chain signal peptide shown in SEQ ID NO. 25 and encoded by the nucleotide sequence shown in SEQ ID NO. 24, or the CD8 alpha signal peptide shown in SEQ ID NO. 26.

In some embodiments, the polynucleotide encoding the recombinant receptor comprises at least one promoter operably linked to control expression of the recombinant receptor. In some examples, the polynucleotide comprises two, three, or more promoters operably linked to control expression of the recombinant receptor.

In certain cases where the nucleic acid molecule encodes two or more different polypeptide chains (e.g., recombinant receptor and tag), each polypeptide chain may be encoded by a separate nucleic acid molecule. For example, two separate nucleic acids are provided, and each may be transferred or introduced into a cell separately for expression in the cell. In some embodiments, the nucleic acid encoding the recombinant receptor and the nucleic acid encoding the marker are operably linked to the same promoter and are optionally separated by an Internal Ribosome Entry Site (IRES) or nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome jump, which is optionally T2A, P2A, E a 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 present or inserted at different locations within the genome of the cell. In some embodiments, the polynucleotide encoding the recombinant receptor is introduced into a composition comprising the cultured cells, such as by retroviral transduction, transfection, or transformation.

In some embodiments, such as those in which the polynucleotide comprises first and second nucleic acid sequences, the coding sequences encoding each of the different polypeptide chains may be operably linked to the same or different promoters. In some embodiments, the nucleic acid molecule may contain a promoter that drives expression of two or more different polypeptide chains. In some embodiments, such nucleic acid molecules may be polycistronic (bicistronic or tricistronic), see, e.g., U.S. patent No. 6,060,273. In some embodiments, the transcription unit may be engineered to contain an IRES (internal ribosome entry site) bicistronic unit that allows for the co-expression of gene products (e.g., coding markers and coding recombinant receptors) by information from a single promoter. Alternatively, in some cases, a single promoter may direct expression of RNAs that contain two or three genes (e.g., encoding a marker and encoding 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). The ORF thus encodes a single polypeptide which is processed either during translation (in the case of 2A) or post-translationally into separate proteins. In some cases, peptides such as T2A may cause ribosomes to skip synthesis of peptide bonds at the C-terminus of the 2A element (ribosome skipping), resulting in separation of the end of the 2A sequence from the next peptide downstream (see, e.g., de Felipe, genetic Vaccines and Ther.2:13 (2004) and de Felipe et al Traffic 5:616-626 (2004)). Various 2A elements are known. Examples of 2A sequences that may be used in the methods and systems disclosed herein include, but are not limited to, 2A sequences from the following viruses: foot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 21), equine rhinitis A virus (E2A, e.g., SEQ ID NO: 20), leptopetalum album beta tetrad virus (T2A, e.g., SEQ ID NO:6 or 17), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO:18 or 19), as described in U.S. patent publication No. 20070116690.

Any of the recombinant receptors described herein may be encoded by a polynucleotide comprising one or more nucleic acid sequences encoding the recombinant receptor in any combination or permutation. For example, one, two, three or more polynucleotides may encode one, two, three or more different polypeptides, such as recombinant receptors. In some embodiments, one vector or construct contains a nucleic acid sequence encoding a marker, and a separate vector or construct contains a nucleic acid sequence encoding a recombinant receptor (e.g., 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 present downstream of the nucleic acid encoding the marker.

In some embodiments, the vector backbone contains a nucleic acid sequence encoding one or more markers. In some embodiments, the one or more markers are transduction markers, surrogate markers, and/or selection markers.

In some embodiments, the marker is a transduction marker or an alternative marker. The transduction markers or surrogate markers can be used to detect cells into which a polynucleotide (e.g., a polynucleotide encoding a recombinant receptor) has been introduced. In some embodiments, the transduction marker may indicate or confirm modification to the cell. In some embodiments, the surrogate marker is a protein that is prepared to co-express with a recombinant receptor (e.g., CAR) on the cell surface. In particular embodiments, such surrogate markers are surface proteins that have been modified to have little or no activity. In some embodiments, the surrogate markers are encoded by the same polynucleotide encoding the recombinant receptor. In some embodiments, the nucleic acid sequence encoding the recombinant receptor is operably linked to a nucleic acid sequence encoding a marker, optionally separated by an Internal Ribosome Entry Site (IRES) or a nucleic acid encoding a self-cleaving peptide or a ribosome-jump-inducing peptide, such as a 2A sequence, e.g., T2A, P2A, E2A or F2A. In some cases, the extrinsic marker gene may be used in conjunction with engineered cells to allow detection or selection of cells, and in some cases may also be used to promote cell suicide.

Exemplary surrogate markers may include truncated forms of a cell surface polypeptide, such as the following: signals that are nonfunctional and do not transduce or are incapable of transducing signals or are generally transduced by full length forms of cell surface polypeptides, and/or are not internalized or are incapable of internalization. Exemplary truncated cell surface polypeptides include truncated forms of a growth factor or other receptor, such as truncated human epidermal growth factor receptor 2 (tHER 2), truncated epidermal growth factor receptor (tEGFR, exemplary tEGFR sequences shown in SEQ ID NO:7 or 16), or Prostate Specific Membrane Antigen (PSMA) or modified forms thereof. tEGFR may contain a polypeptide consisting of the antibody cetuximabOr other therapeutic anti-EGFR antibodies or binding molecules, which can be used to identify or select cells that have been engineered with the tgfr construct and the encoded foreign protein, and/or to eliminate or isolate cells expressing the encoded foreign protein. See U.S. patent No. 8,802,374 and Liu et al, nature biotech.2016, month 4; 34 (4):430-434). In some aspects, the marker (e.g., surrogate marker) comprises all or part (e.g., truncated form) of CD34, NGFR, CD19, or truncated CD19 (e.g., truncated non-human CD 19) or an epidermal growth factor receptor (e.g., tgfr).

In some embodiments, the label is or includes a fluorescent protein, such as Green Fluorescent Protein (GFP), enhanced Green Fluorescent Protein (EGFP) (such as superfolder GFP), red Fluorescent Protein (RFP) (such as tdTomato, mCherry, mStrawberry, asRed, dsRed or DsRed 2), cyan Fluorescent Protein (CFP), blue-green fluorescent protein (BFP), enhanced Blue Fluorescent Protein (EBFP), and Yellow Fluorescent Protein (YFP), and variants thereof, including species variants, monomer variants, and codon optimized and/or enhanced variants of fluorescent proteins. In some embodiments, the label is or includes an enzyme (e.g., luciferase), the lacZ gene from E.coli (E.coli), alkaline phosphatase, secreted Embryonic Alkaline Phosphatase (SEAP), chloramphenicol Acetyl Transferase (CAT). Exemplary luminescent reporter genes include luciferase (luc), beta-galactosidase, chloramphenicol Acetyl Transferase (CAT), beta-Glucuronidase (GUS), or variants thereof.

In some embodiments, the marker is a selectable marker. In some embodiments, the selectable marker is or includes a polypeptide that confers resistance to an exogenous agent or drug. In some embodiments, the selectable marker is an antibiotic resistance gene. In some embodiments, the selectable marker is an antibiotic resistance gene that confers antibiotic resistance to mammalian cells. In some embodiments, the selectable marker is or includes a puromycin resistance gene, a hygromycin resistance gene, a blasticidin resistance gene, a neomycin resistance gene, a geneticin resistance gene, or a gecomycin resistance gene, or a modified version thereof.

In some embodiments, the molecule is a non-self molecule, e.g., a non-self protein, i.e., a molecule that is not recognized as "self" by the host immune system of the adoptively transferred cell.

In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding a linker sequence (e.g., a cleavable linker sequence, e.g., T2A). For example, the tag and optionally the linker sequence may be as in PCT publication No. WO 2014031687. For example, the marker may be truncated EGFR (tEGFR), which is optionally linked to a linker sequence, such as a T2A cleavable linker sequence. Exemplary polypeptides of truncated EGFR (e.g., tEGFR) comprise the amino acid sequence set forth in SEQ ID NO:7 or 16 or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:7 or 16.

In some embodiments, the label is or includes a fluorescent protein, such as Green Fluorescent Protein (GFP), enhanced Green Fluorescent Protein (EGFP) (such as superfolder GFP), red Fluorescent Protein (RFP) (such as tdTomato, mCherry, mStrawberry, asRed, dsRed or DsRed 2), cyan Fluorescent Protein (CFP), blue-green fluorescent protein (BFP), enhanced Blue Fluorescent Protein (EBFP), and Yellow Fluorescent Protein (YFP), and variants thereof, including species variants, monomer variants, and codon optimized and/or enhanced variants of fluorescent proteins. In some embodiments, the label is or includes an enzyme (e.g., luciferase), the lacZ gene from E.coli, alkaline phosphatase, secreted Embryonic Alkaline Phosphatase (SEAP), chloramphenicol Acetyl Transferase (CAT). Exemplary luminescent reporter genes include luciferase (luc), beta-galactosidase, chloramphenicol Acetyl Transferase (CAT), beta-Glucuronidase (GUS), or variants thereof.

In some embodiments, recombinant infectious viral particles (e.g., vectors such as those derived from simian virus 40 (SV 40), adenovirus, adeno-associated virus (AAV)) are used to transfer the recombinant nucleic acid into the cell. In some embodiments, recombinant lentiviral vectors or retroviral vectors (e.g., gamma-retroviral vectors) are used to transfer recombinant nucleic acids into T cells (see, e.g., koste et al (2014) Gene Therapy, month 4, 3. Doi:10.1038/gt.2014.25; carlens et al (2000) exp. Hematol.,28 (10): 1137-46; alonso-Camino et al (2013) mol. Ter. Nucleic. Acids.,2, e93; park et al, trends Biotechnol.,2011, 11, 29 (11): 550-557).

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

In some embodiments, the retroviral vector has a Long Terminal Repeat (LTR), such as a retroviral vector derived from moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine Stem Cell Virus (MSCV), spleen Focus Forming Virus (SFFV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include those derived from any avian or mammalian cell source. Retroviruses are often amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces retroviral gag, pol and/or env sequences. A number of exemplary retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740, 6,207,453, 5,219,740; miller and Rosman (1989) BioTechniques 7:980-990; miller, A.D. (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. Development.3:102-109).

Methods of lentiviral transduction are known. Exemplary methods are described, for example, in the following documents: wang et al (2012) J.Immunother35 (9): 689-701; cooper et al (2003) blood.101:1637-1644; verhoeyen et al (2009) Methods Mol biol.506:97-114; cavalieri et al (2003) blood.102 (2): 497-505.

In some embodiments, the recombinant nucleic acid is transferred into T cells by electroporation (see, e.g., chicaybam et al, (2013) PLoS ONE 8 (3): e60298; and Van Tedeloo et al (2000) Gene Therapy7 (16): 1431-1437). In some embodiments, the recombinant nucleic acid is transferred into T cells by transposition (see, e.g., manuri et al (2010) Hum Gene Ther 21 (4): 427-437; shalma et al (2013) Molec Ther NuclAcids, e74; and Huang et al (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in MolecularBiology, john Wiley & Sons, new york.n.y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-promoted microprojectile bombardment (Johnston, nature,346:776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al, mol. Cell biol.,7:2031-2034 (1987)).

Other methods and vectors for transferring nucleic acids encoding recombinant products are those described, for example, in International patent application publication No. WO 2014055668 and U.S. Pat. No. 7,446,190.

In some embodiments, cells (e.g., T cells) may be transfected during or after expansion, e.g., with a T Cell Receptor (TCR) or Chimeric Antigen Receptor (CAR). For example, such transfection of the gene for introduction of the desired receptor may be performed using any suitable retroviral vector. The genetically modified cell population may then be freed from the initial stimulus (e.g., an anti-CD 3/anti-CD 28 stimulus) and subsequently stimulated with a second type of stimulus, such as by a de novo introduced receptor. This second type of stimulus may include antigenic stimuli in the form of peptide/MHC molecules, homologous (cross-linked) ligands of genetically introduced receptors (e.g. natural ligands of CARs) or any ligand (e.g. antibodies) that binds directly in-frame with the new receptor (e.g. by recognizing constant regions within the receptor). See, e.g., 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, volume 65:333-347 (2014).

In some cases, vectors may be used that do not require activating cells (e.g., T cells). In some such cases, the cells may be selected and/or transduced prior to activation. Thus, the cells may be engineered before or after culturing the cells, and in some cases, at least a portion of the culturing is simultaneous or during.

Additional nucleic acids (e.g., genes for introduction) include those for improving therapeutic efficacy, such as by promoting viability and/or function of the transferred cells; providing a genetic marker for selecting and/or evaluating cells, such as a gene to assess survival or localization in vivo; genes that improve safety, for example, by making cells sensitive to negative selection in vivo, such as Lupton s.d. et al, mol.and Cell biol.,11:6 (1991); and Riddell et al, human Gene Therapy3:319-338 (1992); see also disclosures of PCT/US91/08442 and PCT/US94/05601 to Lupton et al, which describe the use of bifunctional selectable fusion genes obtained by fusing a dominant positive selectable marker with a negative selectable marker. See, e.g., riddell et al, U.S. patent No. 6,040,177, columns 14-17.

C. Cells and preparation of cells for genetic engineering

In some embodiments, the nucleic acid is heterologous, i.e., is not normally present in the cell or in a sample obtained from the cell, such as a nucleic acid obtained from another organism or cell, e.g., the nucleic acid is not normally found in an engineered cell and/or an organism from which such a cell is derived. In some embodiments, the nucleic acid is not naturally occurring, as is not found in nature, including nucleic acids comprising chimeric combinations of nucleic acids encoding various domains from a plurality of different cell types.

The cells are typically eukaryotic cells, such as mammalian cells, and are typically human cells. In some embodiments, the cells are derived from blood, bone marrow, lymph or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., bone marrow or lymphocytes, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as pluripotent stem cells and multipotent stem cells, including induced pluripotent stem cells (ipscs). Cells are typically primary cells such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, cd4+ cells, cd8+ cells, and subpopulations thereof, such as those subpopulations defined by: function, activation status, maturity, likelihood of differentiation, amplification, recycling, localization and/or persistence, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion characteristics and/or degree of differentiation. With respect to the subject to be treated, the cells may be allogeneic and/or autologous. The method includes an off-the-shelf method. In some aspects, as for the prior art, the cells are pluripotent and/or multipotent, such as stem cells, e.g., induced pluripotent stem cells (ipscs). In some embodiments, the methods comprise isolating cells from a subject, preparing, processing, culturing, and/or engineering them, and reintroducing them into the same subject either before or after cryopreservation.

Subtypes and subsets of T cells and/or cd4+ and/or cd8+ T cells include naive T (T) _N ) Cells, effector T cells (T _EFF ) Memory T cells and subtypes thereof (e.g., stem cell memory T (T) _SCM ) Central memory T (T) _CM ) Effect memory T (T) _EM ) Or terminally differentiated effector memory T cells), tumor Infiltrating Lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated constant T (MAIT) cells, naturally occurring and 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.

In some embodiments, the cell is a Natural Killer (NK) cell. In some embodiments, the cells are monocytes or granulocytes, such as bone marrow cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.

In some embodiments, the cells include one or more nucleic acids introduced by genetic engineering, and thereby express recombinant products or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acid is heterologous, i.e., is not normally present in the cell or in a sample obtained from the cell, such as a nucleic acid obtained from another organism or cell, e.g., the nucleic acid is not normally found in an engineered cell and/or an organism from which such a cell is derived. In some embodiments, the nucleic acid is not naturally occurring, as is not found in nature, including nucleic acids comprising chimeric combinations of nucleic acids encoding various domains from a plurality of different cell types.

In some embodiments, the preparation of the engineered cells includes one or more culturing and/or preparation steps. Cells for introducing nucleic acid encoding a transgenic receptor (e.g., CAR) can be isolated from a sample (e.g., a biological sample, e.g., a biological sample obtained from or derived from a subject). In some embodiments, the subject from which the cells are isolated is a subject suffering from a disease or disorder or in need of or to whom cell therapy is to be administered. In some embodiments, the subject is a human in need of a particular therapeutic intervention (e.g., adoptive cell therapy, wherein the cells are isolated, processed, and/or engineered).

Thus, in some embodiments, the cell is a primary cell, such as a primary human cell. Samples include tissues, fluids, and other samples taken directly from a subject, as well as samples from one or more processing steps such as isolation, centrifugation, genetic engineering (e.g., transduction with viral vectors), washing, and/or incubation. The biological sample may be a sample obtained directly from a biological source or a processed sample. Biological samples include, but are not limited to, body fluid (e.g., blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat), tissue and organ samples, including treated samples derived therefrom.

In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is derived from apheresis or a leukocyte apheresis product. Exemplary samples include whole blood, peripheral Blood Mononuclear Cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsies, tumors, leukemias, lymphomas, lymph nodes, intestinal-related lymphoid tissue, mucosa-related lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils, or other organs and/or cells derived therefrom. In the context of cell therapies (e.g., adoptive cell therapies), samples include samples from autologous and allogeneic sources.

In some embodiments, the cells are derived from a cell line, such as a T cell line. In some embodiments, the cells are obtained from a heterologous source, such as from mice, rats, non-human primates, and pigs.

In some embodiments, the separation of cells includes one or more preparation steps 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, e.g., to remove unwanted components, enrich for desired components, lyse, or remove cells that are sensitive to a particular reagent. In some examples, cells are isolated based on one or more characteristics, such as density, adhesion characteristics, size, sensitivity to a particular component, and/or resistance to a drug.

In some examples, cells from the circulating blood of the subject are obtained, for example, by apheresis or leukocyte apheresis. In some aspects, the sample contains lymphocytes (including T cells, monocytes, granulocytes, B cells), other nucleated leukocytes, erythrocytes, and/or platelets, and in some aspects contains cells other than erythrocytes and platelets.

In some embodiments, blood cells collected from a subject are washed, e.g., to remove plasma fractions, and the cells are placed in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with Phosphate Buffered Saline (PBS). In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all divalent cations. In some aspects, the washing step is accomplished by a semi-automated "flow-through" centrifuge (e.g., cobe 2991 cell processor, baxter) according to manufacturer's instructions. In some aspects, according to the manufacturer's instructionsThe washing step is done by Tangential Flow Filtration (TFF). In some embodiments, cells are resuspended in a plurality of biocompatible buffers (e.g., such as Ca-free ⁺⁺ /Mg ⁺⁺ PBS of (x). In certain embodiments, components of the blood cell sample are removed and the cells are resuspended directly in culture medium.

In some embodiments, the methods include density-based cell separation methods, such as the preparation of leukocytes from peripheral blood by lysing erythrocytes and centrifuging through a Percoll or Ficoll gradient.

In some embodiments, the isolation method comprises isolating the different cell types based on the expression or presence of one or more specific molecules, such as surface markers (e.g., surface proteins), intracellular markers, or nucleic acids, in the cell. In some embodiments, any known method for performing separation based on such markers may be used. In some embodiments, the separation is affinity-based or immunoaffinity-based separation. For example, in some aspects, isolating includes isolating cells and cell populations based on the expression or expression level of one or more markers (typically cell surface markers) of the cells, e.g., by incubating with an antibody or binding partner that specifically binds such markers, and then typically performing a washing step and separating cells that have bound to the antibody or binding partner from those cells that have not bound to the antibody or binding partner.

Such isolation steps may be based on positive selection (where cells that have bound the reagent are retained for further use) and/or negative selection (where cells that have not bound the antibody or binding partner are retained). In some examples, both fractions are retained for further use. In some aspects, negative selection may be particularly useful in the absence of antibodies useful for specifically identifying cell types in heterogeneous populations, such that isolation is preferably based on markers expressed by cells other than the desired population.

Isolation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of cells of a particular type (such as those expressing a marker) refers to increasing the number or percentage of such cells, but without the need to have cells that do not express the marker completely absent. Likewise, negative selection, removal, or depletion of particular types of cells (such as those expressing a marker) refers to reducing the number or percentage of such cells, but does not require complete removal of all such cells.

In some examples, multiple rounds of separation steps are performed, wherein fractions from positive or negative selection of one step are subjected to another separation step, such as subsequent positive or negative selection. In some examples, a single isolation step may deplete cells expressing multiple markers simultaneously, such as by incubating the cells with multiple antibodies or binding partners (each antibody or binding partner being specific for a marker targeted for negative selection). Likewise, by incubating the cells with multiple antibodies or binding partners expressed on various cell types, positive selection can be performed on multiple cell types simultaneously.

For example, in some aspects, a particular subset of T cells (e.g., cells positive for or highly expressed on one or more surface markers (e.g., CD28 ⁺ 、CD62L ⁺ 、CCR7 ⁺ 、CD27 ⁺ 、CD127 ⁺ 、CD4 ⁺ 、CD8 ⁺ 、CD45RA ⁺ And/or CD45RO ⁺ T cells)) are isolated by positive or negative selection techniques.

For example, anti-CD 3/anti-CD 28 conjugated magnetic beads may be used (e.g.,m-450CD3/CD28T Cell Expander) positive selection of CD3 ⁺ 、CD28 ⁺ T cells.

In some embodiments, the separation is performed by enriching a specific cell population via positive selection, or depleting a specific cell population via negative selection. In some embodiments, positive or negative selection is accomplished by incubating the cells with one or more antibodies or other binding agents that are expressed on the positively or negatively selected cells (markers ⁺ ) Or at a relatively high levelHorizontal expression (marker) ^{High height} ) Is bound specifically by the one or more surface markers.

In some embodiments, T cells are isolated from a PBMC sample by negative selection of a marker, such as CD14, expressed on non-T cells (such as B cells, monocytes or other leukocytes). In some aspects, CD4 ⁺ Or CD8 ⁺ Selection procedure for isolation of CD4 ⁺ Helper T cells and CD8 ⁺ Cytotoxic T cells. Such CD4 may be selected by positive or negative selection of markers expressed on one or more naive, memory and/or effector T cell subsets or expressed to a relatively high degree ⁺ And CD8 ⁺ The population is further sorted into a plurality of sub-populations.

In some embodiments, CD8 is used ⁺ The cells are further enriched or depleted for naive, central memory, effector memory and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulations. In some embodiments, the central memory T (T _CM ) Cells are enriched to increase efficacy, such as to improve long-term survival, expansion, and/or transplantation after administration, which is particularly robust in some aspects 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, the combination is enriched in T _CM CD8 of (C) ⁺ T cells and CD4 ⁺ T cells further enhance efficacy.

In embodiments, memory T cells are present in CD8 ⁺ CD62L of peripheral blood lymphocytes ⁺ And CD62L ^- Two subsets. PBMC can be directed against CD62L ^- CD8 ⁺ And/or CD62L ⁺ CD8 ⁺ Fractions were enriched or depleted, such as with anti-CD 8 and anti-CD 62L antibodies.

In some embodiments, the centering memory T (T _CM ) Enrichment of cells is based on positive or high surface expression of CD45RO, CD62L, CCR, CD28, CD3 and/or CD 127; in some aspects, it is based on negative selection of cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, is rich in T _CM CD8 of cells ⁺ Group of peopleBy depleting cells expressing CD4, CD14, CD45RA and positive selection or enrichment of cells expressing CD 62L. In one aspect, the central memory T (T _CM ) Enrichment of cells starts with a negative cell fraction selected based on CD4 expression, which negative cell fraction is selected negatively based on CD14 and CD45RA expression and positive based on CD 62L. In some aspects this selection is made simultaneously, while in other aspects it is made sequentially in either order. In some aspects, will be used to make CD8 ⁺ The same selection procedure based on CD4 expression of cell populations or subpopulations is also used to generate CD4 ⁺ A population or subpopulation of cells such that both positive and negative fractions from CD4 based isolation are retained and used in subsequent steps of the method, optionally after one or more other positive or negative selection steps.

In particular examples, a PBMC sample or other leukocyte sample is subjected to CD4 ⁺ Cell selection, wherein both negative and positive fractions are retained. The negative fractions are then negative for the expression of CD14 and CD45RA or CD19 and positive for the characteristic marker of central memory T cells (e.g. CD62L or CCR 7), wherein the positive and negative selections are performed in any order.

CD4 is detected by identifying a population of cells having a cell surface antigen ⁺ T helper cells are classified as naive, central memory and effector cells. CD4 ⁺ Lymphocytes can be obtained by standard methods. In some embodiments, naive CD4 ⁺ T lymphocytes are CD45RO ^- 、CD45RA ⁺ 、CD62L ⁺ 、CD4 ⁺ T cells. In some embodiments, the central memory is CD4 ⁺ The cells were CD62L ⁺ And CD45RO ⁺ . In some embodiments, the effect is CD4 ⁺ The cells were CD62L ^- And CD45RO ^- 。

In one example, to enrich for CD4 by negative selection ⁺ The mixture of cells, monoclonal antibodies typically includes antibodies directed against CD14, CD20, CD11b, CD16, HLA-DR, and CD 8. In some embodiments, the antibody or binding partner is bound to a solid support orA matrix (e.g., magnetic or paramagnetic beads) to allow separation of cells for positive and/or negative selection. For example, in some embodiments immunomagnetic (or affinity magnetic) separation techniques are used to separate or isolate cells and cell populations (reviewed in Methods in Molecular Medicine, volume 58: metastasis Research Protocols, volume 2: cell Behavior In Vitro and In Vivo, pages 17-25 S.A.Brooks and U.S. Schumacher edition) Humana Press Inc.,Totowa,NJ)。

In some aspects, a sample or composition of cells to be isolated is incubated with a small magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., like Dynalbeads or MACS beads). The magnetically responsive material (e.g., particles) is typically directly or indirectly attached to a binding partner (e.g., an antibody) that specifically binds to a molecule (e.g., a surface label) present on a cell, cells, or cell population that is desired to be isolated (e.g., desired to be selected negatively or positively).

In some embodiments, the magnetic particles or beads comprise magnetically responsive material that binds to a specific binding member (e.g., an antibody or other binding partner). There are many well known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773 and european patent specification EP 452342B, which are hereby incorporated by reference. Colloidal sized particles (such as those described in Owen U.S. patent No. 4,795,698 and Liberti et al, U.S. patent No. 5,200,084) are other examples.

Incubation is typically performed under conditions whereby antibodies or binding partners attached to magnetic particles or beads or molecules that specifically bind such antibodies or binding partners (e.g., secondary antibodies or other reagents) specifically bind to cell surface molecules (if present on cells within the sample).

In some aspects, the sample is placed in a magnetic field and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from unlabeled cells. For positive selection, cells attracted by the magnet are retained; for negative selection, cells that were not attracted (unlabeled cells) were retained. In some aspects, a combination of positive and negative selections is performed during the same selection step, wherein the positive and negative fractions are retained and further processed or subjected to additional separation steps.

In certain embodiments, the magnetically responsive particles are coated in a primary or other binding partner, secondary antibody, lectin, enzyme or streptavidin. In certain embodiments, the magnetic particles are attached to the cells via a coating of a primary antibody specific for one or more markers. In certain embodiments, cells are labeled with a primary antibody or binding partner instead of beads, and then magnetic particles coated with a cell type specific secondary antibody or other binding partner (e.g., streptavidin) are added. In certain embodiments, streptavidin-coated magnetic particles are used in combination with biotinylated primary or secondary antibodies.

In some embodiments, the magnetically responsive particles remain attached to cells that are subsequently incubated, cultured, and/or engineered; in some aspects, the particles remain attached to cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods of removing magnetizable particles from cells are known and include, for example, the use of competitive non-labeled antibodies and magnetizable particles or antibodies conjugated with cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

In some embodiments, affinity-based selection is via Magnetic Activated Cell Sorting (MACS) (Miltenyi Biotec, obu, california). Magnetically Activated Cell Sorting (MACS) systems are capable of selecting cells with magnetized particles attached thereto in high purity. In certain embodiments, MACS operates in a mode in which non-target and target species are eluted sequentially after application of an external magnetic field. That is, cells attached to the magnetized particles remain in place, while unattached species are eluted. Then, after the first elution step is completed, the species that are trapped in the magnetic field and prevented from eluting are released in some way so that they can be eluted and recovered. In certain embodiments, non-target cells are labeled and depleted from a heterogeneous cell population.

In certain embodiments, the separation or isolation is performed using a system, apparatus or device that performs one or more of the separation, cell preparation, separation, treatment, incubation, culture and/or preparation steps of the method. In some aspects, the system is used to perform each of these steps in a closed or sterile environment, for example, to minimize errors, user manipulation, and/or contamination. In one example, the system is a system as described in International patent application publication No. WO 2009/072003 or US20110003380A 1.

In some embodiments, the system or apparatus performs one or more (e.g., all) of the separating, processing, engineering, and formulating steps in an integrated or stand-alone system and/or in an automated or programmable manner. In some aspects, a system or apparatus includes a computer and/or computer program in communication with the system or apparatus that allows a user to program, control, assess and/or adjust various aspects of the processing, separation, engineering and formulation steps.

In some aspects, the separation and/or other steps are performed using a clinimmacs system (Miltenyi Biotec), for example, for automatically separating cells at the clinical scale level in closed and sterile systems. The components may include an integrated microcomputer, a magnetic separation unit, a peristaltic pump, and various pinch valves. In some aspects, the integrated computer controls all components of the instrument and instructs the system to perform the iterative procedure in a standardized order. In some aspects, the magnetic separation unit includes a movable permanent magnet and a bracket for selecting the column. Peristaltic pumps control the flow rate of the whole tube set and, together with pinch valves, ensure a controlled flow of buffer through the system and a continuous suspension of cells.

In some aspects, the clinic macs system uses antibody-coupled magnetizable particles supplied in a sterile pyrogen-free solution. In some embodiments, after labeling the cells with magnetic particles, the cells are washed to remove excess particles. The cell preparation bag is then connected to a tube set, which in turn is connected to a bag containing buffer and a cell collection bag. The tube set consists of pre-assembled sterile tubes (including pre-columns and separation columns) and is intended for single use only. After the separation procedure is initiated, the system automatically applies the cell sample to the separation column. The labeled cells remain in the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the population of cells for use with the methods described herein is unlabeled and does not remain in the column. In some embodiments, a population of cells for use with the methods described herein is labeled and retained in a column. In some embodiments, the population of cells for use with the methods described herein elutes from the column after removal of the magnetic field and is collected within a cell collection bag.

In certain embodiments, the isolation and/or other steps are performed using a CliniMACS Prodigy system (Miltenyi Biotec). In some aspects, the CliniMACS Prodigy system is equipped with a cell handling unit that allows for automatic washing and fractionation of cells by centrifugation. CliniMACS Prodigy system may also include an on-board camera and image recognition software that determines the optimal cell fractionation endpoint by discriminating the macroscopic layers of the source cell product. For example, peripheral blood automatically separates into red blood cells, white blood cells, and plasma layers. CliniMACS Prodigy systems may also include integrated cell culture chambers that enable cell culture protocols such as cell differentiation and expansion, antigen loading, and long-term cell culture. The input port may allow sterile removal and replenishment of the culture medium, and the cells may be monitored using an integrated microscope. See, e.g., klebaroff et al (2012) J Immunother.35 (9): 651-660; terakura et al (2012) blood.1:72-82; wang et al (2012) J Immunother.35 (9): 689-701.

In some embodiments, the population of cells described herein is collected and enriched (or depleted) via flow cytometry, wherein the fluid stream carries cells stained for a plurality of cell surface markers. 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) by using microelectromechanical systems (MEMS) chips in combination with FACS-based detection systems (see, e.g., WO 2010/033140; cho et al (2010) Lab Chip 10,1567-1573; and Godin et al (2008) J Biophoton.1 (5): 355-376. In both cases, cells can be labeled with a variety of labels, allowing for the isolation of well-defined subsets of T cells in high purity.

In some embodiments, the antibody or binding partner is labeled with one or more detectable labels to facilitate isolation for positive and/or negative selection. For example, the separation may be based on binding to a fluorescently labeled antibody. In some examples, the cells are isolated for carrying in a fluid stream based on binding of antibodies or other binding partners specific for one or more cell surface markers, such as by Fluorescence Activated Cell Sorting (FACS) (including manufacturing scale (FACS)) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow cell detection system. Such methods allow positive and negative selection based on multiple markers simultaneously.

In some embodiments, the method of preparation comprises the step of freezing (e.g., cryopreserving) the cells before or after isolation, incubation, and/or engineering. In some embodiments, the freezing and subsequent thawing steps remove granulocytes and, to some extent, monocytes from the cell population. In some embodiments, the cells are suspended in a chilled solution to remove plasma and platelets, for example, after a washing step. In some aspects, any of a variety of known freezing solutions and parameters may be used. One example involves the use of PBS containing 20% DMSO and 8% Human Serum Albumin (HSA), or other suitable cell freezing medium. It was then diluted 1:1 with medium such that the final concentrations of DMSO and HSA were 10% and 4%, respectively. The cells are then typically frozen to-80 ℃ at a rate of 1 ℃/min and stored in the gas phase of a liquid nitrogen storage tank.

In some embodiments, the cells are incubated and/or cultured prior to or in conjunction with genetic engineering. The incubation step may include culturing, incubating, stimulating, activating, and/or propagating. Incubation and/or engineering may be performed in culture vessels such as units, chambers, wells, columns, tubes, tube sets, valves, vials, culture dishes, bags, or other vessels used to culture or incubate cells. In some embodiments, the composition or cell is incubated in the presence of a stimulating condition or a stimulating agent. These conditions include those designed for: conditions for inducing proliferation, expansion, activation and/or survival of cells in a population, for mimicking antigen exposure and/or for priming cells for genetic engineering, such as to introduce recombinant antigen receptors.

The conditions may include one or more of the following: specific media, temperature, oxygen content, carbon dioxide content, time, agents (e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors (e.g., cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents intended to activate cells)).

In some embodiments, the stimulation conditions or agents include one or more agents (e.g., ligands) capable of activating or stimulating the intracellular signaling domain of the TCR complex. In some aspects, the agent initiates or initiates a TCR/CD3 intracellular signaling cascade in the T cell. Such agents may include antibodies, such as antibodies specific for TCRs, e.g., anti-CD 3 antibodies. In some embodiments, the stimulation conditions include one or more agents, such as ligands, capable of stimulating a co-stimulatory receptor, such as anti-CD 28. In some embodiments, such agents and/or ligands may be bound to a solid support (e.g., a bead) and/or one or more cytokines. Optionally, the amplification method may further comprise the step of adding anti-CD 3 and/or anti-CD 28 antibodies to the medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulatory agent includes IL-2, IL-15 and/or IL-7. In some aspects, the IL-2 concentration is at least about 10 units/mL.

In some aspects, incubation is performed according to a variety of techniques, such as those described in the following documents: U.S. patent No. 6,040,177 to Riddell et al; klebaroff et al (2012) J Immunother.35 (9): 651-660; terakura et al (2012) blood.1:72-82; and/or Wang et al (2012) J Immunother35 (9): 689-701.

In some embodiments, T cells are expanded by: adding feeder cells (such as non-dividing Peripheral Blood Mononuclear Cells (PBMCs)) to the culture starting composition (e.g., such that for each T lymphocyte in the initial population to be expanded, the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells); and incubating the culture (e.g., for a time sufficient to expand the number of T cells). In some aspects, the non-dividing feeder cells may comprise gamma irradiated PBMC feeder cells. In some embodiments, PBMCs are irradiated with gamma rays ranging from about 3000 to 3600 rads to prevent cell division. In some aspects, feeder cells are added to the medium prior to the addition of the T cell population.

In some embodiments, the stimulation conditions include a temperature suitable for growth of human T lymphocytes, e.g., at least about 25 degrees celsius, typically at least about 30 degrees celsius, and typically at or about 37 degrees celsius. Optionally, the incubating may further comprise adding non-dividing EBV-transformed Lymphoblastoid Cells (LCLs) as feeder cells. The LCL may be irradiated with gamma rays in the range of about 6000 to 10,000 rads. In some aspects, the LCL feeder cells are provided in any suitable amount, such as a ratio of LCL feeder cells to naive T lymphocytes of at least about 10:1.

In embodiments, 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 infected subject and stimulating the cells in vitro with the same antigen.

Exemplary treatment outcomes and methods of assessing same

In some embodiments of the methods, compositions, combinations, uses, kits and articles of manufacture provided herein, the provided combination therapies produce one or more therapeutic outcomes, as characterized by any one or more of the parameters associated with the therapy or treatment, as described below. In some embodiments, the method is any one as described in section I. In some embodiments, the methods further comprise an assessment of exposure, persistence, and proliferation of T cells (e.g., T cells administered in a T cell-based therapy). In some embodiments, in the methods provided herein, exposure, or prolonged expansion and/or persistence of a cell (e.g., a cell administered by immunotherapy (e.g., T cell therapy), and/or a change in the cellular phenotype or functional activity of the cell can be measured by assessing a characteristic of a T cell in vitro or ex vivo. In some embodiments, such assays can be used to determine or confirm the function of T cells (e.g., T cell therapies) prior to, during, or after administration of the combination therapies provided herein.

In some embodiments, the combination therapy may further comprise one or more screening steps for identifying subjects treated with the combination therapy and/or continuing the combination therapy, and/or steps for assessing treatment outcome and/or monitoring treatment outcome. In some embodiments, the step for assessing the outcome of a treatment may comprise a plurality of steps to assess and/or monitor the treatment and/or identify further or remaining steps of the subject for administration of the therapy and/or for repeat therapy. In some embodiments, the screening step and/or evaluation of treatment outcome may be used to determine the dose, frequency, duration, timing, and/or order of combination therapies provided herein.

In some embodiments, any of the screening steps and/or assessments of treatment outcome described herein can be used prior to, during, or after administration of one or more steps of the provided combination therapy (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 any of the methods provided herein. In some embodiments, the evaluation is performed prior to performing the methods provided herein. In some embodiments, the evaluation is performed after performing one or more steps of the methods provided herein. In some embodiments, the evaluation is performed prior to one or more steps of administering the provided combination therapies, e.g., to screen and identify patients suitable for receiving and/or sensitive to the combination therapies. In some embodiments, the evaluation is performed during, or after one or more steps of administering the provided combination therapies, e.g., to evaluate intermediate or final treatment outcome, e.g., to determine treatment efficacy and/or to determine whether to continue or repeat the treatment and/or to determine whether to administer the remaining steps of the combination therapies.

In some embodiments, the therapeutic outcome includes improved immune function, such as immune function of T cells administered by cell-based therapies and/or endogenous T cells in vivo. In some embodiments, exemplary therapeutic outcomes include, but are not limited to, enhanced T cell proliferation, enhanced T cell functional activity, changes in immune cell phenotype marker expression, such as such features associated with engineered T cells (e.g., CAR-T cells) administered to a subject. In some embodiments, exemplary therapeutic outcomes include reduced disease burden (e.g., tumor burden), improved clinical outcomes, and/or enhanced therapeutic efficacy.

In some embodiments, the assessment of the screening step and/or treatment outcome comprises assessing survival and/or function of T cells administered with the cell-based therapy. In some embodiments, the screening step and/or the assessment of treatment outcome comprises assessing the level of a cytokine or growth factor. In some embodiments, the assessment of screening steps and/or treatment outcome comprises assessing disease burden and/or improvement, e.g., assessing tumor burden and/or clinical outcome. In some embodiments, any of the screening steps and/or evaluation of treatment outcome may include any of the evaluation methods and/or assays described herein and/or known in the art, and may be performed one or more times, e.g., prior to, during, or after the administration of one or more steps of the combination therapy. Exemplary parameter sets related to treatment outcome that may be assessed in some embodiments of the methods provided herein include peripheral blood immune cell population profile and/or tumor burden.

In some embodiments, the method affects the efficacy of a cell therapy in a subject. In some embodiments, the persistence, expansion, and/or presence of cells expressing a recombinant receptor (e.g., CAR) in the subject is greater after administration of the cell dose in the method and compound C than is achieved by a method that does not administer compound C. In some embodiments, the expansion and/or persistence of the administered T cell therapy (e.g., CAR expressing T cells) in the subject is assessed as compared to a method in which the T cell therapy is administered to the subject in the absence of compound C. In some embodiments, the method results in the administered T cells exhibiting increased or prolonged expansion and/or persistence in the subject, as compared to a method in which the T cell therapy is administered to the subject in the absence of compound C.

In some embodiments, administration of compound C reduces disease burden (e.g., tumor burden) in the subject as compared to a method in which a dose of cells expressing the recombinant receptor is administered to the subject in the absence of compound C. In some embodiments, administration of compound C reduces embryo marrow (blast marrow) in the subject as compared to a method in which a dose of cells expressing the recombinant receptor is administered to the subject in the absence of compound C. In some embodiments, administration of compound C results in improved clinical outcomes, e.g., objective Response Rate (ORR), progression Free Survival (PFS), and total survival (OS), compared to methods in which a dose of cells expressing a recombinant receptor is administered to a subject in the absence of compound C.

In some embodiments, the subject may be screened prior to one or more steps of administering the combination therapy. For example, the subject may be screened for a disease and/or a characteristic of a disease burden (e.g., tumor burden) prior to administration of the combination therapy to determine suitability, responsiveness, and/or susceptibility to administration of the combination therapy. In some embodiments, the screening step and/or evaluation of treatment outcome may be used to determine the dose, frequency, duration, timing, and/or order of combination therapies provided herein.

In some embodiments, subjects may be screened after one step of administering the combination therapy to determine and identify subjects who received the remaining steps of the combination therapy and/or monitor the efficacy of the therapy. In some embodiments, the number, level or amount of T cells administered and/or proliferation and/or activity of T cells administered is assessed prior to and/or after administration of compound C.

In some embodiments, a change and/or a change (e.g., an increase, a decrease, or a decrease) in a parameter or outcome as compared to a level, value, or measurement of the same parameter or outcome at a different evaluation time point, a different condition, a reference point, and/or a different subject is determined or evaluated. For example, in some embodiments, a fold change (e.g., an increase or decrease) in a particular parameter (e.g., the number of engineered T cells in a sample) can be determined as compared to the same parameter in a different condition (e.g., prior to administration of compound C). In some embodiments, the level, value or measurement of two or more parameters is determined and the relative levels are compared. In some embodiments, the level, value, or measurement of the determined parameter is compared to a level, value, or measurement from a control sample or untreated sample. In some embodiments, the level, value or measurement of the determined parameter is compared to the level of a sample from the same subject at a different point in time. The values obtained in the quantification of the individual parameters may be combined for disease assessment purposes, for example by using multiparameter analysis to form arithmetic or logical operations on the level, value or measured value of the parameter. In some embodiments, the ratio of two or more specific parameters may be calculated.

A.T cell exposure, persistence and proliferation

In some embodiments, the parameters associated with the therapy or treatment outcome (which include parameters that may be evaluated for the screening step and/or to evaluate the treatment outcome and/or to monitor the treatment outcome) are or include an assessment of exposure, persistence, and proliferation of T cells (e.g., T cells administered in a T cell-based therapy). In some embodiments, increased exposure or prolonged expansion and/or persistence of cells, and/or changes in cellular phenotype or functional activity of cells, e.g., cells administered by immunotherapy (e.g., T cell therapy), in a method provided herein can be measured by assessing characteristics of T cells in vitro or ex vivo. In some embodiments, such assays may be used to determine or confirm the function of T cells for immunotherapy (e.g., T cell therapy) before or after one or more steps of the combination therapies provided herein.

In some embodiments, administration of compound C is designed to promote exposure of the subject to cells (e.g., T cells administered in a T cell-based therapy), such as by promoting expansion and/or persistence of the cells over time. In some embodiments, the T cell therapy exhibits increased or prolonged expansion and/or persistence in the subject as compared to a method in which the T cell therapy is administered to the subject in the absence of compound C.

In some embodiments, the provided methods increase the subject's exposure to the administered cells (e.g., increased cell number or duration over time) and/or improve the efficacy and therapeutic outcome of immunotherapy (e.g., T cell therapy). In some aspects, the methods are advantageous in that greater and/or longer exposure of cells expressing recombinant receptors (e.g., CAR-expressing cells) improves treatment outcome as compared to other methods. Such outcomes may include survival and remission of the patient even in individuals with severe tumor burden.

In some embodiments, administration of compound C can increase the maximum, total, and/or duration of exposure to the cells (e.g., T cells administered for T cell-based therapy) in a subject, as compared to administration of T cells alone in the absence of compound C. In some aspects, administration of compound C enhances efficacy in the case of a high disease load (and thus higher amounts of antigen) and/or a more invasive or resistant B cell malignancy, as compared to administration of only T cells in the same case in the absence of compound C (potentially resulting in immunosuppression, disability, and/or depletion, and thus potentially preventing expansion and/or persistence of the cells).

In some embodiments, the presence and/or amount of cells expressing a recombinant receptor (e.g., CAR-expressing cells administered by T cell-based therapy) in the subject is detected after administration of the T cells and before, during, and/or after administration of compound C. In some aspects, quantitative PCR (qPCR) is used to assess the amount of cells expressing recombinant receptors (e.g., CAR-expressing cells administered in T cell-based therapies) in a blood or serum or organ or tissue sample (e.g., a disease site, such as a tumor sample) of a subject. In some aspects, persistence is quantified as a copy of the DNA or plasmid encoding the receptor (e.g., CAR) per microgram of DNA (e.g., total DNA obtained from the sample), or as the number of receptor expressing cells (e.g., CAR expressing cells) per microliter of sample (e.g., blood or serum) or per microliter of total number of Peripheral Blood Mononuclear Cells (PBMCs) or leukocytes or T cells in the sample.

In some embodiments, the cells are detected in the subject at or at least 4, 7, 10, 14, 18, 21, 24, 27, or 28 days after administration of the T cells (e.g., CAR-expressing T cells). In some aspects, the cells are detected at or at least 2, 4, or 6 weeks after administration of the T cells, or 3, 6, or 12, 18, or 24, or 30 or 36 months, or 1, 2, 3, 4, 5 years or more after administration.

In some embodiments, the persistence of the receptor-expressing cells (e.g., CAR-expressing cells) in the subject is greater after administration of the T cells (e.g., CAR-expressing T cells) and/or compound C by an alternative method, such as those involving administration of only immunotherapy (e.g., administration of T cells (e.g., CAR-expressing T cells)) in the absence of compound C, as achieved by the method.

Exposure (e.g., number) of cells (e.g., T cells administered by T cell therapy) indicative of expansion and/or persistence may be stated in terms of: the maximum number of cells exposed to the subject, the duration of detectable cells or cells above a certain number or percentage, the area under the curve of the number of cells versus time, and/or combinations thereof, and indicators thereof. Such outcomes can be assessed using known methods, such as qPCR, to detect the copy number of nucleic acid encoding a recombinant receptor as compared to the total amount of nucleic acid or DNA in a particular sample (e.g., blood, serum, plasma, or tissue (e.g., tumor sample)). And/or flow cytometry assays, which typically use antibodies specific for a receptor to detect cells expressing the receptor. Cell-based assays can also be used to detect the number or percentage of functional cells, such as cells that can bind to and/or neutralize a disease or disorder or cells that express an antigen recognized by a receptor and/or cells that can induce a response (e.g., a cytotoxic response) against the cells.

In some aspects, increased exposure of the subject to the cells comprises increased cell expansion. In some embodiments, the receptor-expressing cells (e.g., CAR-expressing cells) are expanded in the subject after administration of the T cells (e.g., CAR-expressing T cells) and/or after administration of compound C. In some aspects, the method results in greater expansion of the cells as compared to other methods, such as those involving administration of T cells (e.g., CAR expressing T cells) in the absence of administration of compound C.

In some aspects, the methods result in high in vivo proliferation of the administered cells, e.g., as measured by flow cytometry. In some aspects, the cells are detected for a high peak ratio. For example, in some embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells express a recombinant receptor (e.g., CAR) at a peak or maximum level following administration of T cells (e.g., CAR-expressing T cells) and/or compound C in a blood or disease site of a subject or a leukocyte fraction thereof (e.g., PBMC fraction or T cell fraction).

In some embodiments, the method results in a maximum concentration of at least 100, 500, 1000, 1500, 2000, 5000, 10,000, or 15,000 copies of nucleic acid encoding a receptor (e.g., CAR) per microgram of DNA in the blood or serum or other bodily fluid or organ or tissue of the subject, or at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 receptor expressing cells (e.g., CAR expressing cells) in the total number of Peripheral Blood Mononuclear Cells (PBMCs), the total number of mononuclear cells, the total number of T cells, or the total microliter. In some embodiments, the cells expressing the receptor are detected as at least 10%, 20%, 30%, 40%, 50% or 60% of total PBMCs in the blood of the subject, and/or at this level for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48 or 52 weeks after administration of the T cells (e.g., CAR expressing T cells) and/or compound C, or for 1, 2, 3, 4 or 5 years or more after such administration.

In some aspects, the method results in at least a 2-fold, at least a 4-fold, at least a 10-fold, or at least a 20-fold increase in the copy number of nucleic acid encoding a recombinant receptor (e.g., CAR) per microgram of DNA in, for example, serum, plasma, blood, or tissue (e.g., tumor sample) of a subject.

In some embodiments, the receptor-expressing cells are detectable in the serum, plasma, blood, or tissue of the subject (e.g., a tumor sample) for at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days or more after administration of the T cells (e.g., CAR-expressing T cells) or after administration of the compound C (e.g., by a specified method such as qPCR or a flow cytometry-based detection method), which lasts at least or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks or more after administration of the T cells (e.g., CAR-expressing T cells) and/or compound C.

In some aspects, at least about 1x 10 can be detected or present in a subject or body fluid, plasma, serum, tissue, or compartment thereof, such as in blood (e.g., peripheral blood) or a disease site (e.g., tumor) thereof ² At least about 1x 10 ³ At least about 1x 10 ⁴ At least about 1x 10 ⁵ Or at least about 1x 10 ⁶ Or at least about 5x 10 ⁶ Or at least about 1x 10 ⁷ Or at least about 5x 10 ⁷ Or at least about 1x 10 ⁸ And/or at least 10, 25, 50, 100, 200, 300, 400 or 500 or 1000 receptor expressing cells per microliter (e.g., CAR expressing cells), e.g., at least 10 of the cells per microliter. In some embodiments, such number or concentration of cells can be detected in the subject after administration of T cells (e.g., CAR-expressing T cells) and/or after administration of compound C for at least about 20 days, at least about 40 days, or at least about 60 days, or at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 2 or 3 years. Such cell numbers can be detected as by flow cytometry-based or quantitative PCR-based methods and extrapolated to total cell numbers using known methods. See, e.g., brentjens et al, sci TranslMed.2013 (177); park et al Molecular Therapy 15 (4): 825-833 (2007); savoldo et al, JCI121 (5): 1822-1826 (2011); davila et al, (2013) PLoS ONE 8 (4): e61338; davila et al, oncoimmunology 1 (9): 1577-1583 (2012); lamers, blood 2011 117:72-82; jensen et al, biolBlood Marrow Transplant, 9, 2010; 16 (9) 1245-1256; brentjens et al, blood 201118 (18): 4817-4818.

In some aspects, the copy number (e.g., vector copy number) of the nucleic acid encoding the recombinant receptor per 100 cells is at least 0.01, at least 0.1, at least 1, or at least 10, e.g., in the peripheral blood or bone marrow or other compartment, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or at least about 6 weeks, or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 2 or 3 years after administration of the cells (e.g., CAR-expressing T cells) and/or compound C, as measured by immunohistochemistry, PCR, and/or flow cytometry. In some embodiments, the copy number of the vector expressing the receptor (e.g., CAR) per microgram of genomic DNA is at least 100, at least 1000, at least 5000, or at least 10,000, or at least 15,000, or at least 20,000 at about 1 week, about 2 weeks, about 3 weeks, or at least about 4 weeks after administration of a T cell (e.g., CAR expressing T cell) or compound C, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 2 or 3 years after such administration.

In some aspects, the receptor (e.g., CAR) expressed by the cell is detectable in the subject, its plasma, serum, blood, tissue, and/or diseased site (e.g., tumor site) by quantitative PCR (qPCR) or by flow cytometry, at least about 3 months, at least about 6 months, at least about 12 months, at least about 1 year, at least about 2 years, at least about 3 years, or for a period of time exceeding 3 years after administration of the cell (e.g., after initiation of administration of the T cell (e.g., CAR-expressing T cell)) and/or after administration of compound C.

In some embodiments, the area under the curve (AUC) of the concentration of the receptor (e.g., CAR) expressing cells in the body fluid, plasma, serum, blood, tissue, organ, and/or diseased site (e.g., tumor site) of the subject is greater after administration of the T cells (e.g., CAR expressing T cells) and/or compound C than achieved by an alternative dosing regimen in which the T cells (e.g., CAR expressing T cells) are administered to the subject in the absence of administration of compound C.

In some aspects, the methods result in high in vivo proliferation of the administered cells, e.g., as measured by flow cytometry. In some aspects, the cells are detected for a high peak ratio. For example, in some embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells express a recombinant receptor (e.g., CAR) at a peak or maximum level following administration of T cells (e.g., CAR-expressing T cells) and/or compound C in the subject's blood, plasma, serum, tissue, or disease site, or a leukocyte fraction thereof (e.g., PBMC fraction or T cell fraction).

In some aspects, increased or prolonged expansion and/or persistence of the dose of cells in a subject administered compound C is associated with the benefit of a tumor-associated outcome in the subject. In some embodiments, the tumor-associated outcome comprises a decrease in tumor burden or a decrease in embryo marrow in the subject. In some embodiments, the tumor burden is reduced or reduced by at least or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% after administration of the method. In some embodiments, after the cell dose, the disease burden, tumor size, tumor volume, tumor mass, and/or tumor burden or volume is reduced by at least or at least about 50%, 60%, 70%, 80%, 90% or more compared to a subject that has been treated with a method that does not involve administration of compound C.

B.T cell functional Activity

In some embodiments, the parameters associated with the therapy or treatment outcome (which include parameters that may be evaluated for the screening step and/or to evaluate the treatment outcome and/or monitor the treatment outcome) include one or more of T cell activity, phenotype, proliferation, or function. In some embodiments, any assay known in the art for assessing the activity, phenotype, proliferation, and/or function of a T cell (e.g., a T cell administered by a T cell therapy) may be used. The biological activity of the engineered cell population is measured in some embodiments, e.g., by any of a variety of known methods, before and/or after administration of the cells and/or compound C. Parameters to be assessed include specific binding of engineered or natural T cells or other immune cells to an antigen, which is assessed in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of an engineered cell to destroy a target cell can be measured using any suitable method known, such as, for example, cytotoxicity assays described in the following documents: kochenderfer et al, J.Immunotherapy,32 (7): 689-702 (2009); and Herman et al, J.Immunologic Methods,285 (1): 25-40 (2004).

In some embodiments, T cells, such as recombinantly expressed (e.g., CAR) T cells, can be evaluated before and/or after administration of the cells and/or compound C to assess or determine whether the T cells exhibit the characteristics of depletion. In some cases, depletion can be assessed by monitoring loss of T cell function, such as reduced or reduced antigen-specific or antigen receptor-driving activity, such as reduced or reduced ability to produce cytokines or drive cytolytic activity against a target antigen. In some cases, depletion can also be assessed by monitoring expression of surface markers associated with the depletion phenotype on T cells (e.g., CD4 and/or CD4T cells). The depletion markers include inhibitory receptors such as PD-1, CTLA-4, LAG-3 and TIM-3.

In some embodiments, such reduced or decreased activity is observed over time after administration to a subject and/or after prolonged exposure to an antigen.

In particular embodiments, provided methods (i) achieve the increase in antigen specificity or antigen receptor driving activity, and (ii) prevent, inhibit or delay the onset of and/or reverse the depletion phenotype. In some embodiments, the amount, duration, and/or frequency is effective to (i) achieve the increase in antigen specificity or antigen receptor driving activity, and (ii) prevent, inhibit, or delay the onset of the depletion phenotype. In other embodiments, the amount, duration, and/or frequency is effective to (i) achieve the increase in antigen specificity or antigen receptor driving activity, and (ii) prevent, inhibit, or delay the onset of, and reverse the depletion phenotype.

In some embodiments, the depletion phenotype for a T cell or population of T cells comprises: an increase in the level or extent of surface expression of one or more depletion markers, optionally 2, 3, 4, 5 or 6 depletion markers, or an increase in the percentage of the population of T cells exhibiting said surface expression, over a reference population of T cells under the same conditions; or a decrease in the level or extent of activity exhibited by said T cell or population of T cells upon exposure to an antigen or antigen receptor specific agent as compared to a reference population of T cells under the same conditions; an increase in the level or extent of surface expression of one or more depletion markers, optionally 2, 3, 4, 5 or 6 depletion markers, or an increase in the percentage of the population of T cells exhibiting said surface expression, over a reference population of T cells under the same conditions; or a reduction in the level or extent of activity exhibited by the T cell or T cell population when exposed to an antigen or antigen receptor specific agent as compared to a reference T cell population under the same conditions.

In certain embodiments, the biological activity of a cell is measured by assessing the expression and/or secretion of one or more cytokines (e.g., CD107a, IFNγ, IL-2, GM-CSF, and TNF α) and/or by assessing cytolytic activity.

In some embodiments, assays for activity, phenotype, proliferation, and/or function of T cells (e.g., T cells administered by T cell therapy) include, but are not limited to ELISPOT, ELISA, cell proliferation, cytotoxic lymphocyte (CTL) assays, binding to T cell epitopes, antigens or ligands, or intracellular cytokine staining, proliferation assays, lymphokine secretion assays, direct cytotoxicity assays, and limiting dilution assays. In some embodiments, the proliferative response of T cells may be measured, for example, by combining ³ H-thymidine, brdU (5-bromo-2 '-deoxyuridine) or 2' -deoxy-5-ethynyluridine (EdU) is incorporated into the DNA of T cells or measured using dye dilution of dyes such as carboxyfluorescein diacetate succinimidyl ester (CFSE), cellTrace Violet or Membrane dye PKH 26.

In some embodiments, assessing the activity, phenotype, proliferation, and/or function of a T cell (e.g., a T cell administered by a T cell therapy) comprises measuring cytokine production by the T cell, and/or measuring cytokine production in a biological sample (e.g., plasma, serum, blood, and/or tissue sample, e.g., a tumor sample) from a subject. In some cases, such measured cytokines may include, but are not limited to, interleukin-2 (IL-2), interferon-gamma (IFNgamma), interleukin-4 (IL-4), TNF-alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), CD107a, and/or TGF-beta (TGF beta). Assays for measuring cytokines are well known and include, but are not limited to, ELISA, intracellular cytokine staining, flow microbead arrays, RT-PCR, ELISPOT, flow cytometry, and bioassays in which the reactivity (e.g., proliferation) of cells that are responsive to the relevant cytokines are tested in the presence of a test sample.

In some embodiments, assessing the activity, phenotype, proliferation, and/or function of a T cell (e.g., a T cell administered by a T cell therapy) comprises assessing a cell phenotype, e.g., expression of a particular cell surface marker. In some embodiments, expression of a T cell activation marker, a T cell depletion marker, and/or a T cell differentiation marker in a T cell (e.g., a T cell administered by a T cell therapy) is assessed. In some embodiments, the phenotype of the cells is assessed prior to administration. In some embodiments, the cell phenotype is assessed during or after administration of the cell therapy and/or compound C. The T cell activation markers, T cell depletion markers and/or T cell differentiation markers used in the assessment include any markers known for a particular subset of T cells, such as CD25, CD38, human leukocyte antigen-DR (HLA-DR), CD69, CD44, CD137, KLRG1, CD62L ^{Low and low} 、CCR7 ^{Low and low} CD71, CD2, CD54, CD58, CD244, CD160, programmed cell death protein 1 (PD-1), lymphocyte activation gene 3 protein (LAG-3), T cell immunoglobulin domain and mucin domain protein 3 (TIM-3), cytotoxic T lymphocyte antigen-4 (CTLA-4), B and T lymphocyte attenuation factor (BTLA) and/or T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) (see, e.g., liu et al, cell Death and Disease (2015) 6, e 1792). In some embodiments, the depletion marker is any one or more of the following: PD-1, CTLA-4, TIM-3, LAG-3, BTLA, 2B4, CD160, CD39, VISTA and TIGIT. In some embodiments, the cell surface markers assessed are CD25, PD-1 and/or TIM-3. In some embodiments, the cell surface marker assessed is CD25.

In some aspects, detecting the expression level comprises performing an in vitro assay. In some embodiments, the in vitro assay is an immunoassay, an aptamer-based assay, a histological or cytological assay, or an mRNA expression level assay. In some embodiments, the one or more parameters of each of the one or more factors, effectors, enzymes, and/or surface markers are detected by an enzyme-linked immunosorbent assay (ELISA), immunoblot, immunoprecipitation, radioimmunoassay (RIA), immunostaining, flow cytometry assay, surface Plasmon Resonance (SPR), chemiluminescent assay, lateral flow immunoassay, inhibition assay, or affinity assay. In some embodiments, detection of the cytokine and/or surface marker is determined using a binding reagent that specifically binds to at least one biomarker. In some cases, the binding agent is an antibody or antigen binding fragment thereof, an aptamer, or a nucleic acid probe.

In some embodiments, administration of compound C increases the level of circulating CAR T cells.

C. Reaction, efficacy and survival

In some embodiments, the parameters associated with the therapy or treatment outcome (which include parameters that may be evaluated for the screening step and/or to evaluate the treatment outcome and/or monitor the treatment outcome) include tumor or disease burden. Administration of immunotherapy, such as T cell therapy (e.g., CAR expressing T cells) and/or compound C, can reduce or prevent the expansion or burden of a disease or disorder in a subject. For example, where the disease or condition is a tumor, the method generally reduces tumor size, volume, metastasis, percentage of primary cells in bone marrow or molecularly detectable B-cell malignancy, and/or improves prognosis or survival or other symptoms associated with tumor burden.

In some aspects, the enlargement or burden of the disease or disorder in the subject is generally reduced or prevented according to the provided methods and/or with administration of the provided articles or compositions. For example, where the disease or condition is a tumor, the method generally reduces tumor size, volume, metastasis, percentage of primary cells in bone marrow or molecularly detectable B-cell malignancy, and/or improves prognosis or survival or other symptoms associated with tumor burden.

In some embodiments, the provided methods result in reduced tumor burden in the treated subject compared to an alternative method in which immunotherapy, such as T cell therapy (e.g., CAR expressing T cells), is administered without administration of compound C. Tumor burden is not necessarily actually reduced in all subjects receiving the combination therapy, but tumor burden is reduced on average in treated subjects, such as based on clinical data, wherein most subjects treated with such combination therapy exhibit reduced tumor burden, such as at least 50%, 60%, 70%, 80%, 90%, 95% or more of subjects treated with the combination therapy exhibit reduced tumor burden.

The disease burden may encompass the total number of disease cells in the subject or in an organ, tissue or body fluid of the subject (such as an organ or tissue of a tumor or another location that may be indicative of metastasis, for example). For example, tumor cells may be detected and/or quantified in the blood, lymph, or bone marrow in the context of certain hematological malignancies. In some embodiments, the disease burden may include the mass of the tumor, the number or extent of metastases, and/or the percentage of primary cells present in the bone marrow.

In some embodiments, the subject has lymphoma or leukemia. The extent of disease burden can be determined by assessing residual leukemia in blood or bone marrow. In some embodiments, the subject has non-hodgkin's lymphoma (NHL), acute Lymphoblastic Leukemia (ALL), chronic Lymphocytic Leukemia (CLL), or diffuse large B-cell lymphoma (DLBCL). In some embodiments, the subject has MM or DBCBL.

In some aspects, the response rate of a subject (e.g., a subject with NHL) is based on the Lugano standard (Lugano criterion). (Cheson et al, (2014) JCO.,32 (27): 3059-3067; johnson et al, (2015) Radiology 2:323-338; cheson, B.D. (2015) Chin. Clin. Oncol.4 (1): 5). In some aspects, the response assessment utilizes any clinical, hematological, and/or molecular approach. In some aspects, the response assessed using the rugasan standard includes optionally using Positron Emission Tomography (PET) -Computed Tomography (CT) and/or CT. PET-CT evaluation may further include the use of Fluorodeoxyglucose (FDG) for FDG-philic lymphomas. In some aspects, in the case of assessing FDG-philic histological response using PET-CT, a 5-component scale may be used. In some aspects, the 5-component table contains the following criteria: 1, no uptake above background; 2, ingestion is less than or equal to mediastinum; 3, ingestion > mediastinum but not liver; 4, uptake of moderate > liver; 5, uptake is significantly higher than liver and/or new lesions; x, a new uptake region is unlikely to be associated with lymphoma.

In some aspects, the complete response as described using the rukino standard involves a complete metabolic response and a complete radiological response at different measurable sites. In some aspects, these sites include lymph node and extralymph sites, where CR on a 5-score scale is described as scoring 1, 2 or 3 with or without residual tumor when PET-CT is used. In some aspects, in the spleen or bone marrow with high physiological uptake or activation of the Waldeyer's rings or in the extranodular sites (e.g., for chemotherapy or bone marrow colony stimulating factors), uptake may be greater than normal mediastinum and/or liver. In this case, if the uptake at the initial affected site is not greater than that of the surrounding normal tissue, even if the tissue has high physiological uptake, it can be inferred as a complete metabolic response. In some aspects, the response is assessed in lymph nodes using CT, where CR is described as having no diseased extra-lymph sites and the longest transverse diameter (LDi) of the lesions of the target lymph nodes/lymph node tumors must be restored to ∈1.5cm. Other assessment sites include bone marrow, where PET-CT based assessments should indicate evidence of lack of FDG-philic disease in bone marrow, and CT based assessments should indicate normal morphology and if indeterminate should be IHC negative. Other locations may include assessment of organ enlargement, which should return to normal. In some aspects, unmeasured lesions and new lesions are assessed, which should be absent in the case of CR (Cheson et al, (2014) JCO32 (27): 3059-3067; johnson et al, (2015) Radiology 2:323-338; cheson, B.D. (2015) Chin. Clin. Oncol.4 (1): 5).

In some aspects, the Partial Reaction (PR) as described using the rugasan standard involves partial metabolic and/or radiological reactions at different measurable sites. In some aspects, these sites include lymph nodes and extralymph sites, where PR is described as scoring 4 or 5 minutes when PET-CT is used, with reduced uptake and one or more residual masses of any size compared to baseline. In the interim, such findings may be indicative of disease in the reaction. At the end of treatment, such findings may be indicative of residual disease. In some aspects, the response in lymph nodes is assessed using CT, where PR is described as an SPD reduction of ≡50% up to 6 measurable target nodules and extranodular sites. If the lesion is too small to measure on CT, then 5mm by 5mm is designated as the default value; if the lesion is no longer visible, the value is 0mm x 0mm; for >5mm x 5mm but smaller than normal nodules, calculations were performed using actual measurements. Other evaluation sites include bone marrow, where PET-CT based evaluation should indicate residual uptake above uptake in normal bone marrow but reduced compared to baseline (diffuse uptake is compatible with reactive changes from allowed chemotherapy). In some aspects, if there is a sustained focal change in bone marrow in the context of a nodular response, further evaluation should be considered using MRI or biopsy or interval scan. In some aspects, other sites may include an assessment of organ enlargement, wherein the spleen must have been restored by >50% over normal length. In some aspects, the unmeasured lesions and new lesions are assessed, which should be absent/normal, restored but not increased in the case of PR. Non-response/disease Stabilization (SD) or disease Progression (PD) can also be measured using PET-CT and/or CT based assessment. (Cheson et al, (2014) JCO.,32 (27): 3059-3067; johnson et al, (2015) Radiology 2:323-338; cheson, B.D. (2015) Chin. Clin. Oncol.,4 (1): 5).

In some aspects, progression Free Survival (PFS) is described as the length of time a subject survives a disease (e.g., B cell malignancy) during and after treatment of the disease, but the disease does not worsen. In some aspects, an Objective Response (OR) is described as a measurable response. In some aspects, the Objective Response Rate (ORR) is described as the proportion of patients achieving CR or PR. In some aspects, total survival (OS) is described as the length of time that a subject diagnosed with a disease (e.g., a B-cell malignancy) remains alive from the date of diagnosis or the date of treatment initiation. In some aspects, event-free survival (EFS) is described as the length of time that the subject remains free of certain complications or events that the treatment is intended to prevent or delay after the end of B-cell malignancy treatment. These events may include recurrence of a B-cell malignancy or onset of certain symptoms (e.g., bone pain caused by a B-cell malignancy that has spread to bone), or death.

In some embodiments, the measure of duration of response (DOR) comprises the time from recording to tumor response to disease progression. In some embodiments, the parameters used to assess the response may include a persistent response, e.g., a response that persists after a period of time from the initiation of therapy. In some embodiments, the persistent response is indicated by a response rate of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months after initiation of the therapy. In some embodiments, the reaction may last for greater than 3 months or greater than 6 months.

In some aspects, objective tumor response is determined using RECIST criteria. (Eisenhauer et al EuropeanJournal of Cancer 45 (2009) 228-247). In some aspects, objective tumor response to a target lesion is determined using RECIST criteria. In some aspects, a complete response as determined using RECIST criteria is described as the disappearance of all target lesions, and any pathological lymph nodes (whether target or non-target) must be reduced to <10mm on the short axis. In other aspects, the partial response as determined using RECIST criteria is described as a reduction in the sum of diameters of target lesions by at least 30% with reference to the sum of baseline diameters. In other aspects, disease Progression (PD) is described as an increase in the sum of diameters of target lesions by at least 20% with reference to the minimum sum in the study (including the baseline sum if the baseline sum is the minimum in the study). In addition to a relative increase of 20%, the sum must also show an absolute increase of at least 5mm (in some aspects, the appearance of one or more new lesions is also considered to be progressive). In other aspects, disease Stabilization (SD) is described with reference to the minimum overall diameter at study, neither scaled down enough to fit PR, nor increased enough to fit PD.

In the case of MM, exemplary parameters that assess the extent of disease loading include parameters such as: the number of cloned plasma cells (e.g., >10% in bone marrow biopsies or any amount in biopsies from other tissues; plasmacytoma), the presence of monoclonal proteins (accessory proteins) in serum or urine, evidence of sensory end organ damage associated with plasma cell disorders (e.g., hypercalcemia (corrected calcium >2.75 mmol/l), renal insufficiency attributable to myeloma; anemia (hemoglobin <10 g/dl), and/or bone lesions (soluble lesions or osteoporosis with compression fracture)).

In the case of DLBCL, exemplary parameters for assessing the extent of disease burden include parameters such as: cell morphology (e.g., central blasts, immune blasts, and mesogenic cells), gene expression, miRNA expression, and protein expression (e.g., BCL2, BCL6, MUM1, LMO2, MYC, and p21 expression).

In some aspects, the response rate of a subject (e.g., a subject with CLL) is based on the International Infinite lymphocytic leukemia seminar (IWCLL) response standard (Hallek et al, blood 2008, month 6, 15; 111 (12): 5446-5456). In some aspects, these criteria are described as follows: complete Remission (CR), which in some aspects requires the absence of peripheral blood cloned lymphocytes, the absence of lymphadenopathy, the absence of hepatomegaly or splenomegaly, the absence of systemic symptoms and satisfactory blood cell count upon immunophenotyping; complete remission is accompanied by incomplete bone marrow recovery (CRi), which in some aspects is described as CR described above, but without normal blood cell count; partial Remission (PR), which is described in some aspects as a decrease in lymphocyte count of 50% or greater, a decrease in lymphadenopathy of 50% or a decrease in liver or spleen of 50% or greater, and an improvement in peripheral blood cell count; disease Progression (PD), which in some aspects is described as an increase in lymphocyte count of 50% or more to >5x 10 ⁹ 1/L, lymphadenopathy increase of 50% or more, liver or spleen size increase of 50% or more, richtert transformation or new cytopenia due to CLL; and disease stabilization, which in some aspects is described as not meeting the criteria for CR, CRi, PR or PD.

In some embodiments, a subject exhibits CR OR if the subject's lymph node size is less than OR about 20mm, less than OR about 10mm, OR less than OR about 10mm within 1 month of administration of the cell dose.

In some embodiments, no marker clone of CLL is detected in the subject's bone marrow (or in greater than 50%, 60%, 70%, 80%, 90% or more of the subject's bone marrow treated according to the method). In some embodiments, the indicator clone of CLL is assessed by IgH depth sequencing. In some embodiments, the marker clone is not detected at or about or at least about 1, 2, 3, 4, 5, 6, 12, 18, or 24 months after administration of the cell.

In some embodiments, the subject exhibits a morphological disorder if, for example, greater than or equal to 5% of the primary cells are present in the bone marrow, such as greater than or equal to 10% of the primary cells are present in the bone marrow, greater than or equal to 20% of the primary cells are present in the bone marrow, greater than or equal to 30% of the primary cells are present in the bone marrow, greater than or equal to 40% of the primary cells are present in the bone marrow, or greater than or equal to 50% of the primary cells are present in the bone marrow, as determined by light microscopy. In some embodiments, the subject exhibits complete or clinical remission if less than 5% of the primary cells are present in the bone marrow.

In some embodiments, the subject may exhibit complete remission, but a small fraction of morphologically undetectable (by optical microscopy techniques) residual leukemia cells are present. A subject is said to exhibit Minimal Residual Disease (MRD) if the subject exhibits less than 5% primary cells in bone marrow and exhibits a molecularly detectable B cell malignancy. In some embodiments, molecularly detectable B-cell malignancies can be assessed using any of a variety of molecular techniques that allow sensitive detection of small numbers of cells. In some aspects, such techniques include PCR assays that can determine unique Ig/T cell receptor gene rearrangements or fusion transcripts resulting from chromosomal translocations. In some embodiments, B cell malignancy cells can be identified based on an immunophenotype characteristic of leukemia using flow cytometry. In some embodiments, molecular detection of B cell malignancy can detect as few as 1 leukemia cell out of 100,000 normal cells. In some embodiments, the subject exhibits a molecularly detectable MRD if at least or more than 1 leukemia cell out of 100,000 cells is detected, e.g., by PCR or flow cytometry. In some implementations In embodiments, the disease burden of the subject is non-molecularly detectable or MRD ^- Such that leukemia cells in a subject cannot be detected using PCR or flow cytometry techniques in some cases.

In the case of leukemia, the extent of disease burden can be determined by evaluating residual leukemia in blood or bone marrow. In some embodiments, the subject exhibits a morphological disorder if greater than or equal to 5% of the primary cells are present in the bone marrow (e.g., as detected by light microscopy). In some embodiments, the subject exhibits complete or clinical remission if less than 5% of the primary cells are present in the bone marrow.

In some embodiments, for leukemia, the subject may exhibit complete remission, but there are a small fraction of residual leukemia cells that are morphologically undetectable (by optical microscopy techniques). A subject is said to exhibit Minimal Residual Disease (MRD) if the subject exhibits less than 5% primary cells in bone marrow and exhibits a molecularly detectable B cell malignancy. In some embodiments, molecularly detectable B-cell malignancies can be assessed using any of a variety of molecular techniques that allow sensitive detection of small numbers of cells. In some aspects, such techniques include PCR assays that can determine unique Ig/T cell receptor gene rearrangements or fusion transcripts resulting from chromosomal translocations. In some embodiments, B cell malignancy cells can be identified based on an immunophenotype characteristic of leukemia using flow cytometry. In some embodiments, molecular detection of B cell malignancy can detect as few as 1 leukemia cell out of 100,000 normal cells. In some embodiments, the subject exhibits a molecularly detectable MRD if at least or more than 1 leukemia cell out of 100,000 cells is detected, e.g., by PCR or flow cytometry. In some embodiments, the disease burden of the subject is not molecularly detectable or MRD ^- Such that leukemia cells in a subject cannot be detected using PCR or flow cytometry techniques in some cases.

In some embodiments, the method and/or administration of a cell therapy such as a T cell therapy (e.g., CAR expressing T cells) and/or compound C reduces disease burden as compared to disease burden at a time immediately prior to administration of an immunotherapy such as T cell therapy and/or compound C.

In some aspects, immunotherapy, such as T cell therapy and/or administration of compound C, may prevent an increase in disease burden, and this may be evidenced by no change in disease burden.

In some embodiments, the method reduces the burden of the disease or disorder (e.g., the number of tumor cells, the size of the tumor, the duration of patient survival or no event survival) to a greater extent and/or for a longer period of time as compared to the reduction in the burden of the disease or disorder that would be observed using an alternative therapy, such as an alternative therapy in which the subject receives only immunotherapy, e.g., T cell therapy, in the absence of administration of compound C. In some embodiments, the degree of disease burden is reduced to a greater extent or for a longer duration following administration of an immunotherapy, such as a combination therapy of T cell therapy and compound C, than would be achieved by administration of each agent alone (e.g., administration of compound C to a subject that has not received an immunotherapy, such as a T cell therapy; or administration of an immunotherapy, such as a T cell therapy, to a subject that has not received compound C).

In some embodiments, the load of a disease or disorder in a subject is detected, assessed, or measured. In some aspects, the disease burden can be detected by detecting the total number of disease cells or disease-associated cells (e.g., tumor cells) in a subject or in an organ, tissue, or body fluid (e.g., blood or serum) of a subject. In some embodiments, disease burden (e.g., tumor burden) is assessed by measuring the number or extent of metastases. In some aspects, the survival, survival over a specified period of time, degree of survival, presence or duration of event-free or symptom-free survival, or relapse-free survival of a subject is assessed. In some embodiments, any symptom of the disease or disorder is assessed. In some embodiments, a measure of the disease or disorder load is specified. In some embodiments, exemplary parameters for determining include a particular clinical outcome indicative of improvement or amelioration of a disease or disorder (e.g., a tumor). Such parameters include: duration of disease control, including Complete Response (CR), partial Response (PR), or disease Stabilization (SD) (see, e.g., guidelines for response assessment criteria (Response Evaluation Criteria In Solid Tumors, RECIST) in solid tumors); objective Response Rate (ORR); progression Free Survival (PFS); and total survival (OS). Specific thresholds for parameters can be set to determine the efficacy of the combination therapy methods provided herein.

In some aspects, the disease burden is measured or detected prior to administration of an immunotherapy, such as a T cell therapy, after administration of an immunotherapy, such as a T cell therapy, but prior to administration of compound C, and/or after administration of both an immunotherapy, such as a T cell therapy, and compound C. In the case of multiple administrations of one or more steps of a combination therapy, the disease burden may be measured in some embodiments before or after, or at the time between, any of the administration steps, doses, and/or cycles of administration.

In some embodiments, the load is reduced or reduced by at least or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% by the provided methods as compared to immediately prior to administration of compound C and an immunotherapy, such as T cell therapy. In some embodiments, the disease burden, tumor size, tumor volume, tumor mass, and/or tumor burden or volume is reduced by at least or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more after administration of an immunotherapy, such as T cell therapy and/or compound C, as compared to immediately before administration of an immunotherapy, such as T cell therapy and/or compound C.

In some embodiments, reducing disease burden by the method includes inducing morphological complete remission, e.g., as assessed 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or more than 6 months after administration (e.g., initiation) of the combination therapy.

In some aspects, the determination of minimal residual disease (e.g., as measured by multiparameter flow cytometry) is negative, or the level of minimal residual disease is less than about 0.3%, less than about 0.2%, less than about 0.1%, or less than about 0.05%.

In some embodiments, the method increases event-free survival or overall survival of the subject as compared to other methods. For example, in some embodiments, at 6 months after the methods of combination therapy provided herein, the event-free survival or probability of a subject treated by the methods is greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%. In some aspects, the overall survival rate is greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%. In some embodiments, the subject treated with the method exhibits an event-free survival, a relapse-free survival, or a survival of up to at least 6 months or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. In some embodiments, the time to progression is improved, such as the time to progression is greater than or greater than about 6 months or at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 or 10 years.

In some embodiments, the probability of recurrence is reduced after treatment by the method as compared to other methods. For example, in some embodiments, at 6 months after the method of combination therapy, the probability of recurrence is less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10%.

In some cases, the pharmacokinetics of the administered cells (e.g., adoptive transfer cells) are determined to assess the availability, e.g., bioavailability, of the administered cells. Methods for determining the pharmacokinetics of adoptive transfer cells may include withdrawing peripheral blood from a subject to whom engineered cells have been administered, and determining the number or ratio of the engineered cells in the peripheral blood. Methods for selecting and/or isolating cells can include the use of Chimeric Antigen Receptor (CAR) -specific antibodies (e.g., brentjens et al,Sci.Transl.Med.2013, 3; 5 (177): 177ra 38); protein L (Zheng et al, J.Transl. Med.2012, month 2; 10:29); an epitope tag (e.g., strep-tag sequence) introduced directly into a specific site in the CAR, whereby the CAR is directly assessed using Strep-tagged binding reagents (Liu et al (2016) Nature Biotechnology,34:430; international patent application publication No. WO 2015095895); and monoclonal antibodies that specifically bind to the CAR polypeptide (see international patent application publication No. WO 2014190273). In some cases, an external marker gene may be used in conjunction with an engineered cell therapy to allow detection or selection of cells, and in some cases also to promote cell suicide. In some cases, truncated epidermal growth factor receptor (EGFRt) can be co-expressed with a transgene of interest (e.g., CAR) in transduced cells (see, e.g., U.S. patent No. 8,802,374). EGFRt may contain the antibody cetuximab Or other therapeutic anti-EGFR antibodies or binding molecules, which can be used to identify or select cells that have been engineered with an EGFRt construct and another recombinant receptor, such as a Chimeric Antigen Receptor (CAR), and/or to eliminate or isolate cells expressing the receptor. See U.S. patent No. 8,802,374 and Liu et al, nature biotech.2016, month 4; 34 (4):430-434.

In some embodiments, the number of car+ T cells in a biological sample (e.g., blood) obtained from a patient can be determined at a time after administration of a cell therapy, e.g., to determine the pharmacokinetics of the cells. In some embodiments, the number of detectable car+ T cells (optionally car+cd8+ T cells and/or car+cd4+ T cells) in the blood of a subject or in a majority of subjects so treated by the method is greater than 1 cell/μl, greater than 5 cells/μl, or greater than 10 cells/μl.

Toxicity and adverse outcome

In embodiments of the provided methods, toxicity or other adverse outcomes of the subject, including treatment-related outcomes, e.g., development of neutropenia, cytokine Release Syndrome (CRS), or Neurotoxicity (NT), are monitored in the subject administered the provided combination therapies including cell therapy (e.g., T cell therapy) and compound C. In some embodiments, the provided methods are performed to reduce the risk of toxic outcome or symptoms, profile, factor or characteristic of promoting toxicity, such as symptoms or outcomes associated with or indicative of severe neutropenia, severe Cytokine Release Syndrome (CRS), or severe neurotoxicity.

In some embodiments, the provided methods do not result in a high rate or likelihood of toxicity or toxic outcome, or reduce the rate or likelihood of toxicity or toxic outcome, such as severe Neurotoxicity (NT) or severe Cytokine Release Syndrome (CRS), as compared to certain other cell therapies. In some embodiments, the method does not result in or increase the risk of: severe NT (sNT), severe CRS (sccrs), macrophage activation syndrome, tumor lysis syndrome, fever at or about 38 degrees celsius for three or more days, and plasma CRP levels of at least or about 20 mg/dL. In some embodiments, greater than or greater than about 30%, 35%, 40%, 50%, 55%, 60% or more of the subjects treated according to the provided methods do not exhibit any fractionated CRS or any fractionated neurotoxicity. In some embodiments, no more than 50% of the treated subjects (e.g., at least 60%, at least 70%, at least 80%, at least 90% or more of the treated subjects) have a Cytokine Release Syndrome (CRS) higher than grade 2 and/or a neurotoxicity higher than grade 2. In some embodiments, at least 50% of the subjects treated according to the methods (e.g., at least 60%, at least 70%, at least 80%, at least 90% or more of the treated subjects) do not exhibit severe toxicity consequences (e.g., severe CRS or severe neurotoxicity), such as do not exhibit grade 3 or higher graded neurotoxicity and/or do not exhibit severe CRS, or do not exhibit such for some period of time after treatment (e.g., within one week, two weeks, or one month of administration of the cells).

A. Cytokine Release Syndrome (CRS) and neurotoxicity

In some aspects, the toxic outcome is, or is associated with, or is indicative of, cytokine Release Syndrome (CRS) or severe CRS (sccrs). In some cases, CRS, such as sccrs, may occur after adoptive T cell therapy and administration of other biologic products to a subject. See Davila et al, sci trans l Med 6,224 a25 (2014); brentjens et al, sci.Transl.Med.5,177 a38 (2013); grupp et al, N.Engl.J.Med.368,1509-1518 (2013); and Kochenderfer et al, blood 119,2709-2720 (2012); xu et al, cancer letters 343 (2014) 172-78.

Typically, CRS is caused by excessive systemic immune responses mediated, for example, by T cells, B cells, NK cells, monocytes and/or macrophages. Such cells can release a large number of inflammatory mediators, such as cytokines and chemokines. Cytokines may elicit an acute inflammatory response and/or induce endothelial organ damage that may lead to microvascular leakage, heart failure or death. Severe life threatening CRS may lead to lung infiltration and lung injury, renal failure or disseminated intravascular coagulation. Other severe life threatening toxicities may include cardiotoxicity, respiratory distress, neurotoxicity, and/or liver failure. CRS may be treated with anti-inflammatory therapies (e.g., anti-IL-6 therapies, such as anti-IL-6 antibodies, e.g., tolizumab) or antibiotics or other agents as described.

The outcomes, signs, and symptoms of CRS are known and include those described herein. In some embodiments, where a particular dosage regimen or administration achieves or does not achieve a given CRS-related outcome, sign, or symptom, the particular outcome, sign, and symptom and/or amount or extent thereof may be specified.

In the case of CAR-expressing cells, CRS typically occurs 6-20 days after infusion of CAR-expressing cells. See Xu et al, cancer Letters 343 (2014) 172-78. In some cases, CRS occurs less than 6 days or more than 20 days after CAR T cell infusion. The incidence and timing of CRS may be related to baseline cytokine levels or tumor burden at the time of infusion. Typically, CRS includes elevated serum levels of Interferon (IFN) - γ, tumor Necrosis Factor (TNF) - α, and/or Interleukin (IL) -2. Other cytokines that may be rapidly induced in CRS are IL-1β, IL-6, IL-8 and IL-10.

Exemplary outcomes associated with CRS include fever, stiffness, chills, hypotension, dyspnea, acute Respiratory Distress Syndrome (ARDS), encephalopathy, ALT/AST elevation, renal failure, heart disease, hypoxia, neurological disorders, and death. Neurological complications include delirium, seizure-like activity, confusion, difficulty finding words, aphasia, and/or becoming dull. Other consequences associated with CRS include fatigue, nausea, headache, seizures, tachycardia, myalgia, rash, acute vascular leak syndrome, liver function damage, and renal failure. In some aspects, CRS is associated with an increase in one or more factors (e.g., serum ferritin, d-dimer, transaminase, lactate dehydrogenase, and triglyceride), or with hypofibrinogenemia or hepatosplenomegaly.

In some embodiments, the CRS-related outcomes include one or more of the following: sustained heat generation, e.g., heat generation at a specified temperature (e.g., greater than or greater than about 38 degrees celsius) for two or more days, e.g., three or more days, e.g., four or more days or for at least three consecutive days; a heating of greater than or greater than about 38 degrees celsius; elevation of cytokines, such as compared to pre-treatment levels of at least two cytokines (e.g., at least two of the group consisting of interferon gamma (ifnγ), GM-CSF, IL-6, IL-10, flt-3L, irregular chemokines, and IL-5 and/or tumor necrosis factor alpha (tnfa)), a fold maximum change of, e.g., at least or at least about 75, or a fold maximum change of at least one of such cytokines of, e.g., at least or at least about 250; and/or at least one toxic clinical sign, such as hypotension (e.g., as measured by at least one intravenous vasoactive drug); hypoxia (e.g. Plasma Oxygen (PO) ₂ ) A level below or below about 90%); and/or one or more neurological disorders (including mental state changes, dullness, and epilepsy).

Exemplary CRS-related outcomes include increased or high serum levels of one or more factors, including cytokines and chemokines and other factors related to CRS. Exemplary outcomes further include an increase in synthesis or secretion of one or more such factors. Such synthesis or secretion may be performed by T cells or cells that interact with T cells (e.g., innate immune cells or B cells).

In some embodiments, CRS-related serum factors or CRS-related ends include inflammatory cytokines and/or chemokines, including interferon gamma (IFN-gamma), TNF-a, IL-1 beta, IL-2, IL-6, IL-7, IL-8, IL-10, IL-12, sIL-2Ra, granulocyte macrophage colony-stimulating factor (GM-CSF), macrophage Inflammatory Protein (MIP) -1, tumor necrosis factor alpha (TNF alpha), IL-6, and IL-10, IL-1 beta, IL-8, IL-2, MIP-1, flt-3L, irregular chemokines, and/or IL-5. In some embodiments, the factor or outcome comprises C-reactive protein (CRP). In addition to being an early and easily measurable risk factor for CRS, CRP is also a marker for cell expansion. In some embodiments, subjects measured to have high CRP levels (e.g., > 15 mg/dL) suffer from CRS. In some embodiments, the subject measured as having a high CRP level does not suffer from CRS. In some embodiments, the measure of CRS includes a measure of CRP and another factor indicative of CRS.

In some embodiments, one or more inflammatory cytokines or chemokines are monitored before, during, or after CAR treatment and/or compound C treatment. In some aspects, the one or more cytokines or chemokines include IFN-gamma, TNF-alpha, IL-2, IL-1 beta, IL-6, IL-7, IL-8, IL-10, IL-12, sIL-2 Ralpha, granulocyte macrophage colony-stimulating factor (GM-CSF), or Macrophage Inflammatory Protein (MIP). In some embodiments, IFN-gamma, TNF-alpha and IL-6 are monitored.

CRS criteria have been developed which appear to be associated with the onset of CRS for predicting which patients are more likely to be at risk of developing sccrs (see davella et al Science translational media.2014; 6 (224): 224ra 25). Factors include fever, hypoxia, hypotension, altered nervous system, elevated serum levels of inflammatory cytokines such as a panel of seven cytokines (ifnγ, IL-5, IL-6, IL-10, flt-3L, irregular chemokines and GM-CSF), the treatment-induced elevation of which may be closely related to both pre-treatment tumor burden and crs symptoms. Other guidelines for diagnosis and management of CRS are known (see, e.g., lee et al, blood.2014;124 (2): 188-95). In some embodiments, the criteria reflecting CRS classification are those detailed in table 3 below.

In some embodimentsIn this case, the subject is considered to develop "severe CRS" ("sfcrs") in response to or secondary to administration of the cell therapy or cell dose thereof, provided that the subject exhibits after administration: (1) heat at least 38 degrees celsius for at least three days; (2) An elevated cytokine comprising (a) a maximum fold change of at least two of the following seven cytokine groups, as compared to the level immediately after administration, of at least 75: interferon gamma (ifnγ), GM-CSF, IL-6, IL-10, flt-3L, irregular chemokines (fracktalkine) and IL-5, and/or (b) at least one of the following seven groups of cytokines has a maximum fold change of at least 250 as compared to the level immediately after administration: interferon gamma (IFNgamma), GM-CSF, IL-6, IL-10, flt-3L, irregular chemokines, and IL-5; and (c) at least one toxic clinical sign, such as hypotension (requiring at least one intravenous vasoactive drug) or hypoxia (PO) ₂ <90%) or one or more neurological disorders (including mental state change, dullness, and/or epilepsy). In some embodiments, the heavy CRS includes 3-level or higher-rank CRS, as shown in table 4.

In some embodiments, the outcome associated with heavy CRS or CRS of grade 3 or higher, such as grade 4 or higher, includes one or more of the following: sustained heat generation, for example, heat generation at a specified temperature (e.g., greater than or greater than about 38 degrees celsius) for two or more days, for example, three or more days, for example, four or more days, or at least three consecutive days; a heating of greater than or greater than about 38 degrees celsius; elevation of cytokines, such as compared to the pre-treatment levels of at least two cytokines (e.g., at least two of the group consisting of interferon gamma (ifnγ), GM-CSF, IL-6, IL-10, flt-3L, irregular chemokines, and IL-5 and/or tumor necrosis factor alpha (tnfa)), a fold maximum change of, e.g., at least or at least about 75, or a fold maximum change of at least one of such cytokines of, e.g., at least or at least about 250; and/or at least one toxic clinical sign, such as hypotension (e.g., as measured by at least one intravenous vasoactive drug); hypoxia (e.g. Plasma Oxygen (PO) ₂ ) A level below or below about 90%); and/or one or more neurological disorders (including mental state changes,Dullness and seizures). In some embodiments, the heavy CRS includes CRS that are required to be managed or cared for in an Intensive Care Unit (ICU).

In some embodiments, the CRS (e.g., heavy CRS) includes a combination of: (1) Sustained fever (at least 38 degrees celsius for at least three days) and (2) serum levels of CRP of at least or at least about 20mg/dL. In some embodiments, CRS encompasses hypotension requiring the use of two or more vasopressors or respiratory failure requiring mechanical ventilation. In some embodiments, the dose of vasopressor is increased in a second or subsequent administration.

In some embodiments, severe CRS or grade 3 CRS encompasses an increase in alanine aminotransferase, an increase in aspartate aminotransferase, chills, febrile neutropenia, headache, left ventricular dysfunction, encephalopathy, hydrocephalus, and/or tremor.

Methods of measuring or detecting various outcomes may be specified.

In some aspects, the toxic outcome of a therapy (e.g., a cell therapy) is or is associated with or indicative of neurotoxicity or severe neurotoxicity. In some embodiments, symptoms associated with clinical risk of neurotoxicity include confusion, delirium, expressive aphasia, dullness, myoclonus, somnolence, altered mental state, convulsions, epileptic-like activity, epilepsy (optionally as confirmed by electroencephalogram [ EEG ]), elevated beta amyloid (aβ) levels, elevated glutamate levels, and elevated oxygen free radical levels. In some embodiments, neurotoxicity is graded based on severity (e.g., using a grade 1-5 scale) (see, e.g., guilo Cavaletti & Paola Marmiroli Nature Reviews Neurology, 657-666 (month 12 2010); U.S. national institute of cancer—common toxicity standard version 4.03 (NCI-CTCAE v 4.03)).

In some cases, the neurological symptom may be the earliest symptom of crs. In some embodiments, neurological symptoms are observed to begin 5 to 7 days after the infusion of the cell therapy. In some embodiments, the duration of the neurological change may be in the range of 3 to 19 days. In some cases, recovery from neurological changes occurs after resolution of other symptoms of sccrs. In some embodiments, treatment with anti-IL-6 and/or one or more steroids does not accelerate the time or extent of resolution of neurological changes.

In some embodiments, a subject is considered to develop "severe neurotoxicity" in response to or secondary to administration of a cell therapy or a cell dose thereof, provided that the subject exhibits symptoms of limiting self-care (e.g., bathing, dressing and dressing, eating, toileting, taking) after administration of: 1) Symptoms of peripheral motor neuropathy, including inflammation or degeneration of peripheral motor nerves; 2) Symptoms of peripheral sensory neuropathy, including inflammation or degeneration of peripheral sensory nerves, dysesthesias (e.g., sensory perceptual distortion, resulting in abnormal and uncomfortable sensations), neuralgia (e.g., intense pain sensation along the nerve or nerve group), and/or paresthesias (e.g., sensory neuron dysfunction, resulting in abnormal skin sensations of stinging, numbness, compression, coldness, and warmth) in the absence of a stimulus. In some embodiments, the severe neurotoxicity comprises grade 3 or higher neurotoxicity, as shown in table 4. In some embodiments, severe neurotoxicity is considered prolonged grade 3 if symptoms or grade 3 neurotoxicity persist for 10 days or more.

In some embodiments, the method reduces symptoms associated with CRS or neurotoxicity as compared to other methods. In some aspects, the provided methods reduce symptoms, outcomes, or factors associated with CRS, including symptoms, outcomes, or factors associated with severe CRS or 3-grade or higher-grade CRS, as compared to other methods. For example, a subject treated according to the methods of the invention may lack and/or have reduced symptoms, outcomes, or factors of detectable CRS (e.g., severe CRS or grade 3 or higher CRS), such as any of the symptoms, outcomes, or factors described (e.g., as shown in table 3). In some embodiments, a subject treated according to the methods of the invention may have reduced neurotoxic symptoms such as weakness or numbness of limbs, impaired memory, vision and/or intelligence, uncontrollable compulsive and/or compulsive behavior, delusions, headache, cognitive and behavioral problems (including loss of motor control, cognitive degeneration and autonomic nervous system dysfunction), and sexual dysfunction, as compared to subjects treated by other methods. In some embodiments, a subject treated according to the methods of the invention may have reduced symptoms associated with peripheral motor neuropathy, peripheral sensory neuropathy, dysesthesia, neuralgia, or paresthesia.

In some embodiments, the methods reduce the consequences associated with neurotoxicity, including damage to the nervous system and/or brain, such as neuronal death. In some aspects, the methods reduce the levels of factors associated with neurotoxicity, such as beta amyloid (aβ), glutamate, and oxygen radicals.

In some embodiments, the toxic outcome is Dose Limiting Toxicity (DLT). In some embodiments, the toxic outcome is the absence of dose limiting toxicity. In some embodiments, dose Limiting Toxicity (DLT) is defined as any grade 3 or higher fractionated toxicity, as described or assessed by any known or disclosed guideline for assessing specific toxicity, such as any guideline described above and including adverse event common terminology standard (Common Terminology Criteria for Adverse Events, CTCAE) version 4.0 of the National Cancer Institute (NCI). In some embodiments, dose Limiting Toxicity (DLT) is defined when any event discussed below occurs following administration of a cell therapy (e.g., T cell therapy) and/or compound C, including a) febrile neutropenia; b) Grade 4 neutropenia persists for about or more than about 7 days; c) Grade 3 or grade 4 thrombocytopenia is accompanied by clinically significant bleeding; and d) grade 4 thrombocytopenia persists for more than 24 hours.

In some embodiments, the provided embodiments result in a low incidence or risk of occurrence of toxicity (e.g., CRS or neurotoxicity or severe CRS or neurotoxicity, e.g., grade 3 or higher CRS or neurotoxicity) as observed by administering a dose of T cells according to the provided combination therapies and/or as observed by the provided articles or compositions. In some cases, this allows for the administration of the cell therapy on an outpatient basis. In some embodiments, administration of the cell therapy (e.g., a dose of T cells (e.g., car+t cells)) according to the provided methods and/or with the provided articles or compositions is performed on an outpatient basis, or without hospitalization of the subject, such as hospitalization requiring overnight stay.

In some aspects, prior to or concomitant with administration of a cellular dose, no intervention for treating any toxicity is administered to a subject (including a subject treated on an outpatient basis) according to the provided methods and/or with a provided article or composition to administer a cellular therapy (e.g., a dose of T cells (e.g., car+t cells)) unless or until the subject exhibits signs or symptoms of toxicity (e.g., neurotoxicity or CRS).

In some embodiments, if a subject (including a subject treated on an outpatient basis) to whom a cell therapy (e.g., a dose of T cells (e.g., car+t cells)) is administered exhibits fever, the subject is given treatment or instructed to receive or administer treatment to alleviate the fever. In some embodiments, fever in a subject is characterized by a body temperature of the subject that is at or above a certain threshold temperature or level (or measured at a threshold temperature or level). In some aspects, the threshold temperature is a temperature associated with at least low heat generation, with at least medium heat generation, and/or with at least high heat generation. In some embodiments, the threshold temperature is a particular temperature or range. For example, the threshold temperature may be at or about or at least about 38, 39, 40, 41, or 42 degrees celsius, and/or may be in a range of at or about 38 degrees celsius to at or about 39 degrees celsius, in a range of at or about 39 degrees celsius to at or about 40 degrees celsius, in a range of at or about 40 degrees celsius to at or about 41 degrees celsius, or in a range of at or about 41 degrees celsius to at or about 42 degrees celsius.

In some embodiments, the treatment designed to reduce fever comprises treatment with an antipyretic. The antipyretic may include any agent, composition or ingredient that reduces fever, such as one of many agents known to have antipyretic effects, such as NSAIDs (e.g., ibuprofen, naproxen, ketoprofen and nimesulide), salicylates (e.g., aspirin, choline salicylate, magnesium salicylate and sodium salicylate), acetaminophen, analgin, nabumetone, phenone, antipyrine, antipyretics. In some embodiments, the antipyretic is acetaminophen. In some embodiments, acetaminophen may be administered orally or intravenously at a dose of 12.5mg/kg every four hours. In some embodiments, the antipyretic is or comprises ibuprofen or aspirin.

In some embodiments, if the fever is sustained fever, an alternative treatment for treating toxicity is administered to the subject. For subjects treated on an outpatient basis, if the subject has and/or is determined to have or have sustained fever, the subject is instructed to return to the hospital. In some embodiments, if a subject exhibits fever at or above a relative threshold temperature, and the subject suffers from and/or is determined or considered to suffer from sustained fever after a specified treatment (e.g., a treatment designed to reduce fever, such as a treatment with an antipyretic (e.g., an NSAID or salicylate, e.g., ibuprofen, acetaminophen, or aspirin)) or without a decrease in fever or body temperature by a specified amount or more (e.g., more than 1 ℃, and typically without a change of about or more than about 0.5 ℃, 0.4 ℃, 0.3 ℃, or 0.2 ℃) of the subject. For example, a subject is considered to have sustained fever if the subject exhibits or is determined to exhibit fever of at least or at least about 38 or 39 degrees celsius, which fever does not decrease or does not decrease by more than or about 0.5 ℃, 0.4 ℃, 0.3 ℃ or 0.2 ℃, or decreases by about 1%, 2%, 3%, 4% or 5% over a 6 hour period, 8 hour period, or 12 hour period or 24 hour period, even after treatment with an antipyretic such as acetaminophen. In some embodiments, the dose of antipyretic is a dose that is generally effective to reduce fever or a specific type of fever in such subjects, such as fever associated with a bacterial or viral infection (e.g., a local or systemic infection).

In some embodiments, a subject has and/or is determined to have sustained fever if the subject exhibits fever at or above the relevant threshold temperature, and the subject's fever or body temperature does not vary by about or above about 1 ℃, and typically does not vary by about or above about 0.5 ℃, 0.4 ℃, 0.3 ℃, or 0.2 ℃. Typically, such absence of a change of greater than or equal to a certain amount is measured over a given period of time (e.g., over a period of 24 hours, 12 hours, 8 hours, 6 hours, 3 hours, or 1 hour, which may be measured from when the first fever sign or body temperature is first above the indicated threshold). For example, in some embodiments, a subject is considered or determined to exhibit sustained fever if the subject exhibits fever of at least or at least about 38 or 39 degrees celsius, the temperature of which does not vary more than or more than about 0.5 ℃, 0.4 ℃, 0.3 ℃, or 0.2 ℃ over a 6 hour period, 8 hour period, or 12 hour period or 24 hour period.

In some embodiments, the heat generation is sustained heat generation; in some aspects, after an initial therapy (e.g., a cell therapy, such as a dose of T cells (e.g., car+t cells)) that potentially induces the toxicity, the subject is treated when it has been determined that the subject has sustained fever, such as within one, two, three, four, five, six hours or less of such a determination or the first such determination.

In some embodiments, one or more interventions or agents for treating the toxicity (e.g., therapies targeting toxicity) are administered at a time, e.g., as measured according to any of the preceding embodiments, at or immediately prior to determining or confirming (e.g., first determining or confirming) that the subject exhibits sustained fever. In some embodiments, the one or more therapies targeting toxicity are administered within a certain period of time of such confirmation or determination, such as within 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, or 8 hours of such confirmation or determination.

B. Hematological toxicity

In some aspects, the toxic outcome is, or is associated with, or is indicative of, hematologic toxicity (e.g., thrombocytopenia and/or neutropenia). In some cases, hematological toxicity (including thrombocytopenia and neutropenia) is graded according to adverse event common terminology criteria (version 4.03; national cancer institute, besselda, malyland, U.S.). In some cases, hematologic toxicity (e.g., thrombocytopenia and/or neutropenia) is monitored before, during, and after one or more administrations of compound C. In some cases, hematological toxicity (e.g., thrombocytopenia and/or neutropenia) is monitored prior to each administration of compound C. In some cases, hematologic toxicity (e.g., thrombocytopenia and/or neutropenia) is monitored at least every 1, 2, 3, 4, 5, 6, or 7 days after administration of compound C.

In some embodiments, whole blood count is performed to monitor the level of white blood cells (white blood cells), including neutrophils and platelets, in the subject. Whole blood cell (CBC) counts and/or differential white blood cell counts may be performed using a variety of methods. In some embodiments, a blood analyzer is used.

Neutropenia is characterized by a decreased blood neutrophil count, generally resulting in increased susceptibility to bacterial and fungal infections. Common symptoms of neutropenia in patients include, for example, fever, aphtha, and ear infections. Patients with severe neutropenia typically suffer from suppurative infections such as sepsis, cutaneous cellulitis, liver abscess, furunculosis, pneumonia, stomatitis, gingivitis, perirectal inflammation, colitis, sinusitis and otitis media.

In some embodiments, absolute Neutrophil Count (ANC) is used to define the level of neutropenia. ANC may be calculated from the components of the whole blood count. In some embodiments, the severity of neutropenia is categorized based on Absolute Neutrophil Count (ANC) in cells measured per microliter of blood: a) mild neutropenia (1000 to 1500 cells/mL); b) Moderate neutropenia (grade 3; 500 to 1000 cells/mL); c) Severe neutropenia (grade 4; <500 cells/mL). In some embodiments, neutropenia may be graded according to the criteria set forth in table 5.

Subjects with severe neutropenia typically have a risk of severe infection.

In some cases, the neutropenia is febrile neutropenia (also known as neutropenic fever or neutropenic sepsis). Thermal neutropenia occurs when the patient's body temperature is above 38 ℃ and the neutrophil level is low or has a neutropenia.

In some embodiments, febrile neutropenia may be graded according to the criteria set forth in table 6.

In some embodiments, the subject is monitored for thrombocytopenia. Thrombocytopenia is characterized by a platelet count of less than 150,000 cells per microliter (μl). The manifestations of thrombocytopenia, particularly in patients with more severe grades, may include bleeding, ecchymosis, petechiae, purpura and spleen hyperfunction. Thrombocytopenia may be characterized as grade 1 thrombocytopenia (i.e., platelet count of 75,000 to 150,000/μl), grade 2 (i.e., platelet count of 50,000 to <75,000/μl), grade 3 (platelet count of 25,000 to <50,000/μl), or grade 4 (i.e., platelet count of less than 25,000/μl).

In some embodiments of the provided methods, the combination therapy with compound C may be altered if the subject is determined to exhibit hematological toxicity, such as thrombocytopenia and/or neutropenia, or a specific stratification thereof. In some aspects, if the subject has grade 3 or higher thrombocytopenia after administration of compound C; grade 3 neutropenia; sustained (e.g., at least over 3, 5, or 7 days) grade 3 neutropenia; grade 4 neutropenia; grade 3 or higher grade febrile neutropenia, the combination therapy is altered. In some embodiments, administration of compound C is permanently stopped or suspended until the signs or symptoms of toxicity are alleviated, reduced, or reduced. The subject may be continuously monitored to assess one or more signs or symptoms of toxicity, such as by CBC or differential leukocyte analysis. In some cases, if toxicity subsides or decreases, administration of compound C may be resumed at the same dose or dosing regimen prior to suspension of the combination therapy, at a lower or reduced dose, and/or at a dosing regimen involving reduced frequency dosing. In some embodiments, the dose is reduced or reduced by at least or at least about or about 10%, 15%, 20%, 25%, 30%, 40%, 50% or 60% in the event that combination therapy is restarted. In some embodiments, if the dose is 0.3mg prior to suspension of cell therapy, the dose is reduced to 0.2mg. In some aspects, if the severity of hematological toxicity is such that the combination therapy is suspended for greater than 4 weeks, the combination therapy may be permanently discontinued.

In some embodiments, one or more agents may be administered to a subject to treat, ameliorate, or alleviate one or more symptoms associated with hematological toxicity. In some cases, a myeloid growth factor (e.g., G-CSF or GM-CSF) is administered to a subject until hematologic toxicity is improved. Examples of such therapies include febuxostat or pefebuxostat. In some aspects, such agents are administered to subjects experiencing severe or febrile neutropenia (including any grade 3 or higher neutropenia of any duration).

C. Non-hematological toxicity

In some aspects, the toxicity outcome is, or is associated with, or is indicative of, one or more non-hematological toxicities following administration of compound C. Examples of non-hematological toxicities include, but are not limited to, a oncological reaction, an infection, a oncolytic syndrome, a cardiac laboratory abnormality, one or more thromboembolic events (such as deep vein thrombosis and pulmonary embolism), and/or pneumonia.

In some aspects, the non-hematological toxicity is a oncological reaction (TFR) (also sometimes referred to as pseudoprogression). TFR is a sudden increase in the size of the site carrying the disease (including lymph nodes, spleen and/or liver), often accompanied by low fever, tenderness and swelling, diffuse rash and in some cases an increase in peripheral blood lymphocyte count. In some embodiments, TFR is graded according to adverse event common terminology standards (version 3.0; national cancer institute, besseda, malyland, U.S.A.). In some embodiments, TFR is graded as follows: grade 1, mild pain, no interference with function; grade 2, moderate pain, pain or analgesic, interfering with function but not with Activities of Daily Living (ADL); grade 3, severe pain, pain or analgesic, hampering function and hampering ADL; grade 4, disability; grade 5, death. In some embodiments, one or more agents (e.g., corticosteroids, NSAIDs, and/or narcotic analgesics) may be administered to the subject to treat, ameliorate, or reduce one or more symptoms associated with TFR.

In some aspects, the non-hematological toxicity is Tumor Lysis Syndrome (TLS). In some embodiments, TLS may be graded according to criteria specified by the Cairo-Bishop grading system (Cairo and Bishop (2004) Br J Haemato, 127:3-11). In some embodiments, intravenous fluid replacement (hydration) may be administered to a subject to reduce hyperuricemia.

In some embodiments, the subject's cardiotoxicity may be monitored, such as by monitoring ECGS, LVEF, and monitoring the levels of troponin-T and BNP. In some embodiments, if elevated troponin-T and/or BNP levels are observed with one or more cardiac symptoms, cardiotoxicity may potentially occur that may necessitate stopping or halting compound C.

In some embodiments of the provided methods, if the subject is determined to exhibit non-hematological toxicity (e.g., TFR or other non-hematological toxicity or a particular classification thereof), then the combination therapy using compound C may be altered. In some aspects, if the subject has grade 3 or higher non-hematologic toxicity (e.g., grade 3 or higher TFR) after administration of compound C, the combination therapy is altered. In some embodiments, administration of compound C is permanently stopped or suspended until the signs or symptoms of toxicity are alleviated, reduced, or reduced. The subject may be continuously monitored to assess one or more signs or symptoms of toxicity. In some cases, if toxicity subsides or decreases, administration of compound C may be resumed at the same dose or dosing regimen prior to suspension of the combination therapy, at a lower or reduced dose, and/or at a dosing regimen involving reduced frequency dosing. In some embodiments, the dose is reduced or reduced by at least or at least about or about 10%, 15%, 20%, 25%, 30%, 40%, 50% or 60% in the event that combination therapy is restarted. In some embodiments, if the dose is 0.3mg prior to suspension of cell therapy, the dose is reduced to 0.2mg. In some embodiments, the dose may be further reduced if grade 3 toxicity occurs again even after the dose is reduced. In some embodiments, if grade 4 toxicity occurs again even after dose reduction, the combination therapy may be permanently discontinued. In some aspects, if the severity of hematological toxicity is such that the combination therapy is suspended for greater than 4 weeks, the combination therapy may be permanently discontinued.

V. products and kits

Also provided are articles of manufacture containing compound C and components (e.g., antibodies or antigen-binding fragments thereof or T cell therapies, such as engineered cells) and/or compositions thereof for immunotherapy. The article of manufacture may comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials such as glass or plastic. In some embodiments, the container contains a composition that is effective to treat, prevent, and/or diagnose a condition, either by itself or in combination with another composition. In some embodiments, the container has a sterile access port. Exemplary containers include intravenous solution bags, vials (including those having a stopper pierceable by an injection needle), or bottles or vials for oral administration. The label or package insert may indicate that the composition is to be used to treat a disease or disorder.

The article of manufacture may comprise (a) a first container having a composition contained therein, wherein the composition comprises engineered cells for immunotherapy (e.g., T cell therapy); and (b) a second container having a composition contained therein, wherein the composition comprises compound C.

In some embodiments, the first container comprises a first composition and a second composition, wherein the first composition comprises a first population of engineered cells for immunotherapy (e.g., cd4+ T cell therapy) and the second composition comprises a second population of engineered cells, wherein the second population can be engineered separately from the first population (e.g., cd8+ T cell therapy). In some embodiments, the first and second cell compositions contain a defined ratio of engineered cells, such as cd4+ and cd8+ cells (e.g., a 1:1 ratio of cd4+: cd8+ car+ T cells).

The article of manufacture may further comprise package insert indicating that the composition may be used to treat a particular condition. Alternatively or additionally, the article of manufacture may further comprise another or the same container comprising a pharmaceutically acceptable buffer. It may further comprise other materials such as other buffers, diluents, filters, needles and/or syringes.

VI definition of

Unless defined otherwise, all technical, symbolic, and other technical and scientific terms or words used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ease of reference, and such definitions contained herein should not be construed as representing substantial differences from the commonly understood meaning in the art.

As used herein, a "subject" is a mammal, such as a human or other animal, and is typically a human. In some embodiments, the subject (e.g., patient) to whom compound C, the engineered cell, or the composition is administered is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or ape. The subject may be male or female, and may be of any suitable age, including infant, juvenile, adolescent, adult and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent.

As used herein, "treatment" (and grammatical variants thereof such as "treatment") refers to the complete or partial improvement or alleviation of a disease or condition or disorder, or a symptom, adverse effect, or outcome or phenotype associated therewith. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The term does not imply complete cure of the disease or complete elimination of any symptoms or one or more effects on all symptoms or outcomes.

As used herein, "delay of progression of a disease" means delay, impediment, slowing, delaying, stabilizing, inhibiting, and/or delaying the progression of a disease (e.g., a B cell malignancy). This delay may have different lengths of time, depending on the medical history and/or the individual being treated. It is apparent that a sufficient or significant delay may actually cover prophylaxis, as the individual will not suffer from the disease. For example, the onset of late B cell malignancy, such as metastasis, can be delayed.

As used herein, "preventing" includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject who may be susceptible to the disease but who has not yet been diagnosed with the disease. In some embodiments, the provided cells and compositions are used to delay the progression of a disease or delay the progression of a disease.

As used herein, "inhibiting" a function or activity is reducing the function or activity when compared to otherwise identical conditions other than the target condition or parameter, or alternatively, when compared to another instance. For example, cells that inhibit tumor growth reduce the growth rate of a tumor compared to the growth rate of a tumor in the absence of cells.

In the context of administration, an "effective amount" of an agent (e.g., an engineered cell or an anti-PD-L1 or antigen-binding fragment thereof, or a pharmaceutical formulation or composition thereof) refers to an amount effective to achieve a desired result (e.g., a therapeutic or prophylactic result) at the necessary dose/amount and for the necessary period of time.

A "therapeutically effective amount" of an agent (e.g., an engineered cell or an anti-PD-L1 or antigen-binding fragment or pharmaceutical formulation or composition thereof) refers to an amount effective to achieve a desired therapeutic outcome (e.g., for treating a disease, condition, or disorder) and/or a pharmacokinetic or pharmacodynamic effect of the treatment at the necessary dose and for the necessary period of time. The therapeutically effective amount can vary depending on factors such as: disease state, age, sex and weight of the subject, and immunomodulatory polypeptide or engineered cell administered. In some embodiments, provided methods involve administering compound C, an engineered cell (e.g., cell therapy), or a composition in an effective amount (e.g., a therapeutically effective amount).

"prophylactically effective amount" means an amount effective to achieve the desired prophylactic result at the requisite dosage and for the requisite period of time. Typically, but not necessarily, because the prophylactic dose is for the subject prior to or earlier in the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more". It is to be understood that the aspects and variations described herein include "consisting of" and/or "consisting essentially of" the aspects and variations.

Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description of the range format is merely for convenience and brevity and should not be interpreted as a inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to specifically disclose all possible sub-ranges as well as individual values within the range. For example, where a range of values is provided, it is to be understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the width of the range.

The term "about" as used herein refers to a common error range for the corresponding values as readily known to those skilled in the art. References herein to "about" a value or parameter include (and describe) implementations directed to the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

As used herein, recitation of a nucleotide or amino acid position "corresponding to" a nucleotide or amino acid position in a disclosed sequence (as shown in the sequence listing) refers to the identified nucleotide or amino acid position after alignment with the disclosed sequence using a standard alignment algorithm (e.g., the GAP algorithm) to maximize identity. By aligning the sequences, the person skilled in the art can, for example, use conserved and identical amino acid residues as guidance to identify the corresponding residues. Generally, to identify corresponding positions, the amino acid sequences are aligned such that a highest order match is obtained (see, e.g., computationalMolecular Biology, lesk, a.m. edit, oxford University Press, new York,1988;Biocomputing:Informatics and Genome Projects,Smith,D.W. Edit, academic Press, newYork,1993;Computer Analysis of Sequence Data,Part I,Griffin,A.M. And Griffin, h.g. edit, humana Press, new.Jersey,1994;Sequence Analysis in Molecular Biology,von Heinje,G, academic Press,1987; and Sequence Analysis Primer, gribskov, m. and Devereux, j. Edit, MStockton Press, new York,1991; carrello et al (1988) SIAM J Applied Math 48:1073).

As used herein, the term "vector" refers to a nucleic acid molecule capable of transmitting another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and that incorporate into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors". Vectors include viral vectors such as retroviral (e.g., gamma-retrovirus and lentivirus) vectors.

The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells" which include primary transformed cells and progeny derived therefrom, irrespective of the number of passages. The nucleic acid content of the offspring may not be exactly the same as the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as selected or selected in the original transformed cell.

As used herein, a statement that a cell or cell population is "positive" for a particular marker refers to the detectable presence of the particular marker (typically a surface marker) on or in the cell. When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, e.g., by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the staining is detectable by flow cytometry at a level that is substantially higher than that detected by the same procedure with an isotype-matched control under otherwise identical conditions, and/or that is substantially similar to that of cells known to be positive for the marker, and/or that is substantially higher than that of cells known to be negative for the marker.

As used herein, a statement that a cell or cell population is "negative" for a particular marker means that the particular marker (typically a surface marker) is not present on or in the cell in a substantially detectable manner. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, e.g., by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the staining is not detected by flow cytometry at a level that is substantially higher than that detected by the same procedure with an isotype-matched control under otherwise identical conditions, and/or that is substantially lower than that of cells known to be positive for the marker, and/or that is substantially similar to that of cells known to be negative for the marker.

As used herein, "percent amino acid sequence identity (%)" and "percent identity" when used with respect to an amino acid sequence (reference polypeptide sequence) are defined as the percentage of amino acid residues in a candidate sequence (e.g., a subject antibody or fragment) that are identical to amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the percent amino acid sequence identity may be accomplished in a variety of ways well known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences compared.

Amino acid substitutions may include substitution of one amino acid for another amino acid in the polypeptide. Substitutions may be conservative amino acid substitutions or non-conservative amino acid substitutions. Amino acid substitutions can be introduced into a binding molecule of interest (e.g., an antibody) and the products screened for a desired activity (e.g., retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

Amino acids can generally be grouped according to the following common side chain characteristics:

(1) Hydrophobic: norleucine, met, ala, val, leu, ile;

(2) Neutral hydrophilic: cys, ser, thr, asn, gln;

(3) Acidic: asp, glu;

(4) Alkaline: his, lys, arg;

(5) Residues that affect chain orientation: gly, pro;

(6) Aromatic: trp, tyr, phe.

In some embodiments, conservative substitutions may involve replacing a member of one of these classes with another member of the same class. In some embodiments, non-conservative amino acid substitutions may involve exchanging members of one of these classes for another class.

As used herein, a composition refers to any mixture of two or more products, substances, or compounds that target Cereblon, including cells. It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

As used herein, a "subject" is a mammal, such as a human or other animal, and is typically a human.

Exemplary embodiments VII

The provided embodiments include:

1. a method of treating lymphoma, the method comprising administering to a subject having lymphoma a combination therapy comprising:

(i) A cell therapy comprising a dose of engineered cells comprising T cells that express a Chimeric Antigen Receptor (CAR) that binds cluster of differentiation 19 (CD 19), wherein the cell therapy is administered on day 1 of the combination therapy; and

(ii) A compound which is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione having the structure:

2. A method of treating lymphoma comprising administering to a subject suffering from lymphoma a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione having the structure:

or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof, wherein:

administering the compound in combination therapy with a T cell therapy comprising a dose of engineered cells comprising T cells expressing a Chimeric Antigen Receptor (CAR) that binds cluster of differentiation 19 (CD 19), wherein the T cell therapy is administered on day 1 of the combination therapy; and

the compound is administered in a plurality of intermittent doses not exceeding once weekly administration, wherein each dose is between or about 0.1mg and or about 0.6mg (inclusive), and wherein administration of the compound is initiated between days 1 and 29 of the combination therapy (inclusive).

3. The method of embodiment 1 or embodiment 2, wherein administration of the compound begins between day 1 and day 22 (inclusive).

4. The method of embodiment 1 or embodiment 2, wherein the administration of the compound is started between day 1 and day 15 (inclusive).

5. The method of any one of embodiments 1-4, wherein administration of the compound begins between (including the endpoints of) day 8 and day 15.

6. The method of any one of embodiments 1-4, wherein administration of the compound is started at or about day 1.

7. The method of any one of embodiments 1-5, wherein administration of the compound begins at or about day 8.

8. The method of any one of embodiments 1-5, wherein administration of the compound begins at or about day 15.

9. The method of any one of embodiments 1-8, wherein each dose of the plurality of intermittent doses is the same.

10. The method of any one of embodiments 1-9, wherein the compound is administered once a week.

11. The method of any one of embodiments 1-10, wherein the compound is administered once every 7 days (Q7D).

12. The method of any one of embodiments 1-9, wherein the compound is administered once every two weeks.

13. The method of any one of embodiments 1-9 and 12, wherein the compound is administered once every 14 days (Q14D).

14. The method of any one of embodiments 1-13, wherein the compound is administered for at least 12 weeks after administration of the cell therapy.

15. The method of any one of embodiments 1-13, wherein the compound is administered up to 12 weeks after administration of the cell therapy.

16. The method of any one of embodiments 1-4, 6, 9-11, 14, and 15, wherein the compound is administered on days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85.

17. The method of any one of embodiments 1-5, 7, 9-11, 14, and 15, wherein the compound is administered on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85.

18. The method of any one of embodiments 1-5, 8-11, 14 and 15, wherein the compound is administered on days 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85.

19. The method of any one of embodiments 1-5, 7, 9, and 12-15, wherein the compound is administered on days 8, 22, 36, 50, 64, and 78.

20. The method of any of embodiments 1-19, wherein the dose of the compound is between or about 0.2mg and or about 0.4mg inclusive.

21. The method of any one of embodiments 1-20, wherein the dose of the compound is at or about 0.4mg.

22. The method of any one of embodiments 1-20, wherein the dose of the compound is less than 0.4mg.

23. The method of any one of embodiments 1-20 and 22, wherein the dose of the compound is at or about 0.3mg.

24. The method of any one of embodiments 1-20 and 22, wherein the dose of the compound is at or about 0.2mg.

25. A method of treating lymphoma comprising administering to a subject suffering from lymphoma a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione having the structure:

The compound was administered in multiple intermittent doses administered once every seven days (Q7D) and on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85, with each dose being 0.3mg.

26. A method of treating lymphoma comprising administering to a subject suffering from lymphoma a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione having the structure:

the compound was administered in multiple intermittent doses administered once every seven days (Q7D) and on days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85, with each dose being 0.3mg.

27. A method of treating lymphoma comprising administering to a subject suffering from lymphoma a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione having the structure:

the compound was administered in multiple intermittent doses administered once every seven days (Q7D) and on days 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85, with each dose being 0.3mg.

28. A method of treating lymphoma comprising administering to a subject suffering from lymphoma a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione having the structure:

the compound was administered in multiple intermittent doses administered once every seven days (Q7D) and on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85, with each dose being 0.4mg.

29. A method of treating lymphoma comprising administering to a subject suffering from lymphoma a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione having the structure:

The compound was administered in multiple intermittent doses administered once every 14 days (Q14D) and on days 8, 22, 36, 50, 64 and 78, with each dose being 0.3mg.

30. A method of treating lymphoma comprising administering to a subject suffering from lymphoma a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione having the structure:

the compound was administered in multiple intermittent doses administered once every seven days (Q7D) and on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85, with each dose being 0.2mg.

31. The method of any one of embodiments 1-30, wherein the compound is or comprises a pharmaceutically acceptable salt of (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione.

The method of any one of embodiments 1-30, wherein the compound is or comprises a hydrate of (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione.

33. The method of any one of embodiments 1-30, wherein the compound is or comprises a solvate of (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione.

34. The method of any one of embodiments 1-30, wherein the compound is or comprises (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione.

35. The method of any one of embodiments 1-34, wherein at the beginning of administration of the compound, the subject does not exhibit severe toxicity following administration of the cell therapy.

36. The method of embodiment 35, wherein:

the severe toxicity is severe Cytokine Release Syndrome (CRS), optionally a grade 3 or higher CRS, an extended grade 3 or higher CRS, a grade 4 CRS, or a grade 5 CRS; and/or

The severe toxicity is severe neurotoxicity, optionally grade 3 or higher, prolonged grade 3 or higher, grade 4 or grade 5 neurotoxicity.

37. The method of any one of embodiments 1-36, wherein if the subject exhibits toxicity, optionally hematological toxicity, after administration of the compound, administration of the compound is suspended and/or the dosage of the compound is modified, optionally reduced.

38. The method of embodiment 37, wherein the toxicity is severe thrombocytopenia, optionally grade 4 thrombocytopenia or prolonged grade 4 thrombocytopenia.

39. The method of embodiment 37, wherein the toxicity is severe neutropenia, optionally grade 4 neutropenia, prolonged grade 4 neutropenia, or febrile neutropenia, optionally grade 3 or higher grade febrile neutropenia, or prolonged grade 3 or higher grade febrile neutropenia.

40. The method of any one of embodiments 37-39, wherein administration of the compound is resumed after the subject no longer exhibits the toxicity.

41. The method of any one of embodiments 1-40, wherein the lymphoma is a B cell malignancy.

42. The method of any one of embodiments 1-41, wherein the lymphoma is non-hodgkin's lymphoma (NHL), optionally wherein the NHL comprises invasive NHL; diffuse large B-cell lymphoma (DLBCL); DLBCL-NOS, optionally transformed inert; EBV positive DLBCL-NOS; t cell/histiocyte enriched large B cell lymphomas; primary mediastinum large B-cell lymphoma (PMBCL); follicular Lymphoma (FL), optionally grade 3B follicular lymphoma (FL 3B); and/or high grade B cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements with DLBCL histology (double/triple hit).

43. The method of any one of embodiments 1-42, wherein the CD19 is human CD19.

44. The method of any one of embodiments 1-43, wherein the Chimeric Antigen Receptor (CAR) comprises an extracellular antigen recognition domain that specifically binds to CD19 and an intracellular signaling domain comprising ITAM.

45. The method of embodiment 44, wherein the intracellular signaling domain comprises a CD 3-zeta (CD 3 zeta) chain, optionally a signaling domain of a human CD 3-zeta chain.

46. The method of embodiment 44 or embodiment 45, wherein the Chimeric Antigen Receptor (CAR) further comprises a costimulatory signaling region.

47. The method of embodiment 46, wherein the costimulatory signaling region comprises the signaling domain of CD28 or 4-1BB, optionally human CD28 or human 4-1 BB.

48. The method of embodiment 46 or embodiment 47, wherein the costimulatory signaling region comprises the signaling domain of human 4-1 BB.

49. The method of any of embodiments 1-48, wherein:

the CAR comprises an scFv specific for the CD 19; a transmembrane domain; a cytoplasmic signaling domain derived from a costimulatory molecule, optionally being or comprising 4-1BB, optionally human 4-1BB; and a cytoplasmic signaling domain derived from an ITAM-containing primary signaling molecule, the cytoplasmic signaling domain optionally being or comprising a CD3 zeta signaling domain, optionally a human CD3 zeta signaling domain; and optionally wherein the CAR further comprises a spacer between the transmembrane domain and the scFv.

50. The method of any of embodiments 1-48, wherein:

The CAR comprises in order an scFv specific for the CD 19; a transmembrane domain; a cytoplasmic signaling domain derived from a costimulatory molecule, said cytoplasmic signaling domain optionally being or comprising a 4-1BB signaling domain, optionally a human 4-1BB signaling domain; and a cytoplasmic signaling domain derived from an ITAM-containing primary signaling molecule, the cytoplasmic signaling domain optionally being a CD3 zeta signaling domain, optionally a human CD3 zeta signaling domain.

51. The method of any one of embodiments 1-48, wherein the CAR comprises, in order, an scFv specific for the CD 19; a spacer; a transmembrane domain; a cytoplasmic signaling domain derived from a costimulatory molecule, said cytoplasmic signaling domain optionally being a 4-1BB signaling domain; and a cytoplasmic signaling domain derived from an ITAM-containing primary signaling molecule, the cytoplasmic signaling domain optionally being or comprising a CD3 zeta signaling domain.

52. The method of any of embodiments 49-51, wherein

The spacer is a polypeptide spacer comprising or consisting of all or a portion of an immunoglobulin hinge or modified form thereof, or comprising about 15 or fewer amino acids.

53. The method of any one of embodiments 49-52, wherein the spacer comprises or consists of an immunoglobulin hinge, optionally an IgG4 hinge, or all or a portion of a modified form thereof, and/or comprises about 15 or fewer amino acids.

54. The method of embodiment 52 or embodiment 53, wherein the spacer is or about 12 amino acids in length and/or comprises or consists of an immunoglobulin hinge, optionally an IgG4, or a modified form thereof, in whole or in part.

55. The method of any one of embodiments 49-54, wherein the spacer has or consists of: the sequence of SEQ ID NO. 1; the sequence encoded by SEQ ID NO. 2, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34; or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

56. The method according to any one of embodiments 49-55, wherein the cytoplasmic signaling domain derived from a costimulatory molecule comprises SEQ ID No. 12 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

57. The method of any one of embodiments 49-56, wherein the cytoplasmic signaling domain which is derived from an ITAM-containing primary signaling molecule comprises SEQ ID NO 13, 14, or 15 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

58. The method of any one of embodiments 49-57, wherein the scFv comprises the CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 35), the CDRL2 sequence of SRLHSGV (SEQ ID NO: 36) and/or the CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 37); and/or the CDRH1 sequence of DYGVs (SEQ ID NO: 38), the CDRH2 sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39) and/or the CDRH3 sequence of YAMDYWG (SEQ ID NO: 40).

59. The method of any one of embodiments 49-58, wherein the scFv comprises the variable heavy chain region of FMC63 and the variable light chain region of FMC63 and/or 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, and optionally wherein the scFv comprises a VH comprising SEQ ID NO:41 and a VL comprising the amino acid sequence as set forth in SEQ ID NO: 42.

60. The method of any one of embodiments 49-58, wherein the scFv has the amino acid sequence set forth in SEQ ID No. 43.

61. The method of any one of embodiments 1-60, wherein the dose of engineered cells comprises or is from about 1x 10 ⁵ To 5x 10 ⁸ Total CAR expressing T cells, 1x 10 ⁶ To 2.5x10 ⁸ Total CAR expressing T cells, 5x 10 ⁶ To 1x 10 ⁸ Total CAR expressing T cells, 1x 10 ⁷ To 2.5x10 ⁸ Individual total CAR expressing T cells, or 5x 10 ⁷ To 1x 10 ⁸ Each total CAR expressing T cell contains an endpoint.

62. The method of any one of embodiments 1-61, wherein the dose of engineered cells comprises at least or at least about 1x 10 ⁵ Individual CAR expressing cells, at least, or at least about 2.5x 10 ⁵ Individual CAR expressing cells, at least, or at least about 5x 10 ⁵ Individual CAR expressing cells, at least or at least about 1x 10 ⁶ Individual CAR expressing cells, at least, or at least about 2.5x 10 ⁶ Individual CAR expressing cells, at least, or at least about 5x 10 ⁶ Individual CAR expressing cells, at least or at least about 1x 10 ⁷ Individual CAR expressing cells, at least, or at least about 2.5x 10 ⁷ Individual CAR expressing cells, at least, or at least about 5x 10 ⁷ Individual CAR expressing cells, at least or at least about 1x 10 ⁸ Individual CAR expressing cells, at least, or at least about 2.5x 10 ⁸ Individual CAR-expressing cells, or at least about 5x 10 ⁸ And (3) CAR expressing cells.

63. The method of any one of embodiments 1-62, wherein the dose of engineered cells comprises at or about 1x 10 ⁸ And (3) CAR expressing cells.

64. The method of any one of embodiments 1-63, wherein the dose of engineered cells is administered parenterally, optionally intravenously.

65. The method of any one of embodiments 1-64, wherein the T cells are primary T cells obtained from the subject.

66. The method of any one of embodiments 1-65, wherein the T cells are autologous to the subject.

67. The method of any one of embodiments 1-64, wherein the T cells are allogeneic to the subject.

68. The method of any one of embodiments 1-67, wherein the dose of engineered cells comprises cd4+ T cells expressing the CAR and cd8+ T cells expressing the CAR and administration of the dose comprises administering a plurality of separate compositions comprising a first composition comprising one of the cd4+ T cells and the cd8+ T cells and a second composition comprising the other of the cd4+ T cells or the cd8+ T cells.

69. The method of embodiment 68, wherein:

applying the first composition and the second composition 0 to 12 hours apart, 0 to 6 hours apart, or 0 to 2 hours apart, or wherein the application of the first composition and the application of the second composition are performed on the same day, between about 0 and about 12 hours apart, between about 0 and about 6 hours apart, or between about 0 and 2 hours apart; and/or

The initiation of the administration of the first composition and the initiation of the administration of the second composition are between about 1 minute and about 1 hour apart or between about 5 minutes and about 30 minutes apart.

70. The method of embodiment 68 or embodiment 69, wherein the first composition and the second composition are administered no more than 2 hours apart, no more than 1 hour apart, no more than 30 minutes apart, no more than 15 minutes apart, no more than 10 minutes apart, or no more than 5 minutes apart.

71. The method of any of embodiments 68-70, wherein the first composition comprises the cd4+ T cells.

72. The method of any of embodiments 68-70, wherein the first composition comprises the cd8+ T cells.

73. The method according to any of embodiments 68-72, wherein the first composition is administered before the second composition.

74. The method of any one of embodiments 1-73, wherein the subject has been preconditioned with a lymphocyte removal therapy comprising administration of fludarabine and/or cyclophosphamide prior to administration of the cell therapy.

75. The method of any one of embodiments 1-73, further comprising, immediately prior to administering the cell therapy, administering to the subject a lymphocyte removal therapy comprising administration of fludarabine and/or cyclophosphamide.

76. The method of embodiment 74 or embodiment 75, wherein the lymphocyte depletion therapy comprises daily administration of about 200-400mg/m ² Comprising the endpoints, optionally at or about 300mg/m ² Cyclophosphamide of (C), and/or about 20-40mg/m ² Optionally 30mg/m ² Is administered for 2-4 days, optionally for 3 days, or wherein the lymphocyte removal therapy comprises administration of about 500mg/m ² Cyclophosphamide of (c).

77. The method of any of embodiments 74-76, wherein:

the lymphocyte depletion therapy comprises daily administration of at or about 300mg/m ² Cyclophosphamide sum of about 30mg/m ² Fludarabine of (c) for 3 days; and/or

The lymphocyte depletion therapy comprises daily administration of at or about 500mg/m ² Cyclophosphamide sum of about 30mg/m ² Is continued for 3 days.

78. The method of any one of embodiments 1-77, wherein the subject is a human.

79. The method of any one of embodiments 1-78, wherein:

at least 35%, at least 40%, or at least 50% of a subject treated according to the method achieves a Complete Response (CR) that may last or last for 6 months or more or for 9 months or more in at least 60%, 70%, 80%, 90%, or 95% of the subjects who achieved the CR; and/or

Wherein at least 60%, 70%, 80%, 90% or 95% of subjects who achieved CR by six months remain responsive, maintain CR and/or survive without progression for more than or more than 3 months and/or for more than 6 months and/or for more than nine months; and/or

An Objective Response (OR) is achieved by at least 50%, at least 60%, OR at least 70% of the subjects treated according to the method, optionally wherein the OR may last for OR greater than 6 months OR for OR greater than 9 months in at least 60%, 70%, 80%, 90%, OR 95% of the subjects who achieved the OR; and/or

Wherein at least 60%, 70%, 80%, 90% OR 95% of subjects who achieved OR by six months remain responsive OR alive for more than OR more than 3 months and/OR for more than 6 months.

80. The method of any one of embodiments 42-79, wherein, at or immediately prior to administration of the dose of engineered cells, the subject has relapsed after remission or becomes refractory to the following therapy: one, two or three previous therapies for one or more of the NHLs, optionally in addition to another dose of engineered cells expressing the CAR.

81. The method of any one of embodiments 42-80, wherein, at or before administration of the dose of engineered cells:

the subject is identified as or has been identified as having a double/triple hit lymphoma;

the subject is identified as having or has been identified as having a chemotherapeutic refractory lymphoma, optionally a chemotherapeutic refractory DLBCL; and/or

The subject has not achieved Complete Remission (CR) in response to previous therapies.

82. The method of any one of embodiments 1-81, wherein the compound is administered:

Reversing the depletion phenotype in CAR-expressing T cells in the subject;

preventing, inhibiting, or delaying the onset of a depletion phenotype in CAR-expressing T cells in the subject;

reducing the level or extent of a depletion phenotype in CAR-expressing T cells in the subject; or alternatively

Reducing the percentage or total number of CAR-expressing T cells having a depletion phenotype in the subject.

83. The method of any one of embodiments 1-82, wherein the onset of administration of the compound is performed after administration of the T cell therapy, and after administration of the compound or onset thereof, the subject exhibits a restoration or rescue of antigen or tumor specific activity or function of the CAR-expressing T cells in the subject, optionally wherein the onset of restoration, rescue, and/or administration of the compound is a point in time after CAR-expressing T cells in the subject or in the blood of the subject have exhibited a depletion phenotype.

84. The method of any one of embodiments 1-83, wherein administering the compound comprises administering in an amount, frequency, and/or duration effective to:

(a) Achieving an increase in antigen-specific or antigen receptor driving activity of naive or non-depleting T cells in the subject, optionally including T cells expressing the CAR, after exposure of the naive or non-depleting T cells to a CD19 antigen or antigen receptor specific agent, as compared to the case of the administration in the absence of the compound; or (b)

(b) Preventing, inhibiting or delaying the onset of a depletion phenotype in naive or non-depleted T cells in the subject, the naive or non-depleted T cells optionally comprising T cells expressing the CAR, after exposure of the naive or non-depleted T cells to a CD19 antigen or antigen receptor specific agent, as compared to the administration in the absence of the compound; or (b)

(c) Reversing the depletion phenotype in depleted T cells in the subject, the depleted T cells optionally comprising T cells expressing the CAR, as compared to the situation in which the administration is not performed to the subject.

85. The method of embodiment 84, wherein administering the compound comprises administering in an amount, frequency, and/or duration effective to: (i) Achieving the increased activity, and (ii) preventing, inhibiting or delaying the onset of the depletion phenotype and/or reversing the depletion phenotype.

86. The method of embodiment 84 or embodiment 85, wherein the T cells in the subject comprise T cells that express the CAR, and/or the antigen is CD19.

87. The method of any one of embodiments 82-86, wherein the depletion phenotype comprises, with respect to a T cell or population of T cells:

An increase in the level or extent of surface expression of one or more depletion markers, optionally 2, 3, 4, 5 or 6 depletion markers, or an increase in the percentage of the population of T cells exhibiting said surface expression, over a reference population of T cells under the same conditions; or alternatively

A reduction in the level or extent of activity exhibited by a T cell or population of T cells when exposed to a CD19 antigen or antigen receptor specific agent, as compared to a reference population of T cells under the same conditions.

88. The method of embodiment 87, wherein the increase in level, degree, or percentage is greater than or about 1.2-fold, greater than or about 1.5-fold, greater than or about 2.0-fold, greater than or about 3-fold, greater than or about 4-fold, greater than or about 5-fold, greater than or about 6-fold, greater than or about 7-fold, greater than or about 8-fold, greater than or about 9-fold, greater than or about 10-fold, or more.

89. The method of embodiment 87, wherein the reduction in level, degree, or percentage is greater than or about 1.2-fold, greater than or about 1.5-fold, greater than or about 2.0-fold, greater than or about 3-fold, greater than or about 4-fold, greater than or about 5-fold, greater than or about 6-fold, greater than or about 7-fold, greater than or about 8-fold, greater than or about 9-fold, greater than or about 10-fold, or more.

90. The method of any one of embodiments 87-89, wherein the reference T cell population is a T cell population known to have a non-depleting phenotype, is a naive T cell population, is a central memory T cell population, or is a stem cell central memory T cell population, optionally from the same subject or same species as the subject from which the one or more T cells having the depleting phenotype were derived.

91. The method of any one of embodiments 87-90, wherein the reference T cell population (a) is a subject-matched population comprising a plurality of T cells isolated from blood of a subject from which one or more T cells having the depletion phenotype are derived, optionally wherein the plurality of T cells do not express the CAR, and/or (b) is obtained from a subject from which one or more T cells having the depletion phenotype are derived prior to receiving administration of a dose of T cells expressing the CAR.

92. The method of any one of embodiments 87-91, wherein the reference T cell population is a composition comprising a sample of the T cell therapy, or a pharmaceutical composition comprising T cells expressing the CAR, prior to its administration to the subject, optionally wherein the composition is a cryopreserved sample.

93. The method of any one of embodiments 87-92, wherein the one or more exhaustion markers are inhibitory receptors.

94. The method of any one of embodiments 87-93, wherein the one or more depletion markers are selected from the group consisting of PD-1, CTLA-4, TIM-3, LAG-3, BTLA, 2B4, CD160, CD39, VISTA, and TIGIT.

95. The method of any one of embodiments 87-94, wherein the activity is one or more of proliferation, cytotoxicity, or production of one or a combination of inflammatory cytokines, optionally wherein the one or combination of cytokines is selected from IL-2, IFN- γ, and TNF- α.

96. The method according to any one of embodiments 87-95,

wherein exposure to the CD19 antigen or antigen receptor specific agent comprises incubation with the CD19 antigen or antigen receptor specific agent, optionally an agent that binds to an antigen binding domain of the CAR.

97. The method of embodiment 96, wherein exposing the CD19 antigen or antigen receptor specific agent comprises exposing the T cells to target cells expressing CD19 antigen, optionally cells of the B cell malignancy.

VIII. Examples

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1: degradation of Aiolos and Ikaros transcription factors in anti-CD 19 CAR-T cells by compound C

The ability of (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione (compound C) to degrade transcription factors Aiolos and Ikaros in stimulated anti-CD 19 CAR T cells was evaluated.

T cell compositions containing anti-CD 19 CAR expressing T cells were generated from leukapheresis samples from five healthy human adult donors by a process comprising immune affinity-based selection of T cells by individually selecting cd4+ and cd8+ cells from samples from each donor to yield two compositions enriched for cd4+ and cd8+ cells, respectively. Cells enriched for cd4+ and cd8+ compositions were individually activated with anti-CD 3/anti-CD 28 beads and lentivirally transduced with vectors encoding anti-CD 19 CAR. The anti-CD 19 CAR contains an anti-CD 19 single chain variable fragment (scFv) derived from a murine antibody (variable region derived from FMC 63), an immunoglobulin derived spacer, a transmembrane domain derived from CD28, a costimulatory region derived from 4-1BB, and a CD 3-zeta intracellular signaling domain. The expression construct in the viral vector further contains a sequence encoding an alternative surface marker of CAR expression, separated from the CAR coding sequence by a T2A ribosome jump sequence. The transduced population is then incubated alone in the presence of a stimulating agent for cell expansion. Expanded cd4+ cells and cd8+ cells were formulated separately and cryopreserved and stored.

Cryopreserved cd4+ and cd8+ engineered cell compositions were thawed and pooled at a cd4:cd8 ratio of about 1:1 per donor prior to use. The cells of the combined composition were then incubated at 37℃with 5% CO ₂ The following 24 hours of stimulation with 30 μg/mL of CAR-specific anti-idiotype antibody (see, e.g., WO 2018/02100). The stimulus was about 10 in the presence of vehicle control or in the presence of concentration range ^-1 To 10 ⁴ Compound C of nM; (S) -3- [4- (4-morpholin-4-ylmethyl-benzyloxy) -1-oxo-1, 3-dihydro-isoindol-2-yl]-piperidine-2, 6-dione (compound 2); or 3- (5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl) -piperidine-2, 6-dione (compound 1).

After incubation, the stimulated cells were stained with antibodies and analyzed by flow cytometry to assess the intracellular levels of Ikaros and Aiolos in car+ T cells, as measured by Median Fluorescence Intensity (MFI). MFI values for Ikaros and Aiolos were normalized and calculated as a percentage relative to vehicle control.

Concentration-dependent decreases in intracellular Ikaros and Aiolos expression were observed in stimulated CAR-expressing T cells after incubation with compound C, compound 2 and compound 1 (fig. 1). At all tested compound concentration levels, ikaros and Aiolos expression was lower in cells treated with compound C compared to cells treated with compound 2 or compound 1. Furthermore, EC50 values for compounds C, 2 and 1 to reduce expression of Aiolos and Ikaros were calculated as compound concentrations that reduced Aiolos and Ikaros MFI to 50% of their MFI in the absence of compound. These EC50 values are shown as averages in table E1 (95% confidence intervals between five donors are shown in brackets). The results show that compound C is a more potent Ikaros and Aiolos degradation agent in anti-CD 19 CAR T cells than compound 2 or compound 1.

Example 2: effect of Compound C on CAR-T cell function

T cell function of anti-CD 19 CAR T cells treated with compound C was assessed.

anti-CD 19 CAR expressing T cell compositions were generated from three different healthy donors as described in example 1. The cryopreserved cd4+ and cd8+ engineered cell compositions were thawed and combined at a cd4:cd8 ratio of about 1:1 prior to use.

A. Incubation with anti-idiotype antibody and Compound C

To assess viability, proliferation, cell cycle phase and cytokine production, the combined cd4+ and cd8+ CAR-expressing T cell composition is incubated with plate-bound CAR-specific anti-idiotype antibodies (see, e.g., WO 2018/02100) and incubated at 37 ℃ in the presence of compound C, compound 2 or compound 1. After incubation, stimulated CAR T cells were harvested, washed twice in medium, and evaluated.

On day 3 of culture, viability was assessed by flow cytometry using live/dead cell dyes. As shown in figure 2A for three donors (mean ± SEM), the compounds had no effect on cell viability when stimulated with 3 μg/mL anti-idiotype antibody (left panel) or with 30 μg/mL anti-idiotype antibody (right panel).

During incubation with plate-bound anti-idiotype antibodiesThe proliferation of T cells was monitored for seven days by a living cell analysis system (Essen BioScience inc.) and the cell doubling numbers of cells stimulated with 30 μg/mL anti-idiotype antibody for three donors are shown in fig. 2B and fig. 2C (mean ± SEM). As shown in fig. 2B, the presence of the compound reduced the amount of CAR T cell proliferation during CAR-specific stimulation with anti-idiotype antibodies. At all doses tested, cells treated with compound C had the greatest doubling reduction. As shown in fig. 2C, proliferation was reduced (×p) in the presence of 1nM compound C<0.001 And proliferation was further reduced (/ P) in the presence of 10nM of compound C<0.0001)。

During CAR-specific stimulation with plate-bound anti-idiotype antibodies, three days after stimulation, EDU incorporation was used for two hours to determine the cell cycle phase of CAR T cells treated with the compound. For EDU incorporation, CAR T cells were used with Click-iT ^TM EdU cell proliferation kit (Thermo Fisher Scientific) followed by flow cytometry analysis of the prepared cells. The results in FIG. 2D (left panel: stimulation with 3. Mu.g/mL anti-idiotype antibody; right panel: stimulation with 30. Mu.g/mL anti-idiotype antibody) show that treatment with higher doses of compound increased the percentage of CAR T cells at the G1 phase of the cell cycle. Compound C had the greatest effect on the percentage of cells in G1 phase at all doses tested compared to the other compounds tested, at At ten or one hundred times lower doses, there was a similar effect on the percentage of cells in the G1 phase of the cell cycle. As shown in fig. 2E, the percentage of cells in G1 phase increased (×p) in the presence of both 1nM and 10nM compound C<0.001). Without wishing to be bound by theory, accumulation of CAR T cells in the G1 phase may explain one of the mechanisms by which a compound may limit the onset of depletion during chronic stimulation, as shown in example 3 below.

Intracellular cytokine levels in CAR T cells that had been stimulated with 30 μg/mL of anti-idiotype antibody for 72 hours were determined. To this end, supernatants were collected from stimulated CAR T cells and assayed for interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) content using a multiplex electrochemiluminescence assay. For staining for intracellular cytokines (including perforin and granzyme B), cells were fixed and permeabilized using Foxp 3/transcription factor staining buffer set (Thermo Fisher Scientific). Intracellular cytokine levels of ifnγ, perforin, granzyme B and IL-2 were then determined by flow cytometry. As shown in fig. 2F, treatment of cells with the compounds increased intracellular expression of effector cytokines (ifnγ, perforin, granzyme B) as determined by fold change in MFI compared to stimulation with anti-idiotype antibodies in the presence of vehicle controls, but had no significant effect on IL-2 production. In FIG. 2F, log greater than zero ₂ The change times value is indicated with "+". Similar results were observed when the percentage of cytokine-positive cells was measured.

B. Incubation with CD19+ tumor cells and Compound C

To assess the effect of compound C on the cytolytic function of CAR T cells, anti-CD 19 CAR T cells were co-cultured with CAR T cell resistant, CELMoD sensitive RL cd19+ tumor cells at a 1:1 effector to target (E: T) ratio and in the presence of different concentrations of compound C or compound 2. To this end, CAR T cells were plated on poly D-lysine plates. In addition to compound C or vehicle control, then will be usedNucLight Red (Essen BioScience inc.) transduced cd19+ tumor cells were added to CAR T cells. Tumor cell numbers were measured over multiple days.

FIGS. 2G and 2H show the cell number of RL tumor cells treated with anti-CD 19 CAR T cells and Compound C or Compound 2, respectively (left panel: 0.001. Mu.M compound; middle panel: 0.01. Mu.M compound; right panel: 0.1. Mu.M compound). Treatment with CAR T cells in combination with either immunomodulatory compound results in a lower tumor cell number than CAR T cells alone or compounds alone. As shown in fig. 2G, tumor cell numbers were lower for all concentration levels of compound C provided, including 0.001 μm (P < 0.0001) and 0.01 μm (P < 0.0001)). As shown in fig. 2H, at concentration levels of 0.01 μm and 0.1 μm, the tumor cell count was lower when compound 2 was used, but not at 0.001 μm.

C. Summary

Taken together, these results demonstrate that treatment with compound C reduces CAR T cell proliferation, increases the number of cells in the G1 phase, and increases the production of certain cytokines without affecting CAR T cell viability. Furthermore, compound C has a more pronounced effect on CAR T cell proliferation and cell cycle relative to compound 2 or compound 1, and lower doses of compound C improve cell lysis relative to compound 2.

Example 3: evaluation of chronically stimulated CAR-T cells with compound C parallel treatment

Compound C was evaluated for its ability to block or reduce depletion in chronically stimulated CAR T cells.

To mimic the conditions of chronic stimulation, anti-CD 19 CAR expressing T cells generated as described in example 1 were incubated with plate-bound CAR-specific anti-idiotype antibodies (see, e.g., WO 2018/02100) and incubated at 37 ℃ for a period of six days. This six-day stimulation has been shown to induce a hypo-functional state in stimulated CAR T cells. For example, CAR T cells re-stimulated with cd19+ tumor cells after six days of stimulation had reduced ifnγ and IL-2 cytokine production (fig. 3A) and reduced cytolytic function (fig. 3B) compared to freshly thawed CAR T cells that did not undergo six days of stimulation.

Compound C was evaluated for its ability to block or reduce this CAR T cell dysfunction following chronic stimulation. For this, the engineered CAR T cells were treated in parallel with compound C, compound 2 or vehicle control during six days of stimulation. Stimulated CAR T cells were then harvested, washed twice in medium, and re-stimulated with Granta-519 lymphoma tumor spheres (already transduced with IncuCyte NucLight Red) in the absence of compound C, after which cytolytic activity was assessed by loss of red fluorescence over time and average measurements of tumor sphere size at different times after co-culture with CAR-T cells. Cytokine production, ikaros expression and gene expression of CAR T cells were also assessed.

Figure 3C shows a representative image of a Granta-519 tumor sphere co-cultured with CAR T cells stimulated in the presence of compound C on day 9. Figures 3D and 3E show the volume of tumor spheres co-cultured with CAR T cells stimulated in the presence of compound C following chronic stimulation. As shown in fig. 3D and 3E, cytolytic activity during re-excitation was improved by treatment with compound C at a concentration of 1nM during stimulation (< 0.0001). As shown in fig. 3E, treatment with 10nM of compound C during stimulation also resulted in a decrease in tumor size on day 9 of co-culture (< 0.05 with P), but to a degree of less than 1nM of compound C (< 0.0001 with P). Cytokine production of ifnγ in supernatants of co-cultured cells was also assessed five days after re-challenge with tumor cells, and compound C, present at 1nM concentration simultaneously during chronic stimulation, also improved ifnγ production in post-re-challenge treated CAR T cells (fig. 3F, ×p < 0.0001). Treatment with 10nM of compound C during stimulation also increased ifnγ production (< 0.001P), but to a degree lower than 1nM of compound C.

Figure 3G shows Ikaros expression in CAR T cells six days after chronic stimulation in the presence of compound C or compound 2, as determined by MFI. As shown, ikaros expression was lowest in CAR T cells treated with higher concentrations (e.g., 0.01 μm and 0.1 μm) of compound C.

Five days after re-challenge, CAR T cells were sorted from tumor spheres and analyzed for gene expression by RNA sequencing (RNA-seq). The RNA-seq library was sequenced and reads mapped into the genome reference alliance human construct 38 (Genome Reference Consortium Human Build, grch 38) human reference genome. The gene expression level was quantified and differentially expressed using DESeq 2. Gene expression was compared between chronically stimulated CAR T cells treated with compound C and untreated chronically stimulated CAR T cells during stimulation, and between untreated chronically stimulated CAR T cells and unstimulated CAR T cells. Differential expression was calculated based on the effect of treatment with 1nM and 10nM compounds. Differential locus selection cutoff value q.ltoreq.0.05 and absolute log ₂ The change multiple is more than or equal to 0.5.

As shown in fig. 3H, treatment with compound C during chronic stimulation resulted in altered gene expression in chronically stimulated CAR T cells compared to untreated chronically stimulated CAR T cells. For the same set of genes, fig. 3I shows how the expression levels differ between treated and untreated chronically stimulated CAR T cells (y-axis) and between untreated chronically stimulated and unstimulated CAR T cells (x-axis). As shown, genes that were up-regulated by treatment with compound C during stimulation were those down-regulated by chronic stimulation (upper left quadrant), and genes that were down-regulated by treatment with compound C during stimulation were those up-regulated by chronic stimulation (lower right quadrant).

Pathway analysis using CAR T RNA-seq data from the kyoto genes and genome database (Kyoto Encyclopedia of Genes and Genomes, KEGG) was also performed using a clusterifiler. The pathways up-regulated by chronic stimulation, prevented or reduced by treatment with compound C, are shown in fig. 3J and include specific cell proliferation pathways. The gene ratio as shown in fig. 3J was calculated as the percentage of significant gene (significant gene) to total genes in a given pathway.

Taken together, these results demonstrate that the presence of compound C (even at relatively low concentrations) can limit or reduce depletion in chronically stimulated CAR T cells during chronic stimulation conditions. These results also show that Ikaros expression in CAR T cells is reduced by treatment with higher concentrations of compound C, and that pathways involved in T cell function are enriched after chronic stimulation and can be reversed by concurrent treatment of compound C during chronic stimulation.

These results also show that treatment with higher concentrations (10 nM) of compound C during chronic stimulation resulted in less improvement in lytic activity and cytokine production of CAR T cells than treatment with 1nM compound C. Without wishing to be bound by theory, treatment with higher concentrations of compound C may result in sustained or excessive degradation of Ikaros and Aiolos, resulting in CAR T cell dysfunction, limiting the benefits of compound C treatment during chronic stimulation. In contrast, lower concentrations of compound C can improve CAR T cell function without the deleterious effects of sustained or excessive Aiolos and Ikaros degradation.

Example 4: evaluation of chronically stimulated CAR-T cells after rescue treatment with compound C

Compound C was evaluated for its ability to rescue chronically stimulated CAR T cells from depletion.

anti-CD 19 CAR expressing T cells generated as described in example 1 were chronically stimulated as described in example 3, but compound C was not present during stimulation. In contrast, chronically stimulated CAR T cells were treated with compound C during re-challenge with Granta-519 lymphoma tumor spheres or a549 tumor spheres, after which the CAR T cells were evaluated for cytolytic activity, cytokine production, and gene expression.

Figure 4A shows representative images of Granta-519 and a549 tumor spheres on day 9 co-cultured with CAR T cells in the presence of compound C. Fig. 4B and 4C show the volume of spheres co-cultured with anti-CD 19 CAR T cells in the presence of compound C. As shown in fig. 4B, tumor volumes were reduced more by simultaneous treatment with CAR T cells and compound C (1 nM or 10 nM) than by treatment with CAR T cells alone or compound C alone (P <0.0001 for both concentrations). Fig. 4C shows similar results based on tumor size at day 9 of co-culture (P <0.001 for 1nM, P <0.01 for 10 nM). Cytokine production was also assessed five days after re-challenge with tumor cells, and the presence of compound C during re-challenge also improved ifnγ production in chronically stimulated CAR T cells compared to chronically stimulated CAR T cells alone (control; fig. 4D). Cytokine production was highest for CAR T cells re-stimulated in the presence of 1nM compound C (P < 0.0001), and also increased in CAR T cells re-stimulated in the presence of 10nM compound (P < 0.05).

Gene expression in chronically stimulated CAR T cells was also analyzed by RNA sequencing as in example 3. Gene expression was compared between chronically stimulated CAR T cells treated with compound C and untreated chronically stimulated CAR T cells during re-stimulation, and between untreated chronically stimulated CAR T cells and unstimulated CAR T cells. As shown in fig. 4E, treatment with compound C during re-challenge resulted in altered gene expression in chronically stimulated CAR T cells compared to untreated chronically stimulated CAR cells. As shown in fig. 4F, genes that were up-regulated by treatment with compound C during re-excitation were those down-regulated by chronic stimulation (upper left quadrant), and genes that were down-regulated by treatment with compound C during re-excitation were those up-regulated by chronic stimulation (lower right quadrant). The pathway up-regulated by chronic stimulation, reversed by treatment with compound C during re-excitation, is shown in figure 4G.

Taken together, these results demonstrate that the presence of compound C (even at relatively low concentrations) can rescue or restore CAR T cells that have been chronically stimulated from the depletion phenotype. Similar to the results of concurrent stimulation described in example 3, the results revealed that pathways involved in T cell function were enriched following chronic stimulation and could be reversed by rescue treatment with compound C.

Example 5: evaluation of chronically stimulated CAR-T cells treated with different concentration levels of compound C

The effect of different concentrations of compound C on the cytolytic function of CAR T cells after chronic stimulation was tested. During or after chronic stimulation, CAR T cells are treated with various concentrations of compound C.

anti-CD 19 CAR expressing T cells generated as described in example 1 were chronically stimulated as described in example 3 (compound C was present during chronic stimulation) and then re-stimulated with Granta-519 lymphoma tumor spheres in the absence of compound C (parallel treatment group). In addition, anti-CD 19 CAR expressing T cells were chronically stimulated as described in example 4 (no compound C was present during chronic stimulation) and then re-stimulated with Granta-519 lymphoma tumor spheres in the presence of compound C (rescue treatment group). During re-excitation, cytolytic activity was assessed by tumor cell numbers at different times during co-culture with CAR-T cells.

Figure 5A shows the number of tumor cells during re-excitation of chronically stimulated CAR T cells in the presence of compound C (concurrent treatment). Figure 5B shows the number of tumor cells during re-priming of CAR T cells in the presence of compound C (rescue treatment). In both treatment conditions, the number of tumor cells was reduced for CAR T cells treated with compound C at a concentration of 0.001 μm compared to the control conditions. Also in both treatment groups, the number of tumor cells was higher for CAR T cells treated with compound C at concentrations of 0.01 μm and 0.1 μm compared to control conditions.

These results indicate that treatment with higher concentrations of compound C may impair the cytolytic activity of chronically stimulated CAR T cells. Without wishing to be bound by theory, this damage may be due to the sustained or excessive degradation of Ikaros by compound C at higher concentrations, for example as shown in example 3.

Example 6: assessment of intermittent dosing regimen for Compound C

Because of the robust impact of compound C on Ikaros and Aiolos degradation, where excessive or sustained treatment may negatively impact CAR T cell function and/or result in neutropenia secondary to neutrophil maturation arrest, the ability of intermittent dosing regimens for compound C treatment to achieve deep but transient degradation of Ikaros was evaluated. For this, the pharmacokinetics of compound C at a dose of 0.3mg per week (Q7D) were modeled. Based on these results, ikaros degradation in CAR T cells after short (24 hours) and chronic (six days) exposure to compound C was determined. The effect of compound C at a dose of 0.3mg per week on neutrophil development was also modeled.

Fig. 6A shows the expected plasma concentration levels of compound C based on the Q7D dosing regimen. As shown, pharmacokinetic modeling revealed biphasic elimination of compound C, with 20nM C _max Followed by prolonged terminal elimination and about 1nM day 7C _min 。

Fig. 6B and 6C show Ikaros levels in anti-CD 19 CAR T cells generated as described in example 1 after 24 hours (fig. 6B) or six days (fig. 6C) of exposure to compound C. As shown in fig. 6B, dosage levels as low as 0.01 μm of compound C resulted in Ikaros degradation, indicating a 0.3mg dose of compound C (C _max 20 nM) will lead to a large amount of Ikaros degradation after 24 hours. As shown in fig. 6C, six days of exposure to 0.001 μΜ of compound C did not affect Ikaros levels, indicating that Ikaros levels may have an opportunity to return to baseline levels after initial degradation due to the decrease in plasma concentration of compound C over time between doses.

Fig. 6D shows predicted Absolute Neutrophil Count (ANC) spectra generated using four different pharmacodynamic models. These spectra were generated in order to assess the effect of compound C at a dose of 0.3mg per week on neutrophil development, including assessing whether a dose of 0.3mg per week would result in low neutrophil counts (e.g., neutropenia). For all models, at a dose of 0.3mg per week, the average nadir ANC level was predicted to be higher than about 2.2x10 ⁹ Individual cells/L.

Taken together, these results demonstrate that the intermittent dosing regimen of compound C in combination with CAR T cell therapy will allow for deep but transient Ikaros degradation in CAR T cells without substantially affecting neutrophil development or negatively affecting CAR T cell function. Without wishing to be bound by theory, pharmacokinetic elimination using weekly doses of compound C may allow the Ikaros levels to return to baseline values between doses, thereby preventing CART cell dysfunction or neutrophil maturation arrest due to sustained Ikaros degradation.

Example 7: assessment of intermittent dosing regimen of Compound C in chronically stimulated CAR T cells

The effect of intermittent compound C dosing regimen on CAR T cells was evaluated. To simulate biphasic elimination using weekly doses of compound C as shown in example 6, CAR T cells were chronically stimulated for one day at high concentrations of compound C in the presence of compound C, followed by five days at low concentrations of compound C.

In the presence of compound C, anti-CD 19 CAR expressing T cells generated as described in example 1 were chronically stimulated as described in example 3. During chronic stimulation, compound C was present for (i) 50nM for one day and 1nM for the remaining five days, (ii) 25nM for one day and 0.5nM for the remaining five days, (iii) 10nM for one day and five days for the remaining none, or (iv) 1nM for all six days. Stimulated CAR T cells were then re-stimulated with Raji lymphoma tumor spheres in the absence of compound C, followed by assessment of cytolytic activity.

Figure 7A shows tumor cell numbers during re-excitation of chronically stimulated CAR T cells in the presence of compound C as well as fresh (non-chronically stimulated) CAR T cells as described. As shown, during chronic stimulation, compound C was present for one day at high concentration, then for five days at low concentration (one day at 50nM and five days remaining at 1nM, or one day at 25nM and five days remaining at 0.5 nM) resulted in the greatest increase in cytolytic activity of chronically stimulated CAR T cells.

Similar results are shown in fig. 7B for a separate experiment in which CAR T cells were chronically stimulated for 7 days with or without compound C. In this experiment, compound C (i) was present at 50nM for the day of stimulation and at 1nM for the remaining six days, (ii) was present at 25nM for the day of stimulation and at 0.5nM for the remaining six days, (iii) was present at 10nM for all seven days of stimulation, or (iv) was present at 1nM for all seven days of stimulation. The stimulated CAR T cells were then re-stimulated with Raji lymphoma tumor spheres in the absence of compound C, followed by assessment of cytolytic activity over a four day period. As shown in fig. 7B, the presence of compound C at 50nM during the day of stimulation and 1nM during the remaining six days improved the cytolytic activity of chronically stimulated CAR T cells (×× P < 0.0001). Compound C also improved cytolytic activity (× P < 0.0001) for one day of stimulation at 25nM and the remaining six days at 0.5 nM. Phenotypic T cell memory markers of CAR T cells stimulated for seven days in the presence or absence of compound C were also analyzed using flow cytometry after chronic stimulation. As shown in fig. 7C, the cells stimulated in the presence of compound C had a higher percentage of cd27+/ccr7+ cells than CAR expressing CD8T cells stimulated in the absence of compound C. Of the CAR-expressing CD8T cells stimulated with 50nM present on the day of stimulation and 1nM present for the remaining six days, CD27/CCR7 expression (which is a marker of poorly differentiated naive T cells) was highest (< 0.05).

These results indicate that weekly doses of compound C (e.g., as part of an intermittent dosing regimen for compound C) will improve the cytolytic activity of CAR T cells administered in combination with compound C. These results also demonstrate that an intermittent dosing regimen for compound C will result in the retention of the poorly differentiated memory phenotype in CAR T cells.

Example 8: treatment of recurrent/refractory B-cell malignancies with anti-CD 19 CAR T cells and compound C

A combination therapy is administered to a subject having recurrent/refractory invasive B-cell non-hodgkin lymphoma (NHL), the combination therapy comprising compound C and a therapeutic CAR T cell composition comprising autologous T cells that express a CAR specific for CD 19.

Specifically, as described in example 1, autologous anti-CD 19 directed therapeutic T cell compositions were produced in white blood cell apheresis samples from adult human subjects suffering from the following diseases: invasive B-cell NHL, defined as DLBCL NOS, including transformed indolent NHL; grade 3B follicular lymphoma; t cell/histiocyte enriched large B cell lymphomas; EBV positive DLBCL, NOS; primary mediastinal (thymus) large B-cell lymphomas; or high grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements with DLBCL histology (double/triple-hit lymphomas). Invasive B-cell NHL was histologically confirmed at the recent recurrence, otherwise a new tumor biopsy was required.

Qualified subjects have relapsed or refractory to at least two previous normals to systemic therapy, including CD 20-targeting agents and anthracyclines. For a subject with a transformed disease, the subject should have undergone at least two normals to systemic therapy for the transformed disease (i.e., DLBCL) before being eligible. Previous therapy lines do not include those therapy normals given for previous inert conditions (e.g., follicular lymphoma or CLL). Subjects do not have to receive anthracyclines to treat their DLBCL if they receive anthracyclines to treat an inert disease. The subject was not previously treated with compound C.

According to the rukino classification, eligible subjects have Positron Emission Tomography (PET) positive (i.e., a multi-dimensional score of 4 or 5) and Computed Tomography (CT) measurable disease. By CT scanning, the Sum of Products (SPD) of vertical diameters of up to six index lesions of qualified subjects is greater than or equal to 25cm ² 。

For the production of autologous anti-CD 19 CAR T cell compositions, a white blood cell apheresis sample is obtained from the subject about four weeks prior to the planned CAR T cell administration. The subject then received the use of fludarabine (Flu, 30mg/m ² ) And cyclophosphamide (Cy, 300 mg/m) ² ) For three days. Two to seven days after completion of lymphocyte removal chemotherapy, the subject receives a dose of CAR-expressing T cells (e.g., 1x 10 ⁶ Total T cells).

Subjects were assigned to one of seven sub-groups of doses to which compound C was administered. Beginning on day 8, patients assigned to subgroup a received a weekly dosing regimen of 0.3mg of compound C. For subjects with CRS or NTs graded 3 or higher at the beginning of the planned compound C administration, the beginning of compound C administration may be delayed by up to three days. Other dosing regimens may be evaluated based on the safety and tolerability of the dosing regimen of subgroup a. Starting on day 1, subgroup B received a weekly dosing regimen of 0.3mg of compound C. Starting on day 15, subgroup C received a weekly dosing regimen of 0.3mg of compound C. Starting on day 8, subgroup D received a weekly dosing regimen of 0.4mg of compound C. Starting on day 8, subgroup E received a dosing regimen of 0.3mg compound C once every two weeks. Starting on day 8, subgroup F received a weekly dosing regimen of 0.2mg of compound C. Starting on day 8, subgroup G received a weekly dosing regimen of 0.6mg of compound C. For all subgroups, compound C administration was continued until day 85 of the combination therapy.

The safety, pharmacokinetics and pharmacodynamics of the subjects and the clinical response were monitored. Determining a clinical response to the treatment includes assessing whether the subject exhibits disease Progression (PD), partial Response (PR), complete Response (CR), and/or disease stabilization.

The present invention is not intended to be limited in scope by the specific disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods will be apparent from the description and teachings herein. Such changes may be practiced without departing from the true scope and spirit of the disclosure, and are intended to fall within the scope of the disclosure.

Sequence(s)

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<110> Cino therapeutics Co., ltd

<120> combination of CAR T cell therapy and immunomodulatory compounds for treatment of lymphomas

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<150> 63/167,599

<151> 2021-03-29

<150> 63/277,134

<151> 2021-11-08

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Ser His Thr Gln Pro Leu Gly Val Tyr Leu Leu Thr Pro Ala Val Gln

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Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly Lys Val

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Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

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Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

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Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

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Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

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Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe

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Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr

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Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn

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Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile

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Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val

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Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp

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Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn

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Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu

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Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser

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Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu

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Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln

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Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly

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Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro

245 250 255

His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr

260 265 270

Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His

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Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro

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Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala

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Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met

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Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

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Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

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Glu Glu Asn Pro Gly Pro

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Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

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Pro Gly Pro

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Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser

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Asn Pro Gly Pro

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Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val

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Glu Ser Asn Pro Gly Pro

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Pro Gly Gly Gly Ser Gly Gly Gly Gly Pro

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Gly Ser Ala Asp Asp Ala Lys Lys Asp Ala Ala Lys Lys Asp Gly Lys

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Ser

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Ala Phe Leu Leu Ile Pro

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<212> PRT

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Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

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His Ala

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<213> artificial sequence

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Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

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Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro

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<210> 29

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<212> PRT

<213> artificial sequence

<220>

<223> hinge

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Glu Leu Lys Thr Pro Leu Gly Asp Thr His Thr Cys Pro Arg Cys Pro

1 5 10 15

Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu

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Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro

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Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

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<210> 30

<211> 12

<212> PRT

<213> artificial sequence

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Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro

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<210> 31

<211> 12

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<213> artificial sequence

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Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

1 5 10

<210> 32

<211> 9

<212> PRT

<213> artificial sequence

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Tyr Gly Pro Pro Cys Pro Pro Cys Pro

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<212> PRT

<213> artificial sequence

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Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

1 5 10

<210> 34

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> hinge

<400> 34

Glu Val Val Val Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

1 5 10

<210> 35

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> CDR L1

<400> 35

Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn

1 5 10

<210> 36

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> CDR L2

<400> 36

Ser Arg Leu His Ser Gly Val

1 5

<210> 37

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> CDR L3

<400> 37

Gly Asn Thr Leu Pro Tyr Thr Phe Gly

1 5

<210> 38

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> CDR H1

<400> 38

Asp Tyr Gly Val Ser

1 5

<210> 39

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> CDR H2

<400> 39

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

1 5 10 15

<210> 40

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> CDR H3

<400> 40

Tyr Ala Met Asp Tyr Trp Gly

1 5

<210> 41

<211> 120

<212> PRT

<213> artificial sequence

<220>

<223> VH

<400> 41

Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 42

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> VL

<400> 42

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

100 105

<210> 43

<211> 245

<212> PRT

<213> artificial sequence

<220>

<223> scFv

<400> 43

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys

115 120 125

Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser

130 135 140

Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

145 150 155 160

Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile

165 170 175

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu

180 185 190

Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn

195 200 205

Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr

210 215 220

Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

225 230 235 240

Val Thr Val Ser Ser

245

<210> 44

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> CDR L1

<400> 44

Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala

1 5 10

<210> 45

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> CDR L2

<400> 45

Ser Ala Thr Tyr Arg Asn Ser

1 5

<210> 46

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> CDR L3

<400> 46

Gln Gln Tyr Asn Arg Tyr Pro Tyr Thr

1 5

<210> 47

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> CDR H1

<400> 47

Ser Tyr Trp Met Asn

1 5

<210> 48

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> CDR H2

<400> 48

Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys

1 5 10 15

Gly

<210> 49

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> CDR H3

<400> 49

Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe Asp Tyr

1 5 10

<210> 50

<211> 122

<212> PRT

<213> artificial sequence

<220>

<223> VH

<400> 50

Glu Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Gln Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 51

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> VL

<400> 51

Asp Ile Glu Leu Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Thr Tyr Arg Asn Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser

65 70 75 80

Lys Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Arg Tyr Pro Tyr

85 90 95

Thr Ser Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 52

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 52

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 53

<211> 245

<212> PRT

<213> s Artificial sequence

<220>

<223> scFv

<400> 53

Glu Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Gln Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser

130 135 140

Pro Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys

145 150 155 160

Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys

165 170 175

Pro Gly Gln Ser Pro Lys Pro Leu Ile Tyr Ser Ala Thr Tyr Arg Asn

180 185 190

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Thr Asn Val Gln Ser Lys Asp Leu Ala Asp Tyr Phe

210 215 220

Cys Gln Gln Tyr Asn Arg Tyr Pro Tyr Thr Ser Gly Gly Gly Thr Lys

225 230 235 240

Leu Glu Ile Lys Arg

245

<210> 54

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> HC-CDR3

<400> 54

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

1 5 10

<210> 55

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> LC-CDR2

<400> 55

His Thr Ser Arg Leu His Ser

1 5

<210> 56

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LC-CDR3

<400> 56

Gln Gln Gly Asn Thr Leu Pro Tyr Thr

1 5

<210> 57

<211> 735

<212> DNA

<213> artificial sequence

<220>

<223> sequence encoding scFv

<400> 57

gacatccaga tgacccagac cacctccagc ctgagcgcca gcctgggcga ccgggtgacc 60

atcagctgcc gggccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

gacggcaccg tcaagctgct gatctaccac accagccggc tgcacagcgg cgtgcccagc 180

cggtttagcg gcagcggctc cggcaccgac tacagcctga ccatctccaa cctggaacag 240

gaagatatcg ccacctactt ttgccagcag ggcaacacac tgccctacac ctttggcggc 300

ggaacaaagc tggaaatcac cggcagcacc tccggcagcg gcaagcctgg cagcggcgag 360

ggcagcacca agggcgaggt gaagctgcag gaaagcggcc ctggcctggt ggcccccagc 420

cagagcctga gcgtgacctg caccgtgagc ggcgtgagcc tgcccgacta cggcgtgagc 480

tggatccggc agccccccag gaagggcctg gaatggctgg gcgtgatctg gggcagcgag 540

accacctact acaacagcgc cctgaagagc cggctgacca tcatcaagga caacagcaag 600

agccaggtgt tcctgaagat gaacagcctg cagaccgacg acaccgccat ctactactgc 660

gccaagcact actactacgg cggcagctac gccatggact actggggcca gggcaccagc 720

gtgaccgtga gcagc 735

<210> 58

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> hinge

<220>

<221> variant

<222> (1)...(1)

<223> Xaa is glycine, cysteine or arginine

<220>

<221> variant

<222> (4)...(4)

<223> Xaa is cysteine or threonine

<400> 58

Xaa Pro Pro Xaa Pro

1 5

<210> 59

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 59

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr

1 5 10 15

Lys Gly

Claims

1. A method of treating a CD19 expressing cancer, the method comprising administering to a subject having a CD19 expressing cancer a combination therapy comprising:

(i) A T cell therapy comprising a dose of engineered cells comprising T cells expressing a Chimeric Antigen Receptor (CAR) for the cancer, wherein the T cell therapy is administered on day 1 of the combination therapy; and

(ii) A compound which is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione or a pharmaceutically acceptable salt thereof.

2. A method of treating lymphoma, the method comprising administering to a subject having a cancer that is lymphoma a combination therapy comprising:

(i) A T cell therapy comprising a dose of engineered cells comprising T cells expressing a Chimeric Antigen Receptor (CAR) that binds cluster of differentiation 19 (CD 19), wherein the T cell therapy is administered on day 1 of the combination therapy; and

3. The method of claim 1 or claim 2, wherein administration of the compound is initiated after administration of the T cell therapy.

4. A method of treating a CD19 expressing cancer, the method comprising administering to a subject having a CD19 expressing cancer a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione, or a pharmaceutically acceptable salt thereof, wherein:

Administering the compound in combination therapy with a T cell therapy comprising a dose of engineered cells comprising T cells expressing a Chimeric Antigen Receptor (CAR) for the cancer, wherein the T cell therapy is administered on day 1 of the combination therapy and administration of the compound begins after administration of the T cell therapy.

5. A method of treating lymphoma comprising administering to a subject suffering from cancer that is lymphoma a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione, or a pharmaceutically acceptable salt thereof, wherein:

administering the compound in a combination therapy with a T cell therapy comprising a dose of engineered cells comprising T cells expressing a Chimeric Antigen Receptor (CAR) that binds cluster of differentiation 19 (CD 19), wherein the T cell therapy is administered on day 1 of the combination therapy and administration of the compound begins after administration of the T cell therapy.

6. The method of any one of claims 1-5, wherein administration of the compound begins between day 1 and day 29 of the combination therapy, inclusive.

7. The method of any one of claims 1-6, wherein the compound is administered in a plurality of doses, wherein each dose is between or about 0.1mg and or about 0.6mg, inclusive.

8. The method of any one of claims 1-7, wherein the compound is administered in a plurality of intermittent doses that do not exceed once a week administration.

9. A method of treating lymphoma, the method comprising administering to a subject having a cancer that is lymphoma a combination therapy comprising:

(ii) A compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione, or a pharmaceutically acceptable salt thereof, wherein the compound is administered in a plurality of intermittent doses that do not exceed once weekly administration, wherein each dose is between or about 0.1mg and or about 0.6mg inclusive, wherein between day 1 and day 29 of the combination therapy inclusive, administration of the compound is initiated.

10. A method of treating lymphoma comprising administering to a subject suffering from cancer that is lymphoma a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione, or a pharmaceutically acceptable salt thereof, wherein:

the compound is administered in a plurality of intermittent doses not exceeding once weekly administration, wherein each dose is between or about 0.1mg and or about 0.6mg inclusive, and wherein administration of the compound is initiated between days 1 and 29 of the combination therapy, inclusive.

11. The method of any one of claims 1-10, wherein administration of the compound begins between day 1 and day 22, inclusive.

12. The method of any one of claims 1-11, wherein administration of the compound begins between day 1 and day 15, inclusive.

13. The method of any one of claims 1-12, wherein administration of the compound begins between day 8 and day 15, inclusive.

14. The method of any one of claims 1-12, wherein administration of the compound begins at or about day 1.

15. The method of any one of claims 1-13, wherein administration of the compound begins at or about day 8.

16. The method of any one of claims 1-13, wherein administration of the compound begins at or about day 15.

17. The method of any one of claims 1-16, wherein each dose of the plurality of intermittent doses is the same.

18. The method of any one of claims 1-17, wherein the compound is administered once a week.

19. The method of any one of claims 1-18, wherein the compound is administered once every 7 days (Q7D).

20. The method of any one of claims 1-17, wherein the compound is administered once every two weeks.

21. The method of any one of claims 1-17 and 20, wherein the compound is administered once every 14 days (Q14D).

22. The method of any one of claims 1-21, wherein the compound is administered for at least 12 weeks after administration of the T cell therapy.

23. The method of any one of claims 1-21, wherein the compound is administered for up to 12 weeks after administration of the T cell therapy.

24. The method of any one of claims 1-12, 14, 17-19, 22, and 23, wherein the compound is administered on days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85.

25. The method of any one of claims 1-13, 15, 17-19, 22, and 23, wherein the compound is administered on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85.

26. The method of any one of claims 1-13, 16-19, 22, and 23, wherein the compound is administered on days 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85.

27. The method of any one of claims 1-13, 15, 17, and 20-23, wherein the compound is administered on days 8, 22, 36, 50, 64, and 78.

28. The method of any one of claims 1-27, wherein the dose of the compound is between or about 0.3mg and or about 0.6mg inclusive.

29. The method of any one of claims 1-28, wherein the dose of the compound is at or about 0.6mg.

30. The method of any one of claims 1-27, wherein the dose of the compound is between or about 0.2mg and or about 0.4mg inclusive.

31. The method of any one of claims 1-28 and 30, wherein the dose of the compound is at or about 0.4mg.

32. The method of any one of claims 1-28 and 30, wherein the dose of the compound is less than 0.4mg.

33. The method of any one of claims 1-28, 30, and 32, wherein the dose of the compound is at or about 0.3mg.

34. The method of any one of claims 1-28, 30, and 32, wherein the dose of the compound is at or about 0.2mg.

35. A method of treating lymphoma comprising administering to a subject suffering from cancer that is lymphoma a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione, or a pharmaceutically acceptable salt thereof, wherein:

36. A method of treating lymphoma comprising administering to a subject suffering from cancer that is lymphoma a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione, or a pharmaceutically acceptable salt thereof, wherein:

37. A method of treating lymphoma comprising administering to a subject suffering from cancer that is lymphoma a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione, or a pharmaceutically acceptable salt thereof, wherein:

38. A method of treating lymphoma comprising administering to a subject suffering from cancer that is lymphoma a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione, or a pharmaceutically acceptable salt thereof, wherein:

39. A method of treating lymphoma comprising administering to a subject suffering from cancer that is lymphoma a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione, or a pharmaceutically acceptable salt thereof, wherein:

40. A method of treating lymphoma comprising administering to a subject suffering from cancer that is lymphoma a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione, or a pharmaceutically acceptable salt thereof, wherein:

41. A method of treating lymphoma comprising administering to a subject suffering from cancer that is lymphoma a compound that is (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione, or a pharmaceutically acceptable salt thereof, wherein:

The compound was administered in multiple intermittent doses administered once every seven days (Q7D) and on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85, with each dose being 0.6mg.

42. The method of any one of claims 1-41, wherein the compound is or comprises a pharmaceutically acceptable salt of (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione.

43. The method of any one of claims 1-41, wherein the compound is or comprises (S) -2- (2, 6-dioxopiperidin-3-yl) -4- ((2-fluoro-4- ((3-morpholinoazetidin-1-yl) methyl) benzyl) amino) isoindoline-1, 3-dione.

44. The method of any one of claims 1-43, wherein at the beginning of administration of the compound, the subject does not exhibit severe toxicity following administration of the T cell therapy.

45. The method of claim 44, wherein:

46. The method of any one of claims 1-45, wherein if the subject exhibits toxicity, optionally hematological toxicity, after administration of the compound, administration of the compound is suspended and/or the dosage of the compound is modified, optionally reduced.

47. The method of claim 46, wherein the toxicity is severe thrombocytopenia, optionally grade 4 thrombocytopenia or prolonged grade 4 thrombocytopenia.

48. The method of claim 46, wherein the toxicity is severe neutropenia, optionally grade 4 neutropenia, prolonged grade 4 neutropenia, or febrile neutropenia, optionally grade 3 or higher febrile neutropenia, or prolonged grade 3 or higher febrile neutropenia.

49. The method of any one of claims 46-48, wherein administration of the compound is resumed after the subject no longer exhibits the toxicity.

50. The method of any one of claims 1-49, wherein the cancer is a B cell malignancy.

51. The method of any one of claims 1-50, wherein the cancer is non-hodgkin's lymphoma (NHL), optionally wherein the NHL comprises invasive NHL; diffuse large B-cell lymphoma (DLBCL); DLBCL-NOS, optionally transformed inert; EBV positive DLBCL-NOS; t cell/histiocyte enriched large B cell lymphomas; primary mediastinum large B-cell lymphoma (PMBCL); follicular Lymphoma (FL), optionally grade 3B follicular lymphoma (FL 3B); and/or high grade B cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements with DLBCL histology (double/triple hit).

52. The method of any one of claims 1-51, wherein the CD19 is human CD19.

53. The method of any one of claims 1-52, wherein the Chimeric Antigen Receptor (CAR) comprises an extracellular antigen recognition domain that specifically binds to the CD19 and an intracellular signaling domain comprising ITAM.

54. The method of claim 53, wherein the intracellular signaling domain comprises a CD 3-zeta (CD 3 zeta) chain, optionally a signaling domain of a human CD 3-zeta chain.

55. The method of claim 53 or claim 54, wherein the Chimeric Antigen Receptor (CAR) further comprises a costimulatory signaling region.

56. The method of claim 55, wherein the costimulatory signaling region comprises the signaling domain of CD28 or 4-1BB, optionally human CD28 or human 4-1 BB.

57. The method of claim 55 or claim 56, wherein the costimulatory signaling region comprises the signaling domain of human 4-1 BB.

58. The method of any one of claims 1-57, wherein the CAR comprises an scFv specific for the CD 19; a transmembrane domain; a cytoplasmic signaling domain derived from a costimulatory molecule, optionally being or comprising 4-1BB, optionally human 4-1BB; and a cytoplasmic signaling domain derived from an ITAM-containing primary signaling molecule, the cytoplasmic signaling domain optionally being or comprising a CD3 zeta signaling domain, optionally a human CD3 zeta signaling domain; and optionally wherein the CAR further comprises a spacer between the transmembrane domain and the scFv.

59. The method of any one of claims 1-57, wherein the CAR comprises, in order, an scFv specific for the CD 19; a transmembrane domain; a cytoplasmic signaling domain derived from a costimulatory molecule, said cytoplasmic signaling domain optionally being or comprising a 4-1BB signaling domain, optionally a human 4-1BB signaling domain; and a cytoplasmic signaling domain derived from an ITAM-containing primary signaling molecule, the cytoplasmic signaling domain optionally being a CD3 zeta signaling domain, optionally a human CD3 zeta signaling domain; and optionally wherein the CAR further comprises a spacer between the transmembrane domain and the scFv.

60. The method of any one of claims 1-57, wherein the CAR comprises, in order, an scFv specific for the CD 19; a spacer; a transmembrane domain; a cytoplasmic signaling domain derived from a costimulatory molecule, said cytoplasmic signaling domain optionally being a 4-1BB signaling domain; and a cytoplasmic signaling domain derived from an ITAM-containing primary signaling molecule, the cytoplasmic signaling domain optionally being or comprising a CD3 zeta signaling domain.

61. The method of any one of claims 58-60, wherein the spacer is a polypeptide spacer comprising or consisting of all or a portion of an immunoglobulin hinge or a modified form thereof, or comprising about 15 or fewer amino acids.

62. The method of any one of claims 58-61, wherein the spacer comprises or consists of an immunoglobulin hinge, optionally an IgG4 hinge, or all or a portion of a modified form thereof, and/or comprises about 15 or fewer amino acids.

63. The method of any one of claims 58-62, wherein the spacer is or about 12 amino acids in length and/or comprises or consists of an immunoglobulin hinge, optionally IgG4, or a modified version thereof, all or a portion thereof.

64. The method of any one of claims 58-63, wherein the spacer has or consists of: the sequence of SEQ ID NO. 1; the sequence encoded by SEQ ID NO. 2, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34; or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

65. The method of any one of claims 58-64, wherein the cytoplasmic signaling domain which is derived from a costimulatory molecule comprises the sequence set forth in SEQ ID No. 12 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

66. The method of any one of claims 58-65, wherein the cytoplasmic signaling domain which is derived from an ITAM-containing primary signaling molecule comprises the sequence of any one of SEQ ID NOs 13-15 or variants thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

67. The method of any one of claims 58-66, wherein the scFv comprises:

RASQDISKYLN (SEQ ID NO: 35), the CDRL2 sequence of SRLHSGV (SEQ ID NO: 36) and/or the CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 37); and/or

The CDRH1 sequence of DYGVs (SEQ ID NO: 38), the CDRH2 sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39) and/or the CDRH3 sequence of YAMDYWG (SEQ ID NO: 40).

68. The method of any one of claims 58-67, wherein the scFv comprises:

a variable heavy chain region of FMC63 and a variable light chain region of FMC 63; and/or

CDRL1 sequence of FMC63, CDRL2 sequence of FMC63, CDRL3 sequence of FMC63, CDRH1 sequence of FMC63, CDRH2 sequence of FMC63, and sequence CDRH3 of FMC 63; and is also provided with

Optionally wherein the scFv comprises a VH comprising the amino acid sequence shown in SEQ ID NO. 41 and a VL comprising the amino acid sequence shown in SEQ ID NO. 42.

69. The method of any one of claims 58-68, wherein the scFv has the amino acid sequence set forth in SEQ ID No. 43.

70. The method of any one of claims 1-69, wherein the dose of engineered cells comprises at or from about 1x 10 ⁵ To 5x10 ⁸ Total CAR expressing T cells, 1x 10 ⁶ To 2.5x10 ⁸ Total CAR expressing T cells, 5x10 ⁶ To 1x 10 ⁸ Total CAR expressing T cells, 1x 10 ⁷ To 2.5x10 ⁸ Individual total CAR expressing T cells, or 5x10 ⁷ To 1x 10 ⁸ Each total CAR expressing T cell contains an endpoint.

71. The method of any one of claims 1-70, wherein the dose of engineered cells comprises at least or at least about 1x 10 ⁵ Individual CAR expressing cells, at least, or at least about 2.5x 10 ⁵ Individual CAR-expressing cells, at least or at least about 5x10 ⁵ Individual CAR expressing cells, at least or at least about 1x 10 ⁶ Individual CAR expressing cells, at least, or at least about 2.5x 10 ⁶ Individual CAR expressing cells, at least, or at least about 5x10 ⁶ Individual CAR expressing cells, at least or at least about 1x 10 ⁷ Individual CAR expressing cells, at least, or at least about 2.5x 10 ⁷ Individual CAR expressing cells, at least, or at least about 5x10 ⁷ Individual CAR expressing cells, at least or at least about 1x 10 ⁸ Individual CAR expressing cells, at least, or at least about 2.5x 10 ⁸ Individual CAR-expressing cells, or at least about 5x 10 ⁸ And (3) CAR expressing cells.

72. The method of any one of claims 1-71, wherein the dose of engineered cells comprises at or about 1x 10 ⁸ And (3) expressing T cells by the CAR.

73. The method of any one of claims 1-72, wherein the dose of engineered cells is administered parenterally, optionally intravenously.

74. The method of any one of claims 1-73, wherein the T cells are primary T cells obtained from the subject.

75. The method of any one of claims 1-74, wherein the T cells are autologous to the subject.

76. The method of any one of claims 1-73, wherein the T cells are allogeneic to the subject.

77. The method of any one of claims 1-76, wherein the dose of engineered cells comprises cd4+ T cells expressing the CAR and cd8+ T cells expressing the CAR, and administration of the dose comprises administration of a plurality of separate compositions comprising a first composition comprising one of the cd4+ T cells and the cd8+ T cells and a second composition comprising the other of the cd4+ T cells and the cd8+ T cells.

78. The method of claim 77, wherein:

79. The method of claim 77 or claim 78, wherein said first composition and said second composition are administered no more than 2 hours, no more than 1 hour, no more than 30 minutes, no more than 15 minutes, no more than 10 minutes, or no more than 5 minutes apart.

80. The method of any one of claims 77-79, wherein said first composition comprises said cd4+ T cells.

81. The method of any one of claims 77-79, wherein said first composition comprises said cd8+ T cells.

82. The method of any one of claims 77-81, wherein said first composition is administered before said second composition.

83. The method of any one of claims 1-82, wherein the subject has been preconditioned with a lymphocyte removal therapy prior to administration of the T cell therapy, the lymphocyte removal therapy comprising administration of fludarabine and/or cyclophosphamide.

84. The method of any one of claims 1-82, further comprising administering to the subject, immediately prior to administering the T cell therapy, a lymphocyte removal therapy comprising administering fludarabine and/or cyclophosphamide.

85. The method of claim 83 or claim 84, wherein the lymphocyte depletion therapy comprises daily administration of about 200-400mg/m ² Comprising the endpoints, optionally at or about 300mg/m ² Cyclophosphamide of (C), and/or about 20-40mg/m ² Optionally 30mg/m ² Is administered for 2-4 days, optionally for 3 days, or wherein the lymphocyte removal therapy comprises administration of about 500mg/m ² Cyclophosphamide of (c).

86. The method of any one of claims 83-85, wherein

The lymphocyte cleanerThe removal therapy comprises daily administration at or about 300mg/m ² Cyclophosphamide sum of about 30mg/m ² Fludarabine of (c) for 3 days; and/or

87. The method of any one of claims 1-86, wherein the subject is a human.

88. The method of any one of claims 1-87, wherein

At least 60%, 70%, 80%, 90% or 95% of subjects who achieved CR by six months remain responsive, maintain CR and/or survive without progression for 3 months or more and/or for 6 months or more and/or for nine months or more; and/or

At least 60%, 70%, 80%, 90% OR 95% of subjects who achieved OR by six months remain responsive OR viable for 3 months OR more and/OR for 6 months OR more.

89. The method of any one of claims 1-88, wherein at or immediately prior to administration of the dose of engineered cells, the subject has relapsed after remission or becomes refractory to treatment with: one, two or three previous therapies for one or more of the lymphomas, optionally in addition to another dose of engineered cells expressing the CAR.

90. The method of any one of claims 1-89, wherein at or before administration of the dose of engineered cells:

91. The method of any one of claims 1-90, wherein administration of the compound:

Reversing the depletion phenotype in CAR-expressing T cells in the subject;

92. The method of any one of claims 1-91, wherein the onset of administration of the compound is performed after administration of the T cell therapy, and after administration of the compound or onset thereof, the subject exhibits a restoration or rescue of antigen or tumor specific activity or function of the CAR-expressing T cells in the subject, optionally wherein the onset of restoration, rescue, and/or administration of the compound is a point in time after CAR-expressing T cells in the subject or in the blood of the subject have exhibited a depletion phenotype.

93. The method of any one of claims 1-92, wherein administering the compound comprises administering in an amount, frequency, and/or duration effective to:

(a) Achieving an increase in antigen-specific or antigen receptor driving activity of naive or non-depleting T cells in the subject, said naive or non-depleting T cells optionally comprising T cells expressing the CAR, after exposure of said naive or non-depleting T cells to a CD19 antigen or antigen receptor specific agent, as compared to the case of said administration in the absence of said compound; or (b)

(c) Reversing the depletion phenotype in depleted T cells in the subject, optionally comprising T cells expressing the CAR, as compared to the administration in the absence of the compound.

94. The method of claim 93, wherein administering the compound comprises administering in an amount, frequency, and/or duration effective to: (i) Achieving the increased activity, and (ii) preventing, inhibiting or delaying the onset of the depletion phenotype and/or reversing the depletion phenotype.

95. The method of claim 93 or claim 94, wherein the T cells in the subject comprise T cells expressing the CAR and/or the exposure is exposure to CD19 antigen.

96. The method of any one of claims 91-95, wherein the depletion phenotype comprises, with respect to a T cell or population of T cells:

97. The method of claim 96, wherein the increase in level, degree, or percentage is greater than or about 1.2-fold, greater than or about 1.5-fold, greater than or about 2.0-fold, greater than or about 3-fold, greater than or about 4-fold, greater than or about 5-fold, greater than or about 6-fold, greater than or about 7-fold, greater than or about 8-fold, greater than or about 9-fold, greater than or about 10-fold, or more.

98. The method of claim 96, wherein the reduction in level, degree, or percentage is greater than or about 1.2-fold, greater than or about 1.5-fold, greater than or about 2.0-fold, greater than or about 3-fold, greater than or about 4-fold, greater than or about 5-fold, greater than or about 6-fold, greater than or about 7-fold, greater than or about 8-fold, greater than or about 9-fold, greater than or about 10-fold, or more.

99. The method of any one of claims 96-98, wherein the reference T cell population is a T cell population known to have an unconsumed phenotype, is a naive T cell population, is a central memory T cell population, or is a stem cell central memory T cell population, optionally from the same subject or the same species as the subject from which the one or more T cells having the depleted phenotype were derived.

100. The method of any one of claims 96-99, wherein the reference T cell population (a) is a subject-matched population comprising a plurality of T cells isolated from blood of a subject from which one or more T cells having the depletion phenotype are derived, optionally wherein the plurality of T cells do not express the CAR, and/or (b) is obtained from a subject from which one or more T cells having the depletion phenotype are derived prior to receiving administration of a dose of T cells expressing the CAR.

101. The method of any one of claims 96-100, wherein the reference population of T cells is a composition comprising a sample of T cells expressing the CAR, or a pharmaceutical composition comprising T cells expressing the CAR, prior to its administration to the subject, optionally wherein the composition is a cryopreserved sample.

102. The method of any one of claims 96-101, wherein the one or more exhaustion markers are inhibitory receptors.

103. The method of any one of claims 96-102, wherein the one or more depletion markers are selected from the group consisting of PD-1, CTLA-4, TIM-3, LAG-3, BTLA, 2B4, CD160, CD39, VISTA, and TIGIT.

104. The method of any one of claims 96-103, wherein the activity is one or more of proliferation, cytotoxicity, or production of one or a combination of inflammatory cytokines, optionally wherein the one or combination of cytokines is selected from IL-2, IFN- γ, and TNF- α.

105. The method of any one of claims 96-104, wherein exposure to the CD19 antigen or antigen receptor specific agent comprises incubation with the CD19 antigen or antigen receptor specific agent, optionally an agent that binds to an antigen binding domain of the CAR.

106. The method of claim 105, wherein exposing the CD19 antigen or antigen receptor specific agent comprises exposing the T cells to target cells expressing CD19 antigen, optionally cells of the B cell malignancy.