RU2795984C2 - Devices and methods for treatment using adoptive cell therapy - Google Patents

Abstract

FIELD: biotechnology; cell biology; medicine.

SUBSTANCE: invention relates to methods of adoptive cell therapy, including the injection of doses of cells for the treatment of some diseases and conditions, including certain B-cell malignant tumors. The invention relates to a method for treating a patient with CD19-expressing B-cell malignant tumor or suspected of having a CD19-expressing B-cell malignant tumor using adoptive immunotherapy. To implement this method of treatment, a dose of CD4⁺ and CD8⁺ T-cells is injected into a patient in a plurality of separate compositions, namely the first composition, including one of CD4 ⁺ T-cells and CD8⁺ T-cells, and the second composition, including other CD4⁺ T-cells and CD8⁺ T-cells. Each of CD4⁺ and CD8⁺ T-cells, individually, includes a chimeric antigen receptor (CAR) that binds to CD19. And the said CAR contained in CD4+ T-cells and in CD8+ T-cells is the same. In this case, the injection of the first composition and the second composition is carried out with an interval from about 0 to about 12 hours, with an interval from about 0 to about 6 hours, or with an interval from about 0 to about 2 hours.

EFFECT: invention provides improved approaches for the treatment of CD19-expressing B-cell cancers.

50 cl, 63 dwg, 27 tbl, 6 ex

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Application No. 62/514,774, filed on June 2, 2017, entitled "ARTICLES AND METHODS FOR THE TREATMENT USING ADOPTIONAL CELL THERAPY", U.S. Interim Patent Application No. 62/515,530, filed on June 5, 2017, entitled "ARTICLES AND METHODS FOR THE TREATMENT USING ADOPTIONAL CELL THERAPY", U.S. Interim Patent Application No. 62/521,366, filed on June 16, 2017, entitled "ARTICLES AND METHODS FOR THE TREATMENT USING ADOPTIONAL CELL THERAPY", U.S. Interim Patent Application No. 62/527,000, filed on June 29, 2017, entitled "ARTICLES AND METHODS FOR THE TREATMENT USING ADOPTIONAL CELL THERAPY", U.S. Interim Patent Application No. 62/549,938, filed Aug. 24, 2017, entitled "ARTICLES AND METHODS FOR THE TREATMENT USING ADOPTIONAL CELL THERAPY", U.S. Interim Patent Application No. 62/580,425, filed Nov. 1, 2017, entitled "ARTICLES AND METHODS FOR THE TREATMENT USING ADOPTIONAL CELL THERAPY", U.S. Interim Patent Application No. 62/593,871, filed Dec. 1, 2017, entitled "ARTICLES AND METHODS FOR THE TREATMENT USING ADOPTIONAL CELL THERAPY", U.S. Interim Patent Application No. 62/596,764, filed Dec. 8, 2017, entitled "ARTICLES AND METHODS FOR THE TREATMENT USING ADOPTIONAL CELL THERAPY", U.S. Interim Patent Application No. 62/614,957, filed Jan. 8, 2018, entitled “ARTICLES AND METHODS FOR THE TREATMENT USING ADOPTIVIVE CELL THERAPY”, the contents of which are incorporated by reference in their entirety.

Including a sequence listing by reference

[0002] This application is filed with the Sequence Listing in electronic format. The sequence list is presented as a file named 735042012140SeqList.txt, created on June 1, 2018, which is 35306 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

The field to which the invention relates

[0003] The present disclosure relates in some aspects to adoptive cell therapy, including the administration of cell doses for the treatment of patients with disease and conditions, such as certain B-cell malignancies, and related methods, compositions, uses, and articles of manufacture. Cells typically express recombinant receptors such as chimeric antigen receptors (CARs). In some embodiments, the disease or condition is non-Hodgkin's lymphoma (NHL), such as relapsed or refractory NHL, or a specific NHL subtype; in some embodiments, the patient refers to a specific group or subpopulation of NHL patients, such as patients undergoing prior intensive care or patients with a poor prognosis.

State of the art

[0004] Various methods of immunotherapy and/or cell therapy are available for the treatment of diseases and conditions. For example, adoptive cell therapies (including those involving the administration of cells expressing chimeric receptors specific for the disease or disorder of interest, such as chimeric antigen receptors (CARs) and/or other recombinant antigen receptors, as well as other adoptive immunotherapies and adoptive T cell therapies) may be used in the treatment of cancer or other diseases or disorders. Improved approaches are needed. The invention relates to methods and applications that meet such needs.

The essence of the invention

[0005] The invention relates to methods, uses, compositions, compositions and articles for the treatment of patients with or suspected of having a disease or condition such as cancer or tumor, optionally a B-cell malignancy such as NHL or ALL or CLL or their subtype. Methods and other embodiments typically involve administering to a patient T cells, typically engineered T cells, such as cells that express or contain a recombinant receptor, such as a chimeric antigen receptor (CAR) or TCR.

[0006] In some embodiments, the dose of cells or cells administered in connection with any embodiments of the methods, compositions, articles, and uses of the invention contains CD4 ⁺ T cells, or a subtype or phenotype thereof (such as engineered or recombinant receptor-expressing CD4 ⁺ T cells) and/or CD8 ⁺ T cells or a subtype thereof (such as genetically engineered or recombinant CD4 ⁺ receptor-expressing cells). In some embodiments, CD8 ⁺ cells or subtype or phenotype are present in a certain dose or amount or number; in some embodiments, the CD4 ⁺ cells, or subtype or phenotype thereof, are present in a specific dose or amount or number. In some embodiments, CD8 ⁺ cells or a subtype or phenotype thereof and CD4 ⁺ cells or subtype or phenotype are present in an article or composition or combination, or used in methods, in a specific ratio such as, or approximately, 1:1, or from or from about 1:3 to or from about 3:1. In some embodiments, the dose or administration contains or consists of a certain number or number of a single population of cells, and the ratio is a certain ratio or is a natural ratio, such as the ratio in the blood of the patient from which the cells are obtained, or a ratio that occurs without selection or control of a particular relationship.

[0007] In some embodiments, CD4 ⁺ T cells (or a subset thereof) and CD8 ⁺ T cells (or a subset thereof) individually contain a receptor that specifically binds to a target antigen, an expressed disease or condition, or a cell or tissue, and/or which is associated with that disease or condition.

[0008] In some embodiments, CD4 ⁺ and CD8 ⁺ cells are administered and/or combined, eg, in a single formulation and/or from a single container.

[0009] In some embodiments, separate administrations carry CD4 ⁺ and CD8 ⁺ cells in a dose, and/or separate formulations or containers are included, each of which is individually enriched in CD4 ⁺ cells or CD4 ⁺ genetically engineered cells (such as a formulation containing at least a certain percentage, e.g., at least 80%, 85%, 90%, or 95% or more CD4 ⁺ cells and/or not including more than 10% or more than 5% CD8 ⁺ T cells) and CD8 ⁺ cells or CD8 ⁺ genetically engineered cells (such as a composition containing at least a certain percentage, for example, at least 80%, 85%, 90% or 95% or more CD8 ⁺ cells and/or not including more than 10% or more than 5% CD4 ⁺ T cells).

[0010] In some aspects, administration includes administering a plurality of individual compositions, said plurality of individual compositions comprising a first composition comprising either one of CD4 ⁺ T cells and CD8 ⁺ T cells, and a second composition comprising the other of CD4 ⁺ T -cells and CD8 ⁺ T-cells. In some embodiments of any of the methods of the invention, the receptor contained in CD4 ⁺ T cells and/or the receptor contained in CD8 ⁺ T cells comprises a recombinant T cell receptor, and/or wherein CD4 ⁺ T cells and/ or CD8 ⁺ T cells are genetically engineered to express this receptor.

[0011] In some embodiments of any of the embodiments of the invention, the administration of the first composition and the administration of the second composition are carried out on the same day, carried out with an interval of from about 0 to about 12 hours, with an interval of from about 0 to about 6 hours, or from between about 0 and 2 hours; and/or initiating the administration of the first composition and initiating the administration of the second composition is carried out at intervals of from about 1 minute to about 1 hour, or from 5 minutes to about 30 minutes. In some embodiments of any of the methods of the invention, the first composition and the second composition are administered at intervals of 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.

[0012] In some embodiments of any of the embodiments of the invention, the first composition includes CD4 ⁺ T cells. In some embodiments of any of the methods of the invention, the first composition comprises CD8 ⁺ T cells. In some embodiments of any of the methods of the invention, the administration of the first composition is initiated before the administration of the second composition is initiated. In some embodiments of any of the methods of the invention, the cell dose comprises a specific ratio of CD4 ⁺ cells expressing the recombinant receptor to CD8 ⁺ cells expressing the recombinant receptor and/or CD4 ⁺ cells to CD8 ⁺ cells, which ratio is optionally, or approximately, 1:1 or from about 1:3 to about 3:1; and/or CD4 ⁺ T cells comprising the receptor in one of the first and second compositions and CD8 ⁺ T cells comprising the receptor in the other of the first and second compositions are present in a ratio, the ratio being optionally, or approximately, 1 :1 or from about 1:3 to about 3:1; and/or CD4 ⁺ T cells comprising the receptor and CD8 ⁺ T cells comprising the receptor administered in the first and second compositions are present in a ratio, the ratio optionally being or about 1:1 or from about 1:1: 3 to approximately 3:1. In some embodiments of any of the methods of the invention, the specified ratio is, or approximately, 1:1. In some embodiments of any of the methods of the invention, the dose of T cells is administered to the patient 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 some embodiments of any of the methods of the invention, the T cell dose is administered as a double dose comprising a first T cell dose and a subsequent T cell dose, wherein one or both of the first dose and the second dose comprises multiple T cell compositions. In some embodiments of any of the methods of the invention, the subsequent dose is administered at a time point at least or greater than about 7 days, or 14 days after and less than about 28 days after the initiation of the first dose of cells.

[0013] In some embodiments of any of the methods of the invention or embodiments, the cell dose comprises from or from about 1 x 10 ⁵ to or up to about 5 x 10 ⁸ total recombinant receptor-expressing T cells or total T cells, from or from about 1 x 10 ⁵ to or up to about 1 x 10 ⁸ total recombinant receptor-expressing T cells or total T cells, from or from about 5 x 10 ⁵ to or up to about 1 x 10 ⁷ total recombinant receptor expressing T cells or total T cells, or from or from about 1 x 10 ⁶ to 1 x 10 ⁷ total recombinant receptor-expressing T cells or total T cells, inclusive. In some embodiments of any of the methods of the invention, the dose of T cells comprises administering no more than 1 x 10 ⁸ total recombinant receptor-expressing T cells, or total T cells, no more than 1 x 10 ⁷ total recombinant receptor-expressing T cells, or total T cells, not more than 0.5 x 10 ⁷ total recombinant receptor-expressing T cells or total T cells, not more than 1 x 10 ⁶ total recombinant receptor-expressing T cells or total T cells, not more than 0.5 x 10 ⁶ total recombinant receptor-expressing T cells or total T cells. In some embodiments of any of the methods of the invention, the dose of T cells includes from or from about 5 x 10 ⁷ expressing the recombinant receptor T cells to 1 x 10 ⁸ expressing the recombinant receptor T cells, inclusive.

[0014] In some embodiments of any of the methods of the invention, the recombinant receptor specifically binds to an antigen associated with a disease or condition, or expressed in cells in a lesion environment associated with the disease or condition. In some embodiments of any of the methods of the invention, the disease or condition is cancer. In some embodiments of any of the methods of the invention, the disease or condition is myeloma, leukemia, or lymphoma. In some embodiments of any of the methods of the invention, the antigen is ROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2/neu (erbB2 receptor tyrosine kinase), L1-CAM, CD19, CD20, CD22, mesothelin, CEA and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, erb-B2, erb-B3, erb-B4, erbB dimers, EGFR vIII, folta-binding protein (FBP), FCRL5, FCRH5, embryonic acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kinase insertion region receptor (kdr), kappa light chain, Ley antigen, L1 cell adhesion molecule, (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, preferably expressed melanoma antigen (PRAME), survivin, TAG72, B7-H6, IL-13 alpha receptor 2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AI MAGE Al, HLA-A2 NY- ESO-1, PSCA, folic acid receptor alpha, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, embryonic AchR, NKG2D ligands, CD44v6, dual antigen, testicular cancer antigen, mesothelin, mouse CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), Her2/neu, estrogen receptor, progesterone receptor , ephrinB2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms tumor 1 (WT-1), cyclin, cyclin A2, CCL-1, CD138, G-coupled 5D receptor protein (GPCR5D), or a pathogen-specific antigen. In some embodiments of any of the methods of the invention, the antigen is CD19.

[0015] In some embodiments of any of the methods of the invention, the disease or condition is B cell cancer and/or acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin's lymphoma (NHL), and diffuse large B cell lymphoma (DLBCL). In some embodiments of any of the methods of the invention, the disease or condition is NHL and the NHL is selected from the group consisting of aggressive NHL, diffuse large B cell lymphoma (DLBCL), NOS (unspecified) (de novo and transformed from indolent) , primary mediastinal large B-cell lymphoma (PMBCL), T-cell/histocytic large B-cell lymphoma (TCHRBCL), Burkitt's lymphoma, mantle cell lymphoma (MCL) and/or follicular lymphoma (FL), optionally stage 3B follicular lymphoma (FL3B ).

[0016] In some embodiments of any of the methods of the invention, the recombinant receptor includes an extracellular domain containing an antigen-binding domain. In some embodiments, the antigen binding domain is or includes an antibody or an antibody fragment, which is optionally a single chain fragment. In some embodiments of any of the methods of the invention, the fragment comprises the variable regions of an antibody to which a flexible linker is attached. In some embodiments, the implementation of the fragment includes scFv. In some embodiments of any of the methods of the invention, the recombinant receptor also includes a spacer and/or a hinge region.

[0017] In some embodiments of any of the methods of the invention, the recombinant receptor includes an intracellular signaling region. In some embodiments of any of the methods of the invention, the intracellular signaling region includes an intracellular signaling domain. In some embodiments of any of the methods of the invention, the intracellular signaling domain is or includes a primary signaling domain, a signaling domain that is capable of inducing a primary activation signal in a T cell, a T cell receptor (TCR) component signaling domain, and/or a signaling domain, containing an immunoreceptor tyrosine activating motif (ITAM). In some embodiments, the intracellular signaling domain is or includes an intracellular signaling domain of the CD3 chain, optionally the CD3-zeta (CD3ζ) chain, or a signal portion thereof.

[0018] In some embodiments of any of the methods of the invention, the recombinant receptor also includes a transmembrane domain located between the extracellular domain and the intracellular signaling region.

[0019] In some embodiments of any of the methods of the invention, the intracellular signaling domain also includes a costimulatory signaling domain. In some embodiments, the costimulatory signaling region includes an intracellular signaling domain of the costimulatory T cell molecule, or a signaling portion thereof. In some embodiments of any of the methods of the invention, the co-stimulatory signaling region comprises a CD28, 4-1BB, or ICOS intracellular signaling domain, or a signal portion thereof. In some embodiments, the co-stimulatory signaling region is located between the transmembrane domain and the intracellular signaling region.

[0020] In some embodiments of any of the methods of the invention, the recombinant receptor is a chimeric antigen receptor (CAR), optionally the recombinant receptor is a chimeric antigen receptor (CAR), optionally the CAR includes an extracellular antigen-recognition domain specifically binding to an antigen and an intracellular signaling a domain including ITAM, optionally the intracellular signaling domain including the intracellular domain of the CD3-zeta (CD3ζ) chain; and/or wherein the CAR further comprises a co-stimulatory signaling domain, optionally including a CD28 or 4-1BB signaling domain.

[0021] In some embodiments, the articles of manufacture include a container, such as a vial, comprising a composition comprising CD4 ⁺ T cells expressing the recombinant receptor and instructions for administering to a patient having a disease or condition the CD4 ⁺ T cell composition as a plurality of compositions. with a composition comprising CD8 ⁺ T cells expressing the recombinant receptor, or a single dose of cells comprising all or part of a plurality of CD4 ⁺ T cells, and a composition comprising CD8 ⁺ T cells expressing the recombinant receptor. In some embodiments, the article of manufacture includes a container, such as a vial, comprising a composition comprising CD8 ⁺ T cells expressing the recombinant receptor and instructions for administering to a patient having a disease or condition the CD8 ⁺ T cell composition in a plurality of compositions with the composition, comprising CD4 ⁺ T cells expressing the recombinant receptor, or a single dose of cells comprising all or part of a plurality of CD4 ⁺ T cells, and a composition comprising CD8 ⁺ T cells expressing the recombinant receptor.

[0022] In some of the embodiments, the CAR includes, in that order, the CAR includes an scFv antigen-specific transmembrane domain, a cytoplasmic signaling domain derived from a co-stimulatory molecule that optionally is or includes 4-1BB, and a cytoplasmic signaling domain, derived from a primary signaling ITAM-containing molecule, which optionally is or includes a CD3zeta signaling domain and optionally further includes a spacer between the transmembrane domain and scFv;

[0023] In some of the embodiments, the CAR includes, in that order, an scFv antigen-specific transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule that optionally is or includes a 4-1BB signaling domain, and a cytoplasmic signaling domain, derived from a primary signaling ITAM-containing molecule, which is optionally a CD3zeta signaling domain.

[0024] In some of the embodiments, the CAR comprises or consists of, in that order, an antigen-specific scFv, a spacer, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which is optionally a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from a primary ITAM-containing signaling molecule, which optionally is or includes the CD3zeta signaling domain.

[0025] In some aspects, a spacer is a polypeptide spacer that (a) includes or consists of all or part of an immunoglobulin hinge region or a modified version thereof, or is about 15 amino acids or less and does not include CD28 extracellular region or CD8 extracellular region, (b) includes or consists of all or part of the hinge region of an immunoglobulin, optionally the IgG4 core or a modified version thereof, and/or comprises about 15 amino acids or less and does not include CD28 extracellular region or CD8 extracellular region, or (c) is 12 or about 12 amino acids long and/or includes or consists of all or part of the hinge region of an immunoglobulin, optionally IgG4 or a modified version thereof; or (d) has or consists of the sequence 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 above having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or more sequence identity with the specified, or (e) includes or consists of the formula X ₁ PPX ₂ P, where X ₁ is glycine, cysteine or arginine, and X ₂ is cysteine or threonine; and/or the costimulatory region 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 with it; and/or the primary signaling domain comprises SEQ ID NO: 13 or 14 or 15, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with them; and/or scFv comprises a CDRL1 RASQDISKYLN sequence (SEQ ID NO: 35), a CDRL2 SRLHSGV sequence (SEQ ID NO: 36) and/or a CDRL3 GNTLPYTFG sequence (SEQ ID NO: 37), and/or a CDRH1 DYGVS sequence (SEQ ID NO: : 38), the VIWGSETTYYNSALKS CDRH2 sequence (SEQ ID NO: 39) and/or the YAMDYWG CDRH3 sequence (SEQ ID NO: 40), or wherein the scFv comprises an FMC63 heavy chain variable region and an FMC63 light chain variable region and/or an FMC63 CDRL1 sequence, an FMC63 CDRL2 sequence, an FMC63 CDRL3 sequence, an FMC63 CDRH1 sequence, an FMC63 CDRH2 sequence, and an FMC63 CDRH3 sequence, either binds to the same epitope or competes for binding to any of those specified, and optionally the scFv includes, in that order, a V _H , a linker, optionally including SEQ ID NO: 24, and V _L and/or scFv includes a flexible linker and/or includes the amino acid sequence shown as SEQ ID NO: 24.

[0026] In some embodiments, the spacer comprises or consists of SEQ ID NO: 1, the costimulatory region 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 with it, the transmembrane domain has CD28 or includes SEQ ID NO: 9 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 with it, scFv contains a binding domain or CDR, or V _H and V _L FMC63, the primary signaling domain contains SEQ ID NO: 13, 14 or 15 and/or their variant having at least 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with them.

[0027] In some embodiments, the spacer comprises or consists of SEQ ID NO: 30, the costimulatory region 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 with it, the transmembrane domain has CD28 or includes SEQ ID NO: 9 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 with it, scFv contains a binding domain or CDR, or V _H and V _L FMC63, the primary signaling domain contains SEQ ID NO: 13, 14 or 15 and/or their variant having at least 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with them.

[0028] In some embodiments, the spacer comprises or consists of SEQ ID NO: 31, the costimulatory region 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 with it, the transmembrane domain has CD28 or includes SEQ ID NO: 9 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 with it, scFv contains a binding domain or CDR, or V _H and V _L FMC63, the primary signaling domain contains SEQ ID NO: 13, 14 or 15 and/or their variant having at least 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with them.

[0029] In some embodiments, the spacer comprises or consists of SEQ ID NO: 33, the costimulatory region 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 with it, the transmembrane domain has CD28 or includes SEQ ID NO: 9 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 with it, scFv contains a binding domain or CDR, or V _H and V _L FMC63, the primary signaling domain contains SEQ ID NO: 13, 14 or 15 and/or their variant having at least 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with them.

[0030] In some embodiments, the spacer comprises or consists of SEQ ID NO: 34, the costimulatory region 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 with it, the transmembrane domain has CD28 or includes SEQ ID NO: 9 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 with it, scFv contains a binding domain or CDR, or V _H and V _L FMC63, the primary signaling domain contains SEQ ID NO: 13, 14 or 15 and/or their variant having at least 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with them.

[0031] In some embodiments of any of the articles of the invention, the recombinant receptor expressed by CD4 ⁺ cells and the recombinant receptor expressed by CD8 ⁺ T cells are the same or different. In some embodiments of any of the articles of the invention, the vial comprises more than or more than about 10 x 10 ⁶ T cells or expressing a recombinant T cell receptor, more than or more than about 15 x 10 ⁶ T cells or expressing a recombinant T receptor -cells, more than or more than about 25 x 10 ⁶ T cells or expressing a recombinant T cell receptor.

[0032] In some embodiments of any of the articles of the invention, the vial comprises from about 10 million cells per ml to about 70 million cells per ml, from about 10 million cells per ml to about 50 million cells per ml, from about 10 million cells per ml. ml to about 25 million cells per ml, from about 10 million cells per ml to about 15 million cells per ml, from 15 million cells per ml to about 70 million cells per ml, from about 15 million cells per ml to about 50 million cells per ml, from about 15 million cells per ml to about 25 million cells per ml, from about 25 million cells per ml to about 70 million cells per ml, from about 25 million cells per ml to about 50 million cells per ml, and from about 50 million cells per ml to approximately 70 million cells per ml. In some embodiments of any of the articles of the invention, the composition further comprises a cryoprotectant and/or the article further includes instructions for thawing the composition prior to administration to a patient.

[0033] In some embodiments of any of the articles of the invention, a composition or plurality of compositions comprises a cell dose comprising from or about 2 x 10 ⁷ to about 4 x 10 ⁷ CD8 ⁺ cells, e.g., about 2 x 10 ⁷ , 2.5 x 10 ⁷ , 3 x 10 ⁷ , 3.5 x 10 ⁷ or 4 x 10 ⁷ CD8 ⁺ cells, and from or from about 2 x 10 ⁷ to about 4 x 10 ⁷ CD4 ⁺ cells, for example, about 2 x 10 ⁷ , 2.5 x 10 ⁷ , 3 x 10 ⁷ , 3.5 x 10 ⁷ or 4 x 10 ⁷ CD4 ⁺ cells, inclusive. In some embodiments, the implementation of the composition or set of compositions includes a dose of cells, including approximately 3 x 10 ⁷ CD8 ⁺ cells and 3.5 x 10 ⁷ CD4 ⁺ cells.

[0034] In some embodiments of any of the articles of the invention, the plurality of cell compositions comprises a specific ratio of CD4 ⁺ cells expressing the recombinant receptor to CD8 ⁺ cells expressing the recombinant receptor and/or CD4 ⁺ cells to CD8 ⁺ cells, this ratio being optional is about 1:1, or about 1:3 to about 3:1. In some embodiments of any of the articles of the invention, the specific ratio is, or approximately, 1:1. In some embodiments of any of the articles of the invention, the plurality of compositions, collectively, comprises a cell dose comprising from or about 1 x 10 ⁵ to 5 x 10 ⁸ total recombinant receptor-expressing T cells, or total T cells, from 1 x 10 ⁵ up to 1 x 10 ⁸ total recombinant receptor-expressing T cells or total T cells, from or from about 5 x 10 ⁵ to 1 x 10 ⁷ total recombinant receptor-expressing T cells or total T cells, or from or from about 1 x 10 ⁶ to 1 x 10 ⁷ total recombinant receptor-expressing T cells or total T cells, inclusive. In some embodiments of any of the articles of the invention, the plurality of compositions, collectively, comprises a cell dose comprising no more than 1 x 10 ⁸ total recombinant receptor-expressing T cells, or total T cells, no more than 1 x 10 ⁷ total recombinant receptor-expressing T cells or total T cells, not more than 0.5 x 10 ⁷ total recombinant receptor-expressing T cells or total T cells, not more than 1 x 10 ⁶ total recombinant receptor-expressing T cells or total T cells , not more than 0.5 x 10 ⁶ total recombinant receptor-expressing T cells or total T cells. In some embodiments of any of the articles of the invention, the plurality of compositions, collectively, comprises a cell dose comprising from or from about 5 x 10 ⁷ recombinant receptor-expressing T cells and 1 x 10 ⁸ recombinant receptor-expressing T cells, inclusive.

[0035] In some embodiments of any of the articles of the invention, the instructions define the administration of a composition comprising CD4 ⁺ T cells and a composition comprising CD8 ⁺ T cells at 0 to 12 hour intervals, at 0 to 6 hour intervals, or with interval from 0 to 2 hours. In some embodiments of any of the articles of the invention, the instructions specify administering a composition comprising CD4 ⁺ T cells and a composition comprising CD8 ⁺ T cells at intervals of 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. In some embodiments of any of the articles of the invention, the instructions define the administration of the composition comprising CD4 ⁺ T cells prior to the administration of the composition comprising CD8 ⁺ cells. In some embodiments of any of the articles of the invention, the instructions define the administration of the composition comprising CD8 ⁺ T cells prior to the administration of the composition comprising CD4 ⁺ cells.

[0036] In some embodiments of any of the articles of the invention, the recombinant receptor specifically binds to an antigen associated with a disease or condition, or expressed in cells in a lesion environment associated with the disease or condition. In some embodiments of any of the articles of the invention, the disease or condition is cancer. In some embodiments of any of the articles of the invention, the disease or condition is myeloma, leukemia, or lymphoma.

[0037] In some embodiments of any of the articles of the invention, the antigen is ROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2/neu (erbB2 receptor tyrosine kinase), L1-CAM, CD19, CD20 , CD22, mesothelin, CEA and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, erb -B2, erb-B3, erb-B4, erbB dimers, EGFR vIII, folta-binding protein (FBP), FCRL5, FCRH5, embryonic acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R -alpha2, kinase insertion region receptor (kdr), kappa light chain, Ley antigen, L1 cell adhesion molecule, (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, preferably expressed melanoma antigen (PRAME), survivin, TAG72, B7-H6, IL-13 alpha receptor 2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AI MAGE Al, HLA- A2 NY-ESO-1, PSCA, folic acid receptor alpha, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, embryonic AchR, NKG2D ligands, CD44v6, dual antigen, testicular cancer antigen, mesothelin, mouse CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), Her2/neu, estrogen receptor , progesterone receptor, ephrinB2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms tumor 1 (WT-1), cyclin, cyclin A2, CCL-1, CD138, 5D receptor coupled with G-protein (GPCR5D), or pathogen-specific antigen. In some embodiments of any of the articles of the invention, the antigen is CD19.

[0038] In some embodiments of any of the articles of the invention, the disease or condition is B cell cancer and/or acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin's lymphoma (NHL), and diffuse large cell B-cell lymphoma (DLBCL). In some embodiments of any of the methods of the invention, the disease or condition is NHL and the NHL is selected from the group consisting of aggressive NHL, diffuse large B cell lymphoma (DLBCL), NOS (unspecified) (de novo and transformed from indolent) , primary mediastinal large B-cell lymphoma (PMBCL), T-cell/histocytic large B-cell lymphoma (TCHRBCL), Burkitt's lymphoma, mantle cell lymphoma (MCL) and/or follicular lymphoma (FL), optionally stage 3B follicular lymphoma (FL3B ).

[0039] In some embodiments of any of the articles of the invention, the T cells are primary T cells obtained from a patient. In some embodiments of any of the articles of the invention, the T cells are autologous to the patient. In some embodiments of any of the articles of the invention, the T cells are allogeneic to the patient.

[0040] In some embodiments of any of the articles of the invention, the recombinant receptor is or includes a functional non-TCR antigen receptor or TCR or an antigen-binding fragment thereof. In some embodiments, the recombinant receptor is a chimeric antigen receptor (CAR).

[0041] In some embodiments of any of the articles of the invention, the recombinant receptor includes an extracellular domain containing an antigen-binding domain. In some embodiments, the antigen binding domain is or includes an antibody or antibody fragment, which is optionally a single chain fragment. In some embodiments, the implementation of the fragment includes the variable regions of the antibody, which is attached to a flexible linker. In some embodiments, the implementation of the fragment includes scFv.

[0042] In some embodiments of any of the articles or methods of the invention, the recombinant receptor also includes a spacer and/or a hinge region.

[0043] In some embodiments of any of the articles or methods of the invention, the recombinant receptor includes an intracellular signaling region. In some embodiments, the intracellular signaling region includes an intracellular signaling domain. In some embodiments of any of the articles of the invention, the intracellular signaling domain is or includes a primary signaling domain, a signaling domain that is capable of inducing a primary activation signal in a T cell, a T cell receptor (TCR) component signaling domain, and/or a signaling domain, containing an immunoreceptor tyrosine activating motif (ITAM). In some embodiments, the intracellular signaling domain is or includes an intracellular signaling domain of the CD3 chain, optionally the CD3-zeta (CD3ζ) chain, or a signal portion thereof.

[0044] In some embodiments of any of the articles or methods of the invention, the recombinant receptor also includes a transmembrane domain located between the extracellular domain and the intracellular signaling region.

[0045] In some embodiments of any of the articles or methods of the invention, the intracellular signaling domain also includes a costimulatory signaling domain. In some embodiments, the costimulatory signaling region includes the intracellular signaling domain of the costimulatory T cell molecule, or a signaling portion thereof. In some embodiments of any of the articles of the invention, the co-stimulatory signaling region comprises the intracellular signaling domain of CD28, 4-1BB, or ICOS, or a signaling portion thereof. In some embodiments, the costimulatory signaling region is located between the transmembrane domain and the intracellular signaling region.

[0046] In some embodiments, the methods and articles of manufacture are for or capable of treating a patient having non-Hodgkin's lymphoma (NHL). In some aspects, the method includes and/or the article of manufacture defines or includes formulations capable of administering a dose or a plurality of T cells to a patient. In some aspects, T cells, including T cells such as CD8 ⁺ T cells and/or CD4 ⁺ T cells, expressing a chimeric antigen receptor (CAR) that specifically binds to a target antigen expressed by NHL.

[0047] In some embodiments, the dose of T cells includes from or from about 5 x 10 ⁷ CAR-expressing T cells and 1 x 10 ⁸ CAR-expressing T cells, inclusive; and NHL includes diffuse large B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), NOS (de novo or transformed from indolent lymphoma), or stage 3B follicular lymphoma, and wherein the patient is or has been identified as having 0 or 1 according to Eastern Cooperative Oncology Group Performance Status (ECOG).

[0048] In some embodiments of any of the embodiments of the invention, the methods further comprise identifying, or the article of manufacture includes informing, treating a patient having diffuse large B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), NOS ( de novo or transformed from indolent lymphoma) or stage 3B follicular lymphoma having ^an ECOG status of 0 or 1. CD8 ⁺ to CAR expressing cells and/or CD4 ⁺ cells to CD8 ⁺ cells, the ratio being optionally about 1:1 or about 1:3 to about 3:1.

[0049] In some aspects, embodiments are for the treatment of a patient having non-Hodgkin's lymphoma (NHL) and comprise administering to the patient a dose of T cells comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds to a target antigen expressed NHL. In some aspects, the dose of T cells includes a certain ratio of CD4 ⁺ cells expressing CAR to CD8 ⁺ cells expressing CAR, and/or CD4 ⁺ cells to CD8 ⁺ cells, and this ratio in some aspects is approximately or is 1:1. In some aspects, NHL includes diffuse large B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), NOS (unspecified) (de novo or transformed from indolent lymphoma), or stage 3B follicular lymphoma.

[0050] In some embodiments of any of the embodiments of the invention, the patient is or has been identified as, or defined as having status 0, 1 or 2 according to the Eastern Cooperative Oncology Group Performance Status (ECOG). In some embodiments of any of the embodiments of the invention, the patient is or has been identified as, or determined to have an ECOG status of 0 or 1.

[0051] In some embodiments of any of the embodiments of the invention, the methods and/or uses and/or administration of cells according to the products achieve certain results and/or are associated with a certain reduced risk of toxicity, for example, in a population of patients receiving treatment according to the methods or according to information provided in the product. In some aspects, at least 35%, at least 40%, or at least 50% of patients treated according to the method achieve a complete response (CR) and/or a sustained CR; and/or at least 50%, at least 60%, or at least 70% of patients treated according to the method achieve an objective response (OR) and/or sustained OR. In some embodiments of any of the methods of the invention, the response lasts more than 3 months or more than 6 months. In some embodiments, at least 40%, at least 50%, at least 60%, at least 70% of patients who, at the time of administration or prior to administration of the cell dose, had or were identified as having double/triple hit lymphoma or recurrence, optionally relapse within 12 months, after administration of autologous stem cell transplant (ASCT) achieved OR, and optionally OR lasted for or more than 3 months or for 6 months or more. In some embodiments of any of the methods of the invention, greater than or greater than about 50% of patients treated according to the method do not exhibit Grade 3 or greater Cytokine Release Syndrome (CRS) or Grade 3 or greater neurotoxicity. In some embodiments, such patients do not exhibit early onset CRS and/or neurotoxicity.

[0052] The invention relates to methods for assessing the likelihood of response to cell therapy, including: assessing the level, amount or concentration of one or more analytes in a biological sample, and one or more analytes selected from ferritin, LDH, CXCL10, G-CSF and IL-10 wherein: the biological sample is from a patient who is a candidate for cell therapy treatment, said cell therapy comprising a dose of engineered cells expressing the recombinant receptor; and the biological sample was obtained from the patient prior to the use of cell therapy and/or said biological sample does not include a recombinant receptor and/or said genetically engineered cells; and comparing, individually, the level, amount, or concentration of the analyte in the sample with the threshold value, thereby determining the likelihood that the patient will achieve a response to cell therapy. In some embodiments, the methods also include administering cell therapy to a patient if the patient is determined to be likely to achieve a response.

[0053] The invention relates to methods for selecting a patient for treatment, including: assessing the level, amount or concentration of one or more analytes in a biological sample, wherein one or more analytes are selected from ferritin, LDH, CXCL10, G-CSF and IL-10, and : the biological sample is from a patient who is a candidate for cell therapy treatment, said cell therapy comprising a dose of engineered cells expressing the recombinant receptor; and the biological sample was obtained from a patient prior to the use of cell therapy and/or said biological sample does not include a recombinant receptor and/or said genetically engineered cells; and selecting a patient for which a probability of response to treatment is determined based on the results of determining the probability that the patient will achieve a response to cell therapy by comparing, individually, the level, amount or concentration of the analyte in the sample with a threshold value. In some embodiments, the implementation of the methods also include the application of cell therapy to the patient selected for treatment.

[0054] The invention relates to methods of treatment, including: selecting a patient for whom the probability of responding to cell therapy treatment is determined based on the results of determining the probability that the patient will achieve a response to cell therapy, by comparing, individually, the level, amount or concentration of one or more analytes in a biological sample, wherein one or more of the analytes is selected from ferritin, LDH, CXCL10, G-CSF, and IL-10, with a threshold value, wherein: the biological sample comes from a patient who is a candidate for cell therapy treatment, said cell therapy includes a dose of genetically engineered cells expressing the recombinant receptor; and the biological sample was obtained from the patient prior to the use of cell therapy and/or said biological sample does not include a recombinant receptor and/or said genetically engineered cells; and applying cell therapy to the patient selected for treatment.

[0055] In some embodiments, a patient is determined to be likely to achieve a response if the level, amount, or concentration of one or more analytes is below a threshold, and the patient is unlikely to achieve a response if the level, amount, or concentration of one or more analytes is above a threshold. .

[0056] In some embodiments, the threshold is within 25%, 20%, 15%, 10%, or 5% and/or within a standard deviation below the median or mean level, amount or concentration, or equal to or approximately equal to the median or the average level, amount, or concentration of an analyte in a biological sample obtained from a group of patients prior to receiving cell therapy, with each of the patients in the group continuing to achieve a response after administration of a therapeutic composition of cells expressing the recombinant receptor for the treatment of the same disease or condition.

[0057] In some embodiments, the threshold is within 25%, 20%, 15%, 10%, or 5% and/or within a standard deviation above the median or mean level, amount, or concentration of an analyte in a biological sample obtained from a group patients prior to receiving cell therapy, wherein each of the patients in the group continued to show stable disease (SD) and/or progressive disease (PD) after administration of the recombinant receptor-expressing cell composition for the treatment of the same disease or condition.

[0058] In some embodiments, the response includes an objective response. In some embodiments, the objective response includes a complete response (CR) or a partial response (PR).

[0059] The invention relates to methods for assessing the likelihood of a long-term response to cell therapy, including: assessing the level, amount or concentration of one or more analytes in a biological sample, wherein one or more analytes is selected from LDH, ferritin, CRP, D-dimer, SAA- 1, IL-6, IL-10, IL-15, IL-16, TNF-α, IFN-γ, MIP-1α, CXCL-10, IL-8, MCP-1 and MIP-1β, moreover: biological sample is derived from a patient who is a candidate for cell therapy treatment, said cell therapy comprising a dose of engineered cells expressing the recombinant receptor; and the biological sample was obtained from the patient prior to the use of cell therapy and/or said biological sample does not include a recombinant receptor and/or said genetically engineered cells; and comparing, individually, the level, amount, or concentration of the analyte in the sample with a threshold value, thereby determining the likelihood that the patient will achieve a sustained response to cell therapy.

[0060] In some embodiments, the methods also include administering cell therapy to a patient if the patient is shown to be likely to achieve a response.

[0061] The invention relates to methods for selecting a patient for treatment, including: assessing the level, amount or concentration of one or more analytes in a biological sample, wherein one or more analytes are selected from LDH, ferritin, CRP, D-dimer, SAA-1, IL -6, IL-10, IL-15, IL-16, TNF-α, IFN-γ, MIP-1α, CXCL-10, IL-8, MCP-1 and MIP-1β, moreover: the biological sample comes from the patient , which is a candidate for cell therapy treatment, said cell therapy comprising a dose of engineered cells expressing the recombinant receptor; and the biological sample was obtained from the patient prior to the use of cell therapy and/or said biological sample does not include a recombinant receptor and/or said genetically engineered cells; and selecting a patient for which a probability of response to treatment is determined based on the results of determining the probability that the patient will achieve a long-term response to cell therapy by comparing, individually, the level, amount or concentration of the analyte in the sample with a threshold value. In some embodiments, the implementation of the methods also include the application of cell therapy to the patient selected for treatment.

[0062] The invention relates to methods of treatment, including: selecting a patient for whom the probability of responding to treatment with cell therapy is determined based on the results of determining the probability that the patient will achieve a long-term response to cell therapy, by comparing, individually, the level, amount or concentration of one or more than analytes in a biological sample with a threshold value, and one or more analytes is selected from LDH, ferritin, CRP, D-dimer, SAA-1, IL-6, IL-10, IL-15, IL-16, TNF-α, IFN-γ, MIP-1α, CXCL-10, IL-8, MCP-1 and MIP-1β, wherein: the biological sample originates from a patient who is a candidate for cell therapy treatment, said cell therapy comprising a dose of genetically engineered cells expressing the recombinant receptor; and the biological sample was obtained from the patient prior to the use of cell therapy and/or said biological sample does not include a recombinant receptor and/or said genetically engineered cells; and applying cell therapy to the patient selected for treatment.

[0063] In some embodiments, the patient is likely to achieve a sustained response if the level, amount, or concentration of one or more analytes is below a threshold, and the patient is unlikely to achieve a sustained response if the level, amount, or concentration of one or more analytes is above threshold value.

[0064] In some embodiments, the threshold is within 25%, 20%, 15%, 10%, or 5% and/or within a standard deviation below the median or mean level, amount or concentration, or equal to or approximately equal to the median or the average level, amount, or concentration of an analyte in a biological sample obtained from a group of patients prior to receiving cell therapy, with each of the patients in the group continuing to achieve a sustained response after administration of the composition of cells expressing the recombinant receptor for the treatment of the same disease or condition.

[0065] In some embodiments, the threshold is within 25%, 20%, 15%, 11%, or 5% and/or within a standard deviation above the median or mean level, amount, or concentration of an analyte in a biological sample obtained from a group patients before receiving cell therapy, and each of the patients of the group did not achieve a long-term response after administration of a composition of cells expressing the recombinant receptor for the treatment of the same disease or condition.

[0066] In some embodiments, a sustained response includes a complete response (CR) or a partial response (PR) that lasts for 3 months or more, 4 months, 5 months, or 6 months.

[0067] In some embodiments, the long-term response includes a CR or PR that lasts for at least 3 months.

[0068] The invention relates to methods for assessing the risk of developing toxicity after the introduction of cell therapy, including assessing the level, amount or concentration of one or more analytes in a biological sample from a patient or a volumetric parameter of a tumor mass in a patient, wherein one or more analytes are selected from LDH, Ferritin , C-reactive protein (CRP), D-dimer (fibrin breakdown product), IL-6, IL-8, IL-10, IL-15, IL-16 TNF-α, IFN-α2, MCP-1, MIP -1α and MIP-1β, moreover: the patient is a candidate for treatment with cell therapy, and said cell therapy optionally includes a dose or composition of genetically engineered cells expressing the recombinant receptor; and the biological sample was obtained from the patient prior to the use of cell therapy and/or said biological sample does not include a recombinant receptor and/or said genetically engineered cells; and comparing, individually, the level, amount, or concentration of the analyte in the sample, or the volumetric parameter of the tumor mass, with a threshold value, thereby determining the risk of developing toxicity after administration of the cell therapy.

[0069] The invention relates to methods for identifying a patient, including assessing the level, amount or concentration of one or more analytes in a biological sample from a patient or a volumetric parameter of a tumor mass in a patient, wherein one or more analytes are selected from LDH, Ferritin, C-reactive protein ( CRP), D-dimer (fibrin breakdown product), IL-6, IL-8, IL-10, IL-15, IL-16 TNF-α, IFN-α2, MCP-1, MIP-1α and MIP-1β wherein: the patient is a candidate for cell therapy treatment, said cell therapy optionally comprising a dose or composition of engineered cells expressing the recombinant receptor; and the biological sample was obtained from the patient prior to the use of cell therapy and/or said biological sample does not include a recombinant receptor and/or said genetically engineered cells; and identifying a patient at risk of developing toxicity after administration of the cell therapy based on comparison, individually, of the level, amount or concentration of the analyte in the sample or tumor mass volumetric parameter with a threshold value.

[0070] The invention relates to methods of treatment comprising assessing the level, amount or concentration of one or more analytes in a biological sample from a patient or a volumetric parameter of a tumor mass in a patient, wherein one or more analytes are selected from LDH, Ferritin, C-reactive protein (CRP ), D-dimer (fibrin degradation product), IL-6, IL-8, IL-10, IL-15, IL-16 TNF-α, IFN-α2, MCP-1, MIP-1α and MIP-1β, moreover: the patient is a candidate for treatment with cell therapy, and the specified cell therapy optionally includes a dose or composition of genetically engineered cells expressing the recombinant receptor; and the biological sample was obtained from the patient prior to the use of cell therapy and/or said biological sample does not include a recombinant receptor and/or said genetically engineered cells; and comparing, individually, the level, amount or concentration of the analyte in the sample or the volumetric parameter of the tumor mass with a threshold value, thereby determining the risk of developing toxicity after the introduction of cell therapy; and subsequently, or based on the results of the assessment, administering to the patient a cell therapy, and optionally an agent or other treatment capable of treating, preventing, delaying, attenuating, or reducing the development or risk of developing toxicity.

[0071] In some embodiments, the biological sample is a blood or plasma sample.

[0072] In some embodiments, the tumor mass volumetric parameter is the sum of product dimensions (SPD) or is a volumetric measurement based on CT and/or MRI imaging or other body imaging. In some embodiments, the volume parameter of the tumor mass is determined prior to treatment, prior to apheresis, or prior to production of a cell product.

[0073] In some embodiments, the methods also include monitoring the patient for symptoms of toxicity if the patient is receiving cell therapy and identifying the patient as being at risk of developing toxicity.

[0074] In some embodiments, the patient is at risk of developing toxicity if the level, amount, or concentration of one or more analytes or the volumetric parameter of the tumor mass is above a threshold value, and the patient is at low risk of developing toxicity if the level, amount, or concentration of one or more analytes or the volumetric parameter of the tumor mass is below the threshold value.

[0075] In some embodiments, the threshold is within 25%, 20%, 15%, 10%, or 5% and/or within a standard deviation above the median or mean level, amount or concentration, or equal to or approximately equal to the median or mean level, amount, or concentration of an analyte or tumor mass volume parameter in a biological sample obtained from a group of patients prior to receiving cell therapy, and each of the patients in the group continues to develop no toxicity after receiving a composition of cells expressing the recombinant receptor to treat that same disease or condition.

[0076] In some embodiments, the cut-off value is within 25%, 20%, 15%, 10%, or 5% and/or within a standard deviation below the median or mean level, analyte amount or concentration, or volumetric parameter of the tumor mass in the biological a sample obtained from a group of patients prior to receiving cell therapy, and each of the patients of the group continued to develop toxicity after receiving the composition of cells expressing the recombinant receptor for the treatment of the same disease or condition.

[0077] In some embodiments, the toxicity is neurotoxicity or CRS.

[0078] In some embodiments, the toxicity is neurotoxicity or CRS grade 1 or higher.

[0079] In some embodiments, the toxicity is severe neurotoxicity, or is grade 2 or higher neurotoxicity, grade 3 or higher neurotoxicity, at least prolonged grade 3 or grade 4 or higher neurotoxicity, or grade 5 neurotoxicity; or the toxicity is severe CRS or includes CRS grade 2 or higher or grade 3 or higher.

[0080] In some embodiments, the toxicity is neurotoxicity and the tumor bulk volume parameter is SPD and one or more analytes are selected from LDH, IL-10, IL-15, IL-16, TNF-α, and MIP-1β.

[0081] In some embodiments, the toxicity is neurotoxicity and one or more analytes are assessed and the analytes are selected from LDH, Ferritin, CRP, IL-6, IL-8, IL-10, TNF-α, IFN-α2, MCP- 1 and MIP-1β.

[0082] In some embodiments, the toxicity is neurotoxicity, and one or more analytes are assessed, and the analytes are selected from IL-8, IL-10, and CXCL10.

[0083] In some embodiments, the implementation of neurotoxicity is severe neurotoxicity or neurotoxicity of degree 3 or higher.

[0084] In some embodiments, the toxicity is CRS and one or more analytes or tumor mass volume parameter selected from LDH, SPD, CRP, d-dimer, IL-6, IL-15, TNF-α, and MIP-1α.

[0085] In some embodiments, the implementation of the CRS is severe CRS or CRS grade 3 or higher.

[0086] In some embodiments, if the patient is identified as being at risk of developing toxicity, administering to the patient: (1) a treatment agent or other agent capable of treating, preventing, delaying, attenuating, or reducing the development or risk of developing toxicity, and (2) cell therapy, and the introduction of the agent must be entered (i) before, (ii) within one, two or three days, (iii) simultaneously with and/or (iv) at the first increase in temperature after the initiation of the introduction of cell therapy to the patient; and/or cell therapy at a reduced dose or at a dose not associated with a risk of developing toxicity or severe toxicity, or not associated with a risk of developing toxicity or severe toxicity in the majority of patients and/or the majority of patients with a disease or condition that the patient has or which is suspected in him, after the introduction of cell therapy; and/or administering cell therapy to the patient on an inpatient basis and/or hospital admission for one or more days, optionally the cell therapy must otherwise be administered to patients on an outpatient basis or without hospital admission for one or more days.

[0087] In some embodiments, the agent or other treatment is an anti-IL-6 antibody or an antibody to the IL6 receptor.

[0088] In some embodiments, the agent or other treatment is or includes an agent selected from among tocilizumab, siltuximab, clazakizumab, sarilumab, olokizumab (CDP6038), elsilimomab, ALD518/BMS-945429, sirukumab (CNTO 136), CPSI-2634, ARGX-109, FE301 and FM101.

[0089] In some embodiments, the agent or other therapeutic agent is or includes a steroid, optionally dexamethasone.

[0090] In some embodiments, the volumetric parameter is estimated, and the volumetric parameter is SPD, and the threshold value is either about 30 cm ² , or about 40 cm ² , or about 50 cm ² , or about 60 cm ² , or is or is approximately 70 cm ² . In some embodiments, the implementation of the volume parameter is SPD, and the threshold value is or approximately is 50 cm ² .

[0091] In some embodiments, one or more analytes is or includes LDH, and the threshold is or is about 300 units per liter, or is about 400 units per liter, or is about 500 units per liter, or is or is about 600 units per litre. In some embodiments, the analyte is LDH and the threshold is, or is about, 500 units per liter.

[0092] In some embodiments, the implementation of the recombinant receptor specifically binds to an antigen associated with a disease or condition or expressed in the cells of the environment of the lesion associated with the disease or condition. In some embodiments, the disease or condition is cancer. In some embodiments, the disease or condition is myeloma, leukemia, or lymphoma. In some embodiments, the disease or condition is B cell cancer and/or acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin's lymphoma (NHL), and diffuse large B cell lymphoma (DLBCL) .

[0093] In some embodiments, the recombinant receptor is a chimeric antigen receptor (CAR). In some embodiments, the engineered cells include T cells, optionally CD4 ⁺ and/or CD8 ⁺ . In some embodiments, the T cells are primary T cells obtained from the patient or are autologous to the patient.

[0094] The invention relates to methods for treating a patient with non-Hodgkin's lymphoma (NHL), comprising administering to the patient a dose of T cells, including T cells expressing a chimeric antigen receptor (CAR) that specifically binds to a target antigen expressed by NHL, wherein: the dose of T cells includes from or from about 5 x 10 ⁷ expressing the recombinant receptor T cells to 1 x 10 ⁸ expressing the recombinant receptor T cells, inclusive, and the indicated dose includes a certain ratio of CD4 ⁺ cells expressing the recombinant receptor to CD8 ⁺ cells expressing the recombinant receptor and/or CD4 ⁺ cells to CD8 ⁺ cells, and this ratio is approximately or is 1:1; and the method results in (1) a complete response (CR) in at least 35%, at least 40%, or at least 50% of treated patients and/or an objective response (OR) in at least 50%, at least in at least 60% or at least 70% of treated patients and results in (2) no more than 50% of patients developing cytokine release syndrome (CRS) greater than Grade 2 and/or neurotoxicity greater than Grade 2 degree 2.

[0095] In some embodiments of any of the methods of the invention, at least 40%, at least 50%, at least 60%, at least 70% of patients who, at or before the dose of cells had or double/triple hit lymphoma (or well-differentiated B-cell lymphoma with MYC and BCL2 and/or BCL6 permutations with DLBCL (double/triple hit) histology) or recurrence after autologous stem cell transplant (ASCT) was identified, achieved an OR, and not necessarily OR lasted for 3 months or more or for 6 months or more.

[0096] In some embodiments of any of the methods of the invention, the CR or OR lasted for more than 3 months or more than 6 months. In some embodiments of any of the methods of the invention, more than or about more than 50% of patients treated according to the method do not exhibit cytokine release syndrome (CRS) or neurotoxicity of any degree.

[0097] In some embodiments, the CR or OR lasted for more than 3 months or more than 6 months; at least 20%, at least 25%, at least 35%, at least 40%, or at least 50% of patients treated according to the method achieve a CR that is long lasting; at least 60%, 70%, 80%, 90%, or 95% of patients treated according to the method and who achieved CR remain in response or remain in CR, or survive for or more than 3 a month or for 6 months or more or for or more than 9 months; and/or wherein at least 60%, 70%, 80%, 90%, or 95% of patients treated according to the method who achieved a CR in one month and/or three months remain in a state of response, remain in state of CR and/or remain alive or remain alive without progression for or more than 3 months and/or for 6 months or more and/or for more than nine months; and/or at least 50%, at least 60%, or at least 70% of patients treated according to the method achieve an objective response (OR), optionally the OR is long-lasting, or long-term in at least 60%, 70 %, 80%, 90%, or 95% of patients achieving OR for or more than 3 months or for 6 months or more; and/or wherein at least 60%, 70%, 80%, 90%, or 95% of patients treated according to the method and achieving an OR remain in response or survive for or more than 3 months, and/ or for 6 months or more.

[0098] In some embodiments, at or before the cell dose is administered, the patient has or has been identified as having lymphoma associated with or involving central nervous system (CNS) involvement; and/or at least 70%, at least 80%, at least 90%, or at least 95% of patients treated according to the method, in whom, at or before the dose of cells was shown or who were identified as exhibiting lymphoma involving the CNS achieved elimination of the CNS disease.

[0099] The invention relates to methods of treating a patient comprising administering to a patient having lymphoma a dose of T cells comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds to a target antigen expressed by the lymphoma, wherein the patient's lymphoma is associated with or includes involvement of the central nervous system (CNS). In some aspects, at or before the cell dose is administered, the patient has a brain lesion, optionally a temporal lobe lesion. In some examples, the lymphoma is a B-cell malignancy. In some embodiments, the lymphoma is non-Hodgkin's lymphoma (NHL).

[0100] In some of any such embodiments, at least 35%, at least 40%, or at least 50% of patients treated according to the method achieve a complete response (CR) or remission of CNS disease and/or achieve a reduction or clearance of CNS disease, and optionally CR or remission or reduction or clearance of CNS disease is prolonged or lasts in at least 60%, 70%, 80%, 90%, or 95% of patients who achieve CR for or more than 3 months, or for 6 months or more; and/or at least 60%, 70%, 80%, 90%, or 95% of patients who achieve CR or remission or other reduction in CNS disease at one month and/or three months remain in response, remain in remission , for example, in CR, or continue to show signs of reduction or remission, and/or remain alive or remain alive without progression for or more than 3 months and/or for 6 months or more and/or more than nine months; and/or at least 50%, at least 60%, or at least 70% of patients treated according to the method achieve an objective response (OR) or remission of CNS disease, optionally OR or remission of CNS disease being or lasting for at least 60%, 70%, 80%, 90%, or 95% of patients achieving OR for or more than 3 months or for 6 months or more; and/or at least 60%, 70%, 80%, 90%, or 95% of patients who achieve OR or remission of CNS disease remain in response or survive for or more than 3 months and/or for 6 months or more; and/or the brain lesion is reduced in size or volume, optionally by more than or by more than about 25%, 50%, 75%, or more. In some aspects, reduction or remission or clearance of CNS disease is achieved without or without significant signs or symptoms of toxicity, such as neurotoxicity, such as severe neurotoxicity, for example, greater than grade 2 neurotoxicity or greater than grade 3 neurotoxicity, and/or no toxicity, caused by the activation or presence of cell therapy cells in the patient's brain, and/or is achieved without an increased level of toxicity compared to a patient who retains CNS disease and/or is receiving therapy but does not exhibit CNS disease.

[0101] In some embodiments of any of the methods of the invention, more than or more than about 30%, 35%, 40%, or 50% of patients treated according to the method do not show cytokine release syndrome (CRS) or neurotoxicity of any or degrees. In some embodiments of any of the methods of the invention, at least or about 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of patients receiving treatment according to the method, no early onset of CRS or neurotoxicity is shown and/or no onset of CRS is shown earlier than 3 days after initiation of administration and/or no onset of neurotoxicity is shown earlier than 5 days after initiation of administration, and/or with an average onset of neurotoxicity among patients treated according to the method occurs at or after the mean peak, or the average time to resolve CRS in patients treated according to the method and/or the average onset of neurotoxicity among patients treated according to the method is greater than or approximately 8 , 9, 10 or 11 days.

[0102] In some embodiments of any of the methods of the invention, prior to initiating a dose of cells, the patient has not been administered a treatment agent or agent capable of treating, preventing, delaying, attenuating, or reducing the development or risk of developing toxicity. In some embodiments of any of the methods of the invention, the patient has not been administered a treatment agent or agent to treat or prevent or reduce or attenuate neurotoxicity and/or cytokine release syndrome or the risk of it, for a period of time following dosing, the period of time optionally being or is about 1, 2, 3, 4, 5 days, or optionally is or is about 6, 7, 8, 9, 10, 11 days, or optionally is 1, 2, 3, or 4 weeks. In some embodiments of any of the methods of the invention, the patient has not been administered a therapeutic agent or agent to treat or prevent or reduce or attenuate neurotoxicity and/or cytokine release syndrome or the risk thereof, post-dose, before or if the patient does not show a sign or a symptom of toxicity and/or before or if the patient does not show a sign or symptom of toxicity other than fever, optionally the fever is not a prolonged fever, or the fever has been reduced or reduced by more than 1°C after treatment with an antipyretic agent. In some embodiments of any of the methods of the invention, administration and any monitoring is performed on an outpatient basis and/or without admitting the patient to the hospital and/or without overnight stay in the hospital and/or without requiring admission or overnight stay in the hospital, optionally if or until the patient shows a prolonged fever or fever that is or has not been reduced by or has not been reduced by more than 1°C after treatment with an antipyretic.

[0103] In some embodiments of any of the methods of the invention, prior to initiating a cell dose, the patient was not administered an anti-IL-6 or anti-IL-6R antibody, optionally tocilizumab or situximab, and/or was not administered a steroid, optionally dexamethasone. In some embodiments of any of the methods of the invention, the patient has not received an anti-IL-6 or anti-IL-6R antibody, optionally tocilizumab or siltuximab, and/or has not received a steroid, optionally dexamethasone, for a period of time post-dose, wherein the period time is optionally or approximately 1, 2, 3, 4, 5 days, or optionally or approximately 6, 7, 8, 9, 10, 11 days, or optionally 1, 2, 3, or 4 weeks. In some embodiments of any of the methods of the invention, the patient was not administered an anti-IL-6 or anti-IL-6R antibody, optionally tocilizumab or siltuximab, and/or was not administered a steroid, optionally dexamethasone, following cell dosing, before, or if, the patient does not show a sign or symptom of toxicity, optionally neurotoxicity, or CRS, and/or before, or if, the patient does not show a sign or symptom of toxicity, optionally neurotoxicity, or CRS, other than a temperature increase, and optionally, the temperature increase is not a sustained increase temperature, or the temperature rise is or has been reduced or reduced by more than 1°C after treatment with an antipyretic. In some embodiments of any of the methods of the invention, administration and any monitoring is performed on an outpatient basis and/or without admitting the patient to the hospital and/or without overnight stay in the hospital and/or without requiring admission or overnight stay in the hospital, optionally if or until the patient shows a prolonged fever or a fever that is or has not been reduced or reduced by more than 1°C after treatment with an antipyretic.

[0104] In some embodiments of any of the methods of the invention, administration is on an outpatient basis and/or without requiring admission or overnight stay in the hospital. In some embodiments of any of the methods of the invention, if a patient who is receiving or has received treatment on an outpatient basis exhibits a prolonged fever, or a fever that is or has not been reduced or reduced by more than 1°C following treatment with an antipyretic agent, the patient is hospitalized or overnight in the hospital and/or the agent or agent is administered to treat or prevent or reduce or reduce neurotoxicity and/or cytokine release syndrome or the risk thereof.

[0105] In some embodiments of any of the methods of the invention, the NHL is selected from the group consisting of aggressive NHL, diffuse large B cell lymphoma (DLBCL), NOS (de novo and transformed from indolent), primary mediastinal large B cell lymphoma ( PMBCL), mantle cell lymphoma (MCL) and/or follicular lymphoma (FL), optionally stage 3B follicular lymphoma (FL3B). In some embodiments of any of the methods of the invention, NHL includes diffuse large B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), NOS (de novo or transformed from indolent lymphoma), or stage 3B follicular lymphoma. In some examples, NHL includes DLBCL. In some embodiments of any of the methods of the invention, the DLBCL is de novo or transformed from follicular lymphoma (FL) and/or does not include DLBCL transformed from MZL and CLL (Richter).

[0106] In some embodiments of any of the methods of the invention, the patient is or has been identified as having an Eastern Cooperative Oncology Group Performance Status (ECOG) status of 0, 1, or 2. In some embodiments of any of the methods of the invention, the patient is or has been identified as having an ECOG status of 0 or 1. In some embodiments of any of the methods of the invention, at or just prior to administration of the cell dose, the patient experienced a relapse after remission after treatment with, or refractoriness to one or more prior NHL therapies, optionally one, two or three prior therapies other than another dose of CAR-expressing cells.

[0107] In some embodiments of any of the methods of the invention, at or prior to administration of a dose of cells, the patient is or has been identified as having a lymphoma associated with or involving the central nervous system (CNS). In some embodiments of any of the methods of the invention, at least 70%, at least 80%, at least 90%, or at least 95% of patients receiving treatment according to the method, in which, at or before a dose of cells, lymphoma involving the CNS has been shown or identified, the CNS disease has been eliminated.

[0108] In some embodiments of any of the methods of the invention, at or before a dose of cells: the patient is or has been identified as having double/triple hit lymphoma (or well-differentiated B-cell lymphoma with MYC and BCL2 and/or BCL6 permutations with histology DLBCL (double/triple hit)); the patient is or has been identified as having a chemo-refractory lymphoma, optionally a chemo-refractory DLBCL; the patient has not achieved complete remission (CR) in response to prior therapy; and/or the patient had a relapse within 1 year or less than 1 year after receiving an autologous stem cell transplant (ASCT).

[0109] In some embodiments of any of the methods of the invention, the method includes, prior to administering a cell dose, identifying or selecting a patient for cell dose administration having double/triple hit lymphoma (or well-differentiated B-cell lymphoma, with MYC and BCL2 permutations and/ or BCL6 with histology DLBCL (double/triple hit)), chemorefractive lymphoma, optionally chemorefractive DLBCL, has not achieved complete remission (CR) in response to prior cancer therapy, optionally NHL; and/or had a relapse within 1 year or less than 1 year after receiving an autologous stem cell transplant (ASCT); and/or has lymphoma associated with or involving central nervous system (CNS) involvement.

[0110] In some embodiments of any of the methods of the invention, the method further comprises administering an additional therapeutic agent or therapy, optionally other than cell therapy, optionally other than CAR ⁺ T cell therapy. In some embodiments, the additional therapeutic agent or therapy is for the treatment of NHL or cancer and/or increases the persistence, activity and/or efficacy of a cell dose. In some embodiments, an additional therapeutic agent or therapy is administered if the patient does not respond, optionally does not demonstrate a CR or OR, to cell therapy within 1 month, within 2 months, or within 3 months of the cell dose. In some embodiments, an additional therapeutic agent or therapy is administered to a patient: who is or has been identified as having stable or progressive disease (SD/PD) following treatment with prior therapy, optionally prior therapy with a chemotherapeutic agent, who is or has been identified as having status 2 according to Eastern A Cooperative Oncology Group Performance Status (ECOG) which is or has been identified as having transformed follicular lymphoma (tFL) and/or which is or has been identified as having DLBCL transformed from MZL and CLL. In some embodiments, prior to administration of a cell dose or additional therapeutic agent or therapy, the method comprises identifying or selecting a patient for cell dose administration having stable or progressive disease (SD/PD) after treatment with prior therapy, optionally prior chemotherapy agent therapy, status 2 according to Eastern Cooperative Oncology Group Performance Status (ECOG), transformed follicular lymphoma (tFL) and/or DLBCL transformed from MZL and CLL. In some of these embodiments, the additional therapeutic agent or therapy is administered before, with or simultaneously with and/or after initiation of the cell dose.

[0111] In some embodiments of any of the methods of the invention, the CAR comprises an scFv antigen-specific transmembrane domain, a cytoplasmic signaling domain derived from a co-stimulatory molecule, which is optionally 4-1BB, and a cytoplasmic signaling domain derived from the primary signaling ITAM -containing molecule, which is not necessarily CD3zeta. In some embodiments of any of the methods of the invention, the antigen is a B cell antigen, which is optionally CD19.

[0112] In some embodiments of any of the methods of the invention, prior to administration, the patient has previously received antilymphocyte therapy, including administration of fludarabine and/or cyclophosphamide. Some embodiments of any of the methods of the invention further comprise, immediately prior to administration, administering anti-lymphocyte therapy to the patient, including administration of fludarabine and/or cyclophosphamide. In some embodiments of any of the methods of the invention, antilymphocyte therapy comprises administering cyclophosphamide at about 200-400 mg/m ² , optionally at or about 300 mg/m ² inclusive, and/or fludarabine at about 20-40 mg/m ² , optionally 30 mg/m ² , daily for 2-4 days, optionally for 3 days. In some embodiments of any of the methods of the invention, antilymphocyte therapy comprises administering cyclophosphamide at or about 300 mg/m ² and fludarabine at about 30 mg/m ² daily for 3 days.

[0113] In some embodiments of any of the methods of the invention, cell dosing and/or antilymphocyte therapy is performed on an outpatient basis. In some embodiments of any of the methods of the invention, the dose of cells is administered parenterally, optionally intravenously.

[0114] In some embodiments of any of the methods of the invention: at least 40% or at least 50% of patients treated according to the method achieve complete remission (CR), demonstrate progression-free survival (PFS), and/or complete survival (OS) for more than or approximately 3 months, 6 months, or 12 months; on average, patients treated according to the method show an average of PFS or OS for more than or about 6 months, 12 months, or 18 months; and/or the patient shows PFS or OS after therapy for at least or about 6, 12, 18 or more months. In some embodiments of any of the methods of the invention, at or about 14 or 28 days after initiation of cell dosing, the number of CAR ⁺ T cells, optionally CAR ⁺ CD8 ⁺ T cells and/or CAR ⁺ CD4 ⁺ T cells detectable in the patient's blood, or in most patients treated according to the method, is greater than 1 cell per μl, greater than 5 cells per μl, or greater than 10 cells per μl.

[0115] In some embodiments of any of the methods of the invention, the T cells are primary T cells obtained from a patient. In some embodiments of any of the methods of the invention, the T cells are autologous to the patient. In some embodiments of any of the methods of the invention, the T cells are allogeneic to the patient. In some embodiments of any of the methods of the invention, the T cells comprise CD4 ⁺ and CD8 ⁺ T cells administered as a plurality of compositions, said plurality of compositions comprising administering a first composition comprising CD4 ⁺ T cells or CD8 ⁺ T cells, and the introduction of a second composition, including others from among CD4 ⁺ T cells or CD8 ⁺ T cells.

[0116] In some embodiments of any of the methods of the invention, the first composition and the second composition are administered at 0 to 12 hour intervals, 0 to 6 hour intervals, or 0 to 2 hour intervals. In some embodiments of any of the methods of the invention, the first composition and the second composition are administered at intervals of 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. In some embodiments of any of the methods of the invention, the first composition comprises CD4 ⁺ T cells. In some embodiments of any of the methods of the invention, the first composition comprises CD8 ⁺ T cells. In some embodiments of any of the methods of the invention, the first composition is administered prior to the second composition.

[0117] In some embodiments of any of the methods of the invention, the dose of T cells is administered to the patient as a single dose or administered only once over a period of two weeks, one month, three months, six months, 1 year, or more. In some embodiments of any of the methods of the invention, the dose of T cells is administered as a double dose, comprising a first dose of T cells and a subsequent dose of T cells, wherein one or both of the first dose and the second dose comprises administering a plurality of T cell compositions. In some embodiments of any of the methods of the invention, the subsequent dose is administered at a time point at least or greater than about 7 days, or 14 days and less than about 28 days after initiation of the first dose of cells.

[0118] The invention relates to a cell therapy product, comprising a dose or composition of genetically engineered cells expressing a chimeric antigen receptor (CAR), and instructions for administering the cell therapy, wherein the instructions define: the dose of cells is intended to be administered to a patient having or identified as having non-Hodgkin's lymphoma (NHL), wherein the NHL is selected from diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma (PMBCL), NOS (de novo or transformed from indolent lymphoma), or stage 3B follicular lymphoma, wherein the patient is or has been identified as having a status of 0 or 1 according to the Eastern Cooperative Oncology Group Performance Status (ECOG); and the dose of T cells to be administered includes from or from about 5 x 10 ⁷ CAR-expressing T cells to 1 x 10 ⁸ CAR-expressing T cells, inclusive. In some embodiments of any of the articles of the invention, the instructions define the administration of a dose of T cells in a certain ratio of CD4 ⁺ cells expressing CAR to CD8 ⁺ cells expressing CAR and/or CD4 ⁺ cells to CD8 ⁺ cells, and this ratio is not necessarily approximately is 1:1 or from about 1:3 to about 3:1.

[0119] The invention relates to a product comprising cell therapy, including a dose or composition of genetically engineered cells expressing a chimeric antigen receptor (CAR), and instructions for administering cell therapy, and the instructions define: the dose of T cells is intended to be administered in a certain ratio CD4 ⁺ cells expressing CAR to CD8 ⁺ cells expressing CAR, and/or CD4 ⁺ cells to CD8 ⁺ cells, and this ratio is approximately or is 1:1; and the cell dose is intended to be administered to a patient having or identified as having non-Hodgkin's lymphoma (NHL), wherein the NHL is selected from diffuse large B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), NOS (de novo or transformed from indolent lymphoma) or stage 3B follicular lymphoma.

[0120] The invention relates to a product comprising cell therapy, including a dose or composition of genetically engineered cells expressing a chimeric antigen receptor (CAR), and instructions for administering cell therapy, and the instructions define: the dose of cells is intended to be administered to a patient having or identified as having non-Hodgkin's lymphoma (NHL), optionally NHL selected from aggressive NHL, diffuse large B-cell lymphoma (DLBCL), NOS (de novo and transformed from indolent), primary mediastinal large B-cell lymphoma (PMBCL), mantle cell lymphoma ( MCL) and/or follicular lymphoma (FL), optionally stage 3B follicular lymphoma (FL3B), the dose of T cells to be administered comprises from or from about 5 x 10 ⁷ CAR-expressing T cells to 1 x 10 ⁸ CAR-expressing T - cells, inclusive; and the dose of T cells is intended to be administered in a specific ratio of CAR-expressing CD4+ cells to CAR-expressing CD8+ cells and/or CD4+ cells to CD8+ cells, the ratio being approximately or equal to 1:1.

[0121] In some embodiments of any of the articles of the invention, the instructions further specify that the dose of cells is intended to be administered to a patient who is or has been identified as having an Eastern Cooperative Oncology Group Performance Status (ECOG) status of 0, 1, or 2, optionally ECOG 0 or 1. In some embodiments of any of the articles of the invention, the instructions specify that administration is to a patient who has not received, immediately prior to cell dose administration, or before or up to about 1 month prior to cell dose administration, a treatment agent or agent capable of treating, preventing, delaying, attenuating or reducing the development or risk of developing toxicity. In some embodiments of any of the articles of the invention, the agent is or includes an anti-IL-6 or anti-IL-6R antibody, optionally tocilizumab or siltuximab, and/or a steroid, optionally dexamethasone. In some embodiments of any of the articles of the invention, the instructions specify that the dose of cells is not intended to be administered to a patient having DLBCL transformed from MZL and CLL (Richter) and/or to a patient having DLBCL that is de novo or transformed from indolent diseases. In some embodiments of any of the articles of the invention, the instructions specify that the patient does not have DLBCL transformed from MZL and CLL (Richter).

[0122] In some embodiments of any of the articles of the invention, the instructions specify that the administration of cell therapy is administered to a patient who is or has been identified as having double/triple hit lymphoma (or well-differentiated B-cell lymphoma with MYC and BCL2 and/or BCL6 permutations). with histology DLBCL (double/triple hit)), is or has been identified as having chemorefractive lymphoma, optionally chemorefractive DLBCL; and/or has not achieved complete remission (CR) in response to prior therapy. In some embodiments of any of the articles of the invention, the CAR comprises an scFv antigen-specific transmembrane domain, a cytoplasmic signaling domain derived from a co-stimulatory molecule, which is optionally 4-1BB, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule. , which is not necessarily CD3zeta. In some embodiments of any of the articles of the invention, the antigen is a B cell antigen, which is optionally CD19.

[0123] Some embodiments of any of the articles of the invention further include instructions for use with, after, or in connection with antilymphocyte therapy, the antilymphocyte therapy optionally including fludarabine and/or cyclophosphamide. In some embodiments of any of the articles of the invention, the anti-lymphocyte therapy comprises administering cyclophosphamide at about 200-400 mg/m ² , optionally at or about 300 mg/m ² inclusive, and/or fludarabine at about 20-40 mg/m ² , optionally 30 mg/m ² , daily for 2-4 days, optionally for 3 days. In some embodiments of any of the articles of the invention, anti-lymphocyte therapy comprises administering cyclophosphamide at or about 300 mg/m ² and fludarabine at about 30 mg/m ² daily for 3 days.

[0124] In some embodiments of any of the articles of the invention, the instructions further specify that the administration of cell therapy should be or may be administered to the patient on an outpatient basis and/or without the patient being admitted to the hospital overnight or for one or more consecutive days and/or without admitting the patient to the hospital for one or more days. In some embodiments of any of the articles of the invention, the instructions further specify that the cell therapy is for parenteral administration, optionally intravenous administration. In some embodiments of any of the articles of the invention, the cell therapy comprises primary T cells obtained from a patient. In some embodiments of any of the articles of the invention, the T cells are autologous to the patient. In some embodiments of any of the articles of the invention, the T cells are allogeneic to the patient.

[0125] In some embodiments of any of the articles of the invention, the article includes a plurality of cell therapy compositions, the plurality of compositions comprising a first composition of engineered cells comprising CD4 ⁺ T cells or CD8 ⁺ T cells, the instructions specifying that the first the composition is for use with a second composition comprising others from CD4 ⁺ T cells or CD8 ⁺ T cells, optionally the cells of the first composition and the cells of the same composition are from the same patient.

[0126] In some embodiments of any of the articles of the invention, the instructions specify that the first composition and the second composition are intended to be administered at a specific ratio of CD4+ cells expressing the recombinant receptor to CD8+ cells expressing the recombinant receptor and/or CD4+ cells to CD8+ cells, wherein the ratio is optionally about 1:1, or about 1:3 to about 3:1. In some embodiments of any of the articles of the invention, the specific ratio is, or is approximately, 1:1. In some embodiments of any of the articles of the invention, the composition further comprises a cryoprotectant and/or the article further includes instructions for thawing the composition prior to administration to a patient.

[0127] In some embodiments of any of the articles of the invention, the instructions define administering a composition comprising CD4 ⁺ T cells and a composition comprising CD8 ⁺ T cells at 0 to 12 hour intervals, at 0 to 6 hour intervals, or at intervals of 0 to 2 hours. In some embodiments of any of the articles of the invention, the instructions specify administering a composition comprising CD4 ⁺ T cells and a composition comprising CD8 ⁺ T cells at intervals of 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. In some embodiments of any of the articles of the invention, the instructions define the administration of the composition comprising CD4 ⁺ T cells prior to the administration of the composition comprising CD8 ⁺ cells. In some embodiments of any of the articles of the invention, the instructions define the administration of the composition comprising CD8 ⁺ T cells prior to the administration of the composition comprising CD4 ⁺ cells.

[0128] The invention relates to articles comprising one or more reagents capable of detecting one or more analytes and instructions for using the reagent to test a biological sample from a patient who is a candidate for treatment, optionally with cell therapy, said cell therapy optionally comprising dose or composition of engineered cells expressing the recombinant receptor, wherein one or more analytes are selected from LDH, ferritin, CRP, IL-6, IL-7, IL-8, IL-10, IL-15, IL-16, TNF -alpha, IFN-gamma, MCP-1, MIP-1beta, eotaxin, G-CSF, IL-1Ralpha, IL-1Rbeta, IP-10, perforin and D-dimer (fibrin breakdown product).

[0129] Some embodiments of any of the articles of the invention further include cell therapy and/or further include instructions for use with, before and/or in connection with cell therapy treatment. Some embodiments of any of the articles of the invention further comprise one or more therapeutic agents or agents for treating, preventing, delaying, attenuating, or reducing the development or risk of developing toxicity, and/or instructions for administering one or more therapeutic agents or agents for treating, preventing, delaying, attenuating or reducing the development or risk of developing toxicity in a patient.

[0130] In some embodiments of any of the articles of the invention, the instructions further define if the level, amount, or concentration of the analyte in the sample is equal to or greater than a threshold for the analyte: administering to the patient an agent or other treatment capable of treating, preventing, delaying, easing or reducing the development or risk of developing toxicity (i) up to, (ii) within one, two or three days, (iii) simultaneously with and / or (iv) at the first increase in temperature after the initiation of the introduction of cell therapy to the patient; and/or administering cell therapy to the patient at a reduced dose or at a dose not associated with a risk of developing toxicity or severe toxicity, or not associated with a risk of developing toxicity or severe toxicity in the majority of patients and/or the majority of patients with a disease or condition that the patient has or is suspected of having it, after the introduction of cell therapy; and/or administering cell therapy to the patient on an inpatient basis and/or with hospital admission for one or more days, optionally the cell therapy must otherwise be administered to patients on an outpatient basis or without hospital admission for one or more days.

[0131] In some embodiments of any of the devices of the invention, the instructions further specify if the analyte level, amount, or concentration is below an analyte threshold, administering cell therapy to the patient, optionally at a non-reduced dose, optionally on an outpatient basis, or without hospital admission for one or more days.

[0132] In some embodiments of any of the articles of the invention, the instructions further define the administration of a cell therapy to a patient, and wherein the instructions further specify if the level, amount, or concentration of the analyte is below a threshold: administration of the cell therapy does not include administration of, prior to or simultaneously with the administration of the cell therapy. therapy and/or prior to the development of a sign or symptom of toxicity other than fever, a treatment agent or agent capable of treating, preventing, delaying or reducing the development of toxicity; and/or the administration of the cell therapy should be or may be administered to the patient on an outpatient basis and/or without admitting the patient to the hospital overnight or for one or more consecutive days and/or without admitting the patient to the hospital for one or more days.

[0133] In some embodiments of any of the articles of the invention, the threshold is within 25%, 20%, 15%, 10%, or 5% of the mean level, amount, or concentration, and/or within the standard deviation of the mean level, amount, or concentration, analyte in a biological sample obtained from a group of patients prior to the production of a therapeutic composition of cells expressing the recombinant receptor, and each of the patients of the group continued to develop toxicity after receiving the composition of cells expressing the recombinant receptor for the treatment of the same disease or condition. The invention relates to a product that includes cell therapy, and said cell therapy optionally includes a dose or composition of genetically engineered cells expressing a recombinant receptor, and instructions for administering cell therapy after or based on the results of an assessment, in a biological sample, of a level or amount, or concentration one or more analytes in a biological sample, wherein said biological sample is obtained from a patient prior to the application of cell therapy, and/or said biological sample does not include a recombinant receptor and/or said genetically engineered cells, wherein one or more analytes are selected from LDH, ferritin, CRP, IL-6, IL-7, IL-8, IL-10, IL-15, IL-16, TNF-alpha, IFN-gamma, MCP-1, MIP-1beta, eotaxin, G-CSF, IL- 1Ralpha, IL-1Rbeta, IP-10, perforin and D-dimer (fibrin degradation product).

[0134] In some embodiments of any of the articles of the invention, said evaluation includes detection, optionally including contacting a reagent capable of directly or indirectly detecting an analyte with a biological sample and determining the level, amount, or concentration of the analyte in the biological sample. Some embodiments of any of the articles of the invention further include a reagent and/or further include instructions for use with, before and/or in connection with an analyte detection reagent. Some embodiments of any of the articles of the invention further comprise one or more therapeutic agents or agents for treating, preventing, delaying, attenuating, or reducing the development or risk of developing toxicity, and/or instructions for administering one or more therapeutic agents or agents for treating, preventing, delaying, attenuating or reducing the development or risk of developing toxicity in a patient.

[0135] In some embodiments of any of the articles of the invention, instructions for administering cell therapy determine if the level, amount, or concentration of an analyte in a sample is equal to or greater than a threshold: administering to the patient an agent or other treatment capable of treating, preventing, delaying , weakening or reducing the development or risk of developing toxicity (i) up to, (ii) within one, two or three days, (iii) simultaneously with and / or (iv) at the first increase in temperature after initiation of the introduction of a therapeutic composition of cells or genetically engineering cells; and/or administering cell therapy to the patient at a reduced dose or at a dose not associated with a risk of developing toxicity or severe toxicity, or not associated with a risk of developing toxicity or severe toxicity in the majority of patients and/or the majority of patients with a disease or condition that the patient has or is suspected of having, after the introduction of cell therapy; and/or administering cell therapy to the patient on an inpatient basis and/or with hospital admission for one or more days, optionally the cell therapy must otherwise be administered to patients on an outpatient basis or without hospital admission for one or more days.

[0136] In some embodiments of any of the devices of the invention, instructions for administering cell therapy determine if the level, amount, or concentration of an analyte in a sample is below a threshold, administering cell therapy to a patient, optionally at a non-reduced dose, optionally on an outpatient basis, or without admission to hospital for one or more days. In some embodiments of any of the articles of the invention, the instructions further define the administration of the cell therapy to the patient, and wherein the instructions further specify if the level, amount, or concentration of the analyte is below a threshold: no administration, before or simultaneously with the administration of the cell therapy, and/or before the development of the symptom or a symptom of toxicity other than fever, a treatment agent or agent capable of treating, preventing, delaying or reducing the development of toxicity; and/or the administration of the cell therapy must or may be administered to the patient on an outpatient basis and/or without admission of the patient to the hospital overnight or for one or more consecutive days and/or without admission of the patient to the hospital for one or more days.

[0137] In some embodiments of any of the articles of the invention, the threshold is within 25%, 20%, 15%, 10%, or 5% of the mean level, amount, or concentration, and/or within the standard deviation of the mean level, amount or the concentration of an analyte in a biological sample obtained from a group of patients prior to the production of a therapeutic composition of cells expressing the recombinant receptor, and each of the patients of the group continued to develop toxicity after receiving the composition of cells expressing the recombinant receptor for the treatment of the same disease or condition.

[0138] The invention relates to a product that includes an agent capable of treating, preventing, delaying, attenuating or reducing the development or risk of developing toxicity and instructions for administering the agent after or based on the results of evaluating the level, amount or concentration of one or more analytes in a biological sample in a biological sample, with one or more analytes selected from LDH, ferritin, CRP, IL-6, IL-7, IL-8, IL-10, IL-15, IL-16, TNF-alpha, IFN-gamma, MCP -1, MIP-1beta, eotaxin, G-CSF, IL-1Ralpha, IL-1Rbeta, IP-10, perforin and D-dimer (fibrin breakdown product). In some embodiments of any of the articles of the invention, said evaluation includes detection, optionally including contacting a reagent capable of directly or indirectly detecting an analyte with a biological sample and determining the level, amount, or concentration of the analyte in the biological sample.

[0139] In some embodiments of any of the articles of the invention, the instructions specify that the agent is to be administered i) before, (ii) within one, two, or three days, (iii) simultaneously with and/or (iv) at the first rise temperature after initiating the administration of cell therapy to the patient and/or further includes instructions for use with, before and/or in connection with cell therapy treatment. In some embodiments of any of the articles of the invention, said biological sample is obtained from a patient prior to administration of the cell therapy or agent. In some embodiments of any of the articles of the invention, the reagent is a binding molecule that specifically binds to a marker or cells in a myeloid cell population. In some embodiments of any of the articles of the invention, the reagent is an antibody or an antigen-binding fragment thereof. In some embodiments of any of the articles of the invention, the biological sample is or is derived from a blood, plasma, or serum sample. In some embodiments of any of the articles of the invention comprising an analyte detection reagent and/or further comprising instructions for use with, before and/or in connection with the analyte detection reagent. Some embodiments of any of the articles of the invention further include cell therapy and/or further include instructions for use with, before and/or in connection with cell therapy treatment.

[0140] In some embodiments of any of the articles of the invention, the instructions for administering the agent determine if the level, amount, or concentration of the analyte in the sample is equal to or above a threshold, administering the agent to the patient. In some embodiments of any of the articles of the invention, the instruction further defines the administration of the cell therapy to the patient, wherein the administration of the agent must be (i) prior to, (ii) within one, two or three days, (iii) concurrently with and/or (iv ) at the first rise in temperature after the initiation of the administration of cell therapy to the patient. In some embodiments of any of the articles of the invention, the instructions for administering the agent specify, if the level, amount, or concentration is below a threshold, administering the cell therapy to the patient, optionally the instructions specifying that the cell therapy should or may be administered to the patient on an outpatient basis, and/ or without admission of the patient to the hospital overnight or for one or more consecutive days and/or without admission of the patient to the hospital for one or more days.

[0141] In some embodiments of any of the articles of the invention, the threshold is within 25%, 20%, 15%, 10%, or 5% of the mean level, amount, or concentration, and/or within the standard deviation of the mean level, amount or the concentration of an analyte in a biological sample obtained from a group of patients prior to the production of a therapeutic composition of cells expressing the recombinant receptor, and each of the patients of the group continued to develop toxicity after receiving the composition of cells expressing the recombinant receptor for the treatment of the same disease or condition. In some embodiments of any of the articles of the invention, testing or evaluating a cell for an analyte is performed by an immunological assay.

[0142] In some embodiments of any of the articles of the invention, toxicity includes neurotoxicity or cytokine release syndrome (CRS), optionally neurotoxicity or CRS grade 1 or higher. In some embodiments of any of the articles of the invention: the toxicity includes severe neurotoxicity and/or includes grade 2 or higher neurotoxicity, grade 3 or higher neurotoxicity, at least prolonged grade 3 neurotoxicity, or grade 4 or grade 5 or higher neurotoxicity; and/or toxicity includes severe CRS and/or includes CRS grade 2 or higher or grade 3 or higher. In some embodiments of any of the articles of the invention, toxicity is associated with cerebral edema.

[0143] In some embodiments of any of the articles of the invention, the agent or other therapeutic agent is or includes one or more steroids; antagonist or inhibitor of a cytokine or cytokine receptor selected from IL-10, IL-10R, IL-6, IL-6 receptor, IFNγ, IFNGR, IL-2, IL-2R/CD25, MCP-1, CCR2, CCR4, MIP1β, CCR5, TNFalpha, TNFR1, IL-1, and IL-1Ralpha/IL-1beta; or an agent capable of preventing, blocking or attenuating the activity or function of microglial cells. In some embodiments of any of the articles of the invention, the antagonist or inhibitor is or includes an agent selected from an antibody or antigen-binding fragment, a small molecule, a protein or peptide, and a nucleic acid. In some embodiments of any of the articles of the invention, the agent or other treatment is an anti-IL-6 antibody or an anti-IL6 receptor antibody.

[0144] In some embodiments of any of the articles of the invention, the agent or other therapeutic agent is or includes an agent selected from among tocilizumab, siltuximab, clazakizumab, sarilumab, olokizumab (CDP6038), elsilimomab, ALD518/BMS-945429, sirukumab (CNTO 136), CPSI-2634, ARGX-109, FE301 and FM101. In some embodiments of any of the articles of the invention, the agent or other therapeutic agent is or includes tocilizumab. In some embodiments of any of the articles of the invention, the agent or other treatment is or includes siltuximab. In some embodiments of any of the articles of the invention, the steroid is or includes dexamethasone.

[0145] In some embodiments of any of the articles of the invention, the agent capable of preventing, blocking, or attenuating microglial cell activity or function is selected from an anti-inflammatory agent, a NADPH oxidase (NOX ₂ ) inhibitor, a calcium channel blocker, a sodium channel blocker, inhibits GM -CSF, inhibits CSF1R, binds specifically to CSF-1, binds specifically to IL-34, inhibits nuclear factor kappa-B (NF-κB) activation, activates the CB2 receptor and/or is a CB2 agonist, phosphodiesterase inhibitor, inhibits miRNA-155 ((miR-155) or up-regulates miRNA-124 ((miR-124). In some embodiments of any of the articles of the invention, the agent capable of preventing, blocking, or attenuating microglial cell activation or function is a small molecule, peptide, protein , an antibody or antigen-binding fragment thereof, an antibody mimetic, an aptamer, or a nucleic acid molecule.

[0146] In some embodiments of any of the articles of the invention, the agent is selected from minocycline, naloxone, nimodipine, Riluzole, MOR103, lenalidomide, cannabinoid (optionally WIN55 or 212-2), intravenous immunoglobulin (IVIg), ibudilast, anti-miR-155 closed nucleic acid (LNA), MCS110, PLX-3397, PLX647, PLX108-D1, PLX7486, JNJ-40346527, JNJ28312141, ARRY-382, AC-708, DCC-3014, 5-(3-methoxy-4-(4 -methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine (GW2580), AZD6495, Ki20227, BLZ945, emaktuzumab, IMC-CS4, FPA008, LY-3022855, AMG-820 and TG-3003. In some embodiments of any of the articles of the invention, the agent is a colony stimulating factor 1 receptor (CSF1R) inhibitor. In some embodiments of any of the articles of the invention, the inhibitor is selected from: PLX-3397, PLX647, PLX108-D1, PLX7486, JNJ-40346527, JNJ28312141, ARRY-382, AC-708, DCC-3014, 5-(3-methoxy- 4-(4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine (GW2580), AZD6495, Ki20227, BLZ945 or a pharmaceutical salt or prodrug thereof; emaktuzumab, IMC-CS4, FPA008, LY-3022855, AMG-820 and TG-3003, or an antigen-binding fragment thereof; or combinations of any of the foregoing. In some embodiments of any of the articles of the invention, the inhibitor is PLX-3397.

[0147] In some embodiments of any of the articles of the invention, the disease or condition is cancer. In some embodiments of any of the articles of the invention, the disease or condition is myeloma, leukemia, or lymphoma. In some embodiments of any of the articles of the invention, the disease or condition is B-cell malignancy and/or acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin's lymphoma (NHL), and diffuse large B-cell cellular lymphoma (DLBCL).

[0148] In some embodiments of any of the articles of the invention, the antigen is ROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2/neu (erbB2 receptor tyrosine kinase), L1-CAM, CD19, CD20 , CD22, mesothelin, CEA and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, erb -B2, erb-B3, erb-B4, erbB dimers, EGFR vIII, folate binding protein (FBP), FCRL5, FCRH5, embryonic acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R -alpha2, receptor containing kinase insertion domain (kdr), kappa light chain, Ley antigen, L1 cell adhesion molecule, (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6 , preferably expressed melanoma antigen (PRAME), survivin, TAG72, B7-H6, IL-13 receptor alpha 2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AI MAGE Al, HLA -A2 NY-ESO-1, PSCA, folic acid receptor alpha, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, embryonic AchR, NKG2D ligands, CD44v6, dual antigen, testicular cancer antigen, mesothelin , mouse CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms tumor 1 (WT-1), cyclin, cyclin A2, CCL-1, CD138, 5D receptor, G-protein-coupled (GPCR5D), or pathogen-specific antigen.

[0149] In some embodiments of any of the articles of the invention, the recombinant receptor is a T cell receptor or a functional non-T cell receptor. In some embodiments of any of the articles of the invention, the recombinant receptor is a chimeric antigen receptor (CAR). In some embodiments of any of the articles of the invention, the CAR comprises an extracellular antigen-recognition domain specifically binding to an antigen and an intracellular signaling domain comprising ITAM, optionally the intracellular signaling domain including the intracellular domain of the CD3-zeta (CD3ζ) chain; and/or wherein the CAR further comprises a co-stimulatory signaling domain, optionally including a CD28 or 4-1BB signaling domain.

[0150] In some embodiments of any of the articles of the invention, the engineered cells include T cells, optionally CD4 ⁺ and/or CD8 ⁺ . In some embodiments of any of the articles of the invention, the T cells are primary T cells obtained from a patient. In some embodiments of any of the articles of the invention, the dose not associated with a risk of toxicity or severe toxicity is or comprises less than or less than about 5 x 10 ⁷ total recombinant receptor expressing cells, optionally CAR ⁺ cells, total T cells or total peripheral blood mononuclear cells (PBMC), e.g., less than or less than about 2.5 x 10 ⁷ , less than or less than about 1.0 x 10 ⁷ , less than or less than about 5.0 x 10 ⁶ , less than or less than about 1.0 x 10 ⁶ , less than or less than about 5.0 x 10 ⁵ , or less than or less than about 1 x 10 ⁵ total recombinant receptor-expressing cells, optionally CAR ⁺ cells, total T cells or common peripheral blood mononuclear cells (PBMC). In some embodiments of any of the articles of the invention, the dose not associated with a risk of toxicity or severe toxicity is or includes from or about 1 x 10 ⁵ to 5 x 10 ⁷ total recombinant receptor expressing cells, optionally CAR ⁺ cells, total T ^-cells or ^{total peripheral} blood mononuclear cells (PBMC) ^{, e.g.} 10 ⁶ , 1 x 10 ⁵ to 1.0 x 10 ⁶ , 1.0 x 10 ⁵ to 5.0 x 10 ⁵ , 5.0 x 10 ⁵ to 5 x 10 ⁷ , 5 x 10 ⁵ to 2.5 x 10 ⁷ , 5 x 10 ⁵ to 1.0 x 10 ⁷ , 5 x 10 ⁵ to 5.0 x 10 ⁶ , 5 x 10 ⁵ to 1.0 x 10 ⁶ , from 1.0 x 10 ⁶ to 5 x 10 ⁷ , 1 x 10 ⁶ to 2.5 x 10 ⁷ , 1 x 10 ⁶ to 1.0 x 10 ⁷ , 1 x 10 ⁶ to 5.0 x 10 ⁶ , from 5 .0 x 10 ⁶ to 5 x 10 ⁷ , 5 x 10 ⁶ to 2.5 x 10 ⁷ , 5 x 10 ⁶ to 1.0 x 10 ⁷ , 1.0 x 10 ⁷ to 5 x 10 ⁷ , 1 x 10 ⁷ to 2.5 x 10 ⁷ or 2.5 x 10 ⁷ to 5 x 10 ⁷ total recombinant receptor expressing cells, optionally CAR ⁺ cells, total T cells, or total peripheral blood mononuclear cells (PBMC).

[0151] In some embodiments of any of the articles of the invention, the reagent is labeled so that it can be detected, optionally fluorescently labeled. In some embodiments of any of the articles of the invention, one or more of the analytes is LDH, ferritin, CRP, IL-6, IL-8, IL-10, TNF-alpha, IFN-alpha2, MCP-1, and MCP-1beta. In some embodiments of any of the articles of the invention, one or more of the analytes is or includes LDH.

[0152] The invention relates to methods for selecting a patient for treatment, comprising: (a) contacting a biological sample with one or more reagents that can be detected or that are specific for one or more analytes, and one or more analytes are selected from LDH, ferritin , CRP, IL-6, IL-7, IL-8, IL-10, IL-15, IL-16, TNF-alpha, IFN-gamma, MCP-1, MIP-1beta, eotaxin, G-CSF, IL -1Ralpha, IL-1Rbeta, IP-10, perforin and D-dimer (fibrin breakdown product), moreover: the biological sample is obtained from a patient who is a candidate for treatment with cell therapy, and said cell therapy optionally includes a dose or composition of genetically engineered cells expressing the recombinant receptor; and the biological sample was obtained from the patient before the introduction of cell therapy and/or the specified biological sample does not include the recombinant receptor and/or the specified genetically engineered cells; and (b) selecting a patient in whom: (i) the analyte level, amount, or concentration in the samples is equal to or greater than a cut-off value, thereby identifying a patient at risk of developing cell therapy toxicity; or (ii) the level, amount or concentration of the analyte is below a threshold value.

[0153] In some embodiments of any of the methods of the invention: (a) the patient in (i) is selected to administer to the patient (1) a treatment agent or other agent capable of treating, preventing, delaying, attenuating, or reducing the development or risk of developing toxicity and (2) cell therapy, wherein the administration of the agent must be carried out (i) before, (ii) within one, two or three days, (iii) simultaneously with and/or (iv) at the first increase in temperature after initiation of the administration of the cell patient therapy; and/or (c) the patient in (i) is selected to administer the cell therapy to the patient at a reduced dose or at a dose not associated with a risk of toxicity or severe toxicity, or not associated with a risk of toxicity or severe toxicity in the majority of patients, and/or the majority of patients having a disease or condition that the patient has or is suspected of having, after the introduction of cell therapy; and/or (b) the patient in (i) is selected to administer cell therapy to the patient on an inpatient basis and/or with hospital admission for one or more days, optionally the cell therapy must otherwise be administered to patients on an outpatient basis or without admission to the hospital for one or more days.

[0154] In some embodiments of any of the methods of the invention, the patient in (i) is selected, and the method further comprises: (a) administering to the patient (1) a treatment agent or other agent capable of treating, preventing, delaying, attenuating, or reducing development or risk of development of toxicity, and (2) cell therapy, and the introduction of the agent is carried out (i) before, (ii) within one, two or three days, (iii) simultaneously with and/or (iv) at the first increase in temperature after initiating the administration of cell therapy to the patient; and/or (b) administering the cell therapy to the patient at a reduced dose or at a dose not associated with a risk of developing toxicity or severe toxicity, or not associated with a risk of developing toxicity or severe toxicity in the majority of patients and/or the majority of patients with the disease or condition , which the patient has or is suspected of having, after the introduction of cell therapy; and/or (c) administering to the patient a cell therapy or a dose of genetically engineered cells of a cell therapy not associated with a risk of developing toxicity or severe toxicity, or not associated with a risk of developing toxicity or severe toxicity in the majority of patients and/or the majority of patients with the disease or a condition that the patient has or is suspected of having following the administration of cell therapy; and/or (d) administering cell therapy to the patient on an inpatient basis and/or with admission to a hospital for one or more days, optionally the cell therapy must otherwise be administered to patients on an outpatient basis or without hospital admission for one or more days. more days.

[0155] In some embodiments of any of the methods of the invention: (a) the patient in (ii) is selected to administer cell therapy to the patient, optionally at an unreduced dose, optionally on an outpatient basis or without hospital admission for one or more days; (b) the patient in (ii) is selected to administer the cell therapy to the patient, wherein the cell therapy does not include administering, prior to or concurrently with the use of the cell therapy and/or prior to the development of a sign or symptom of toxicity other than fever, a treatment agent or agent capable of treating, preventing, delaying or reducing the development of toxicity; and/or the patient in (ii) is selected to receive cell therapy on an outpatient basis and/or without admission of the patient to the hospital overnight or for one or more consecutive days and/or without admission of the patient to the hospital for one or more days.

[0156] In some embodiments of any of the methods of the invention, the patient in (ii) is selected, and the method further comprises administering cell therapy to the patient, optionally at an unreduced dose, optionally on an outpatient basis, or without hospital admission for one or more days. In some embodiments of any of the methods of the invention, the patient in (ii) is selected, and the method further comprises administering the cell therapy to the patient, wherein: administering the cell therapy does not include administering, before or simultaneously with the administration of the cell therapy and/or prior to the development of a sign or symptom of toxicity other than fever, a therapeutic agent or agent capable of treating, preventing, delaying or reducing the development of toxicity; and/or the administration of the cell therapy to the patient should or may be performed on an outpatient basis and/or without the patient being admitted to the hospital overnight or for one or more consecutive days and/or without the patient being admitted to the hospital for one or more days.

[0157] The invention relates to a method of treatment, including: (a) testing a biological sample in relation to the level, amount or concentration of one or more analytes, and the biological sample comes from a patient who is a candidate for treatment, optionally cell therapy, and the specified cell therapy optionally includes a dose or composition of engineered cells expressing a recombinant receptor for treating a disease or condition, wherein one or more analytes are selected from LDH, ferritin, CRP, IL-6, IL-7, IL-8, IL-10, IL- 15, IL-16, TNF-alpha, IFN-gamma, MCP-1, MIP-1beta, eotaxin, G-CSF, IL-1Ralpha, IL-1Rbeta, IP-10, perforin and D-dimer (fibrin breakdown product) ; and (b) following or based on the results of the test, administering to the patient a cell therapy, and optionally a treatment agent or other agent capable of treating, preventing, delaying, attenuating or reducing the development or risk of developing toxicity.

[0158] The invention relates to a method of treatment comprising, after or based on the results of testing a biological sample from a patient in relation to the level, amount or concentration of one or more analytes, administering to the patient (i) a cell therapy, optionally including a dose or composition of genetically engineered cells expressing the recombinant receptor for the treatment of a disease or condition, and optionally (ii) a treatment agent or other agent capable of treating, preventing, delaying, attenuating, or reducing the development or risk of developing toxicity, wherein: a biological sample is obtained from a patient prior to applications of cell therapy; and one or more analytes selected from LDH, ferritin, CRP, IL-6, IL-7, IL-8, IL-10, IL-15, IL-16, TNF-alpha, IFN-gamma, MCP-1, MIP -1beta, eotaxin, G-CSF, IL-1Ralpha, IL-1Rbeta, IP-10, perforin and D-dimer (fibrin degradation product).

[0159] In some embodiments of any of the methods of the invention, said testing includes detection, optionally comprising contacting a reagent capable of directly or indirectly detecting an analyte with a biological sample, and determining the level, amount, or concentration of the analyte in the biological sample. In some embodiments of any of the methods of the invention, if the level, amount, or concentration of the analyte in the sample is equal to or greater than a threshold value: administering to the patient an agent or other treatment capable of treating, preventing, delaying, attenuating, or reducing the development or risk of developing toxicity (i) before, (ii) within one, two or three days, (iii) simultaneously with and/or (iv) at the first rise in temperature after the initiation of the administration of cell therapy to the patient; and/or administering cell therapy to the patient at a reduced dose or at a dose not associated with a risk of developing toxicity or severe toxicity, or not associated with a risk of developing toxicity or severe toxicity in the majority of patients and/or the majority of patients with a disease or condition that the patient has or is suspected of having, after the introduction of cell therapy; and/or administering cell therapy to the patient on an inpatient basis and/or with hospital admission for one or more days, optionally the cell therapy must otherwise be administered to patients on an outpatient basis or without hospital admission for one or more days.

[0160] In some embodiments of any of the methods of the invention, if the level, amount, or concentration of the analyte is equal to or greater than a threshold: administering cell therapy does not include administering, before or concurrently with cellular therapy, and/or until a sign or symptom of toxicity develops other than fever, a therapeutic agent or agent capable of treating, preventing, delaying or reducing the development of toxicity; and/or the administration of cell therapy to the patient should be or may be performed on an outpatient basis and/or without admission of the patient to the hospital overnight or for one or more consecutive days and/or without admission of the patient to the hospital for one or more days.

[0161] In some embodiments of any of the methods of the invention, administering to a patient a treatment agent or other agent capable of treating, preventing, delaying, attenuating, or reducing the development or risk of developing toxicity, wherein: the patient is a candidate for treatment, optionally with cell therapy, wherein said cell therapy optionally includes a dose or composition of genetically engineered cells expressing the recombinant receptor for the treatment of a disease or condition; and the patient has been identified as being at risk of developing toxicity after or based on the results of testing a biological sample from the patient in relation to the level, amount, or concentration of one or more analytes, and said biological sample was obtained from the patient prior to the use of cell therapy, and/or said biological sample is not includes a recombinant receptor and/or said genetically engineered cells, wherein one or more analytes are selected from LDH, ferritin, CRP, IL-6, IL-7, IL-8, IL-10, IL-15, IL-16, TNF -alpha, IFN-gamma, MCP-1, MIP-1beta, eotaxin, G-CSF, IL-1Ralpha, IL-1Rbeta, IP-10, perforin and D-dimer (fibrin breakdown product).

[0162] In some embodiments of any of the methods of the invention, said testing includes detection, optionally including contacting a reagent capable of direct or indirect detection of an analyte with a biological sample and determining the level, amount, or concentration of the analyte in the biological sample. In some embodiments of any of the methods of the invention, an agent is administered to a patient if the level, amount, or concentration of the analyte in the sample is equal to or greater than a threshold value.

[0163] In some embodiments of any of the methods of the invention, the agent is administered (i) prior to, (ii) within one, two, or three days, (iii) simultaneously with and/or (iv) at the first temperature rise after initiation of the administration of the cell therapy to the patient. In some embodiments of any of the methods of the invention, the threshold is within 25%, 20%, 15%, 10%, or 5% of the average percentage or number, and/or within a standard deviation of the average percentage or number of cells with a surface positive with respect to a myeloid marker in a biological sample obtained from a group of patients prior to receiving a therapeutic composition of cells expressing the recombinant receptor, and each of the patients of the group continued to develop toxicity after receiving the composition of cells expressing the recombinant receptor for the treatment of the same disease or condition.

[0164] In some embodiments of any of the methods of the invention, the threshold is within 25%, 20%, 15%, 10%, or 5% of the mean level, amount, or concentration, and/or within the standard deviation of the mean level, amount or the concentration of an analyte in a biological sample obtained from a group of patients prior to the production of a therapeutic composition of cells expressing the recombinant receptor, and each of the patients of the group continued to develop toxicity after receiving the composition of cells expressing the recombinant receptor for the treatment of the same disease or condition. In some embodiments of any of the methods of the invention, the reagent is a binding molecule that specifically binds to a marker or cells in a myeloid cell population. In some embodiments of any of the methods of the invention, the reagent is an antibody or an antigen-binding fragment thereof. In some embodiments of any of the methods of the invention, the biological sample is or is derived from a blood, plasma, or serum sample. In some embodiments of any of the methods of the invention, testing or evaluating the analyte cell includes an immunological assay.

[0165] In some embodiments of any of the methods of the invention, toxicity includes neurotoxicity or cytokine release syndrome (CRS), optionally neurotoxicity or CRS grade 1 or higher. In some embodiments of any of the methods of the invention: the toxicity includes severe neurotoxicity and/or includes grade 2 or higher neurotoxicity, grade 3 or higher neurotoxicity, at least prolonged grade 3 neurotoxicity, or grade 4 or grade 5 or higher neurotoxicity; and/or toxicity includes severe CRS and/or includes CRS grade 2 or higher or grade 3 or higher. In some embodiments of any of the methods of the invention, the toxicity is associated with cerebral edema.

[0166] In some embodiments of any of the methods of the invention, the agent or other therapeutic agent is or includes one or more steroids; antagonists or inhibitors of a cytokine or cytokine receptor selected from IL-10, IL-10R, IL-6, IL-6 receptor, IFNγ, IFNGR, IL-2, IL-2R/CD25, MCP-1, CCR2, CCR4, MIP1β, CCR5, TNFalpha, TNFR1, IL-1, and IL-1Ralpha/IL-1beta; or agents capable of preventing, blocking or attenuating the activity or function of microglial cells. In some embodiments of any of the methods of the invention, the antagonist or inhibitor is or includes an agent selected from an antibody or antigen-binding fragment, a small molecule, a protein or peptide, and a nucleic acid.

[0167] In some embodiments of any of the methods of the invention, the agent or other treatment is an anti-IL-6 or anti-IL6 receptor antibody. In some embodiments of any of the methods of the invention, the agent or other therapeutic agent is or includes an agent selected from among tocilizumab, siltuximab, clazakizumab, sarilumab, olokizumab (CDP6038), elsilimomab, ALD518/BMS-945429, sirukumab (CNTO 136 ), CPSI-2634, ARGX-109, FE301 and FM101. In some embodiments of any of the methods of the invention, the agent or other treatment is or includes tocilizumab. In some embodiments of any of the methods of the invention, the agent or other treatment is or includes siltuximab.

[0168] In some embodiments of any of the methods of the invention, the steroid is or includes dexamethasone. In some embodiments of any of the methods of the invention, the agent capable of preventing, blocking, or attenuating microglial cell activity or function is selected from an anti-inflammatory agent, a NADPH oxidase (NOX ₂ ) inhibitor, a calcium channel blocker, a sodium channel blocker, inhibits GM-CSF, inhibits CSF1R, binds specifically to CSF-1, binds specifically to IL-34, inhibits nuclear factor kappa-B (NF-κB) activation, activates CB2 receptor and/or is a CB2 agonist, phosphodiesterase inhibitor, inhibits microRNA-155 ((miR -155) or upregulates microRNA-124 ((miR-124.

[0169] In some embodiments of any of the methods of the invention, the agent capable of preventing, blocking, or attenuating microglial cell activation or function is a small molecule, peptide, protein, antibody, or antigen-binding fragment thereof, antibody mimetic, aptamer, or nucleic acid molecule. In some embodiments of any of the methods of the invention, the agent is selected from minocycline, naloxone, nimodipine, Riluzole, MOR103, lenalidomide, cannabinoid (optionally WIN55 or 212-2), intravenous immunoglobulin (IVIg), ibudilast, anti-miR-155 gated nucleic acid (LNA) hydroxy)benzyl)pyrimidine-2,4-diamine (GW2580), AZD6495, Ki20227, BLZ945, emactuzumab, IMC-CS4, FPA008, LY-3022855, AMG-820 and TG-3003.

[0170] In some embodiments of any of the methods of the invention, the agent is a colony stimulating factor 1 receptor (CSF1R) inhibitor. In some embodiments of any of the methods of the invention, the inhibitor is selected from: PLX-3397, PLX647, PLX108-D1, PLX7486, JNJ-40346527, JNJ28312141, ARRY-382, AC-708, DCC-3014, 5-(3-methoxy- 4-(4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine (GW2580), AZD6495, Ki20227, BLZ945 or a pharmaceutical salt or prodrug thereof; emaktuzumab, IMC-CS4, FPA008, LY-3022855, AMG-820 and TG-3003, or an antigen-binding fragment thereof; or combinations of any of the foregoing.

[0171] In some embodiments of any of the methods of the invention, the inhibitor is PLX-3397. In some embodiments of any of the methods of the invention, the recombinant receptor specifically binds to an antigen associated with a disease or condition, or expressed in cells in a lesion environment associated with the disease or condition. In some embodiments of any of the methods of the invention, the disease or condition is cancer. In some embodiments of any of the methods of the invention, the disease or condition is myeloma, leukemia, or lymphoma. In some embodiments of any of the methods of the invention, the disease or condition is B-cell malignancy and/or acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin's lymphoma (NHL), and diffuse large B-cell lymphoma (DLBCL).

[0172] In some embodiments of any of the methods of the invention, the antigen is ROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2/neu (erbB2 receptor tyrosine kinase), L1-CAM, CD19, CD20 , CD22, mesothelin, CEA, and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, erb-B2, erb-B3, erb-B4, erbB dimers, EGFR vIII, folate binding protein (FBP), FCRL5, FCRH5, embryonic acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL- 13R-alpha2, receptor containing kinase insertion domain (kdr), kappa light chain, Ley antigen, L1 cell adhesion molecule, (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE- A6, preferably expressed melanoma antigen (PRAME), survivin, TAG72, B7-H6, IL-13 receptor alpha 2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AI MAGE Al, HLA-A2 NY-ESO-1, PSCA, folic acid receptor alpha, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, Embryonic AchR, NKG2D ligands, CD44v6, dual antigen, testicular cancer antigen, mesothelin, mouse CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms tumor 1 (WT-1), cyclin, cyclin A2, CCL-1, CD138, 5D receptor , associated with G-protein (GPCR5D), or pathogen-specific antigen.

[0173] In some embodiments of any of the methods of the invention, the recombinant receptor is a T cell receptor or a functional non-T cell receptor. In some embodiments of any of the methods of the invention, the recombinant receptor is a chimeric antigen receptor (CAR). In some embodiments of any of the methods of the invention, the CAR comprises an extracellular antigen-recognition domain specifically binding to an antigen and an intracellular signaling domain comprising ITAM, optionally the intracellular signaling domain including the intracellular domain of the CD3-zeta (CD3ζ) chain; and/or wherein the CAR further comprises a co-stimulatory signaling domain, optionally including a CD28 or 4-1BB signaling domain.

[0174] In some embodiments of any of the methods of the invention, the engineered cells include T cells, optionally CD4 ⁺ and/or CD8 ⁺ . In some embodiments of any of the methods of the invention, the T cells are primary T cells obtained from a patient. In some embodiments of any of the methods of the invention, cell therapy comprises administering from or from about 1 x 10 ⁵ to 1 x 10 ⁸ total recombinant receptor expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMC), from or from about 5 x 10 ⁵ to 1 x 10 ⁷ total recombinant receptor-expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMCs) or from or about 1 x 10 ⁶ to 1 x 10 ⁷ total recombinant receptor-expressing cells, total T -cells or total peripheral blood mononuclear cells (PBMC), inclusive.

[0175] In some embodiments of any of the methods of the invention, cell therapy comprises administering no more than 1 x 10 ⁸ total recombinant receptor-expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMCs), no more than 1 x 10 ⁷ total recombinant receptor-expressing cells, total T-cells, or total peripheral blood mononuclear cells (PBMC), not more than 0.5 x 10 ⁷ total recombinant receptor-expressing cells, total T-cells, or total peripheral blood mononuclear cells (PBMC), not more than 1 x 10 ⁶ total recombinant receptor-expressing cells, total T-cells, or total peripheral blood mononuclear cells (PBMC), not more than 0.5 x 10 ⁶ total recombinant receptor-expressing cells, total T-cells, or total peripheral blood mononuclear cells blood (PBMC).

[0176] In some embodiments of any of the methods of the invention, the dose not associated with a risk of developing toxicity or severe toxicity is or includes less than or less than about 5 x 10 ⁷ total recombinant receptor expressing cells, optionally CAR ⁺ cells, total T cells or total peripheral blood mononuclear cells (PBMC), e.g., less than or less than about 2.5 x 10 ⁷ , less than or less than about 1.0 x 10 ⁷ , less than or less than about 5.0 x 10 ⁶ , less than or less than about 1.0 x 10 ⁶ , less than or less than about 5.0 x 10 ⁵ , or less than or less than about 1 x 10 ⁵ total recombinant receptor-expressing cells, optionally CAR ⁺ cells, total T cells, or total peripheral blood mononuclear cells (PBMC).

[0177] In some embodiments of any of the methods of the invention, the dose not associated with a risk of developing toxicity or severe toxicity is or includes from or about 1 x 10 ⁵ to 5 x 10 ⁷ total recombinant receptor expressing cells, optionally CAR ⁺ cells , total T cells or total peripheral blood mononuclear cells (PBMC), e.g. 1 x ¹⁰⁵ to 2.5 x ¹⁰⁷ , 1 x ¹⁰⁵ to 1.0 x ¹⁰⁷ , 1 x ¹⁰⁵ to 5 .0 x 10 ⁶ , 1 x 10 ⁵ to 1.0 x 10 ⁶ , 1.0 x 10 ⁵ to 5.0 x 10 ⁵ , 5.0 x 10 ⁵ to 5 x 10 ⁷ , from 5 x ¹⁰⁵ to ^2.5x107 , ^5x105 to ^{1.0x107 ,} 5x105 ^to 5.0x106, ^5x105 to ^1.0x106 , from 1.0 x 10 ⁶ to 5 x 10 ⁷ , 1 x 10 ⁶ to 2.5 x 10 ⁷ , 1 x 10 ⁶ to 1.0 x 10 ⁷ , 1 x 10 ⁶ to 5.0 x 10 ⁶ , 5.0 x 10 ⁶ to 5 x 10 ⁷ , 5 x 10 ⁶ to 2.5 x 10 ⁷ , 5 x 10 ⁶ to 1.0 x 10 ⁷ , 1.0 x 10 ⁷ to 5 x 10 ⁷ , 1 x 10 ⁷ to 2.5 x 10 ⁷ or 2.5 x 10 ⁷ to 5 x 10 ⁷ total recombinant receptor-expressing cells, optionally CAR ⁺ cells, total T cells, or total peripheral blood mononuclear cells ( PBMC). In some embodiments of any of the methods of the invention, the genetically engineered cells are autologous to the patient. In some embodiments of any of the methods of the invention, the genetically engineered cells are allogeneic to the patient. In some embodiments of any of the methods of the invention, the reagent is labeled so that it can be detected, optionally fluorescently labeled.

[0178] In some embodiments, the instructions provide information about a threshold value, individually for each one or more analytes, that is an indicator of the likelihood that a patient will demonstrate a response to cell therapy treatment. In some embodiments, the instructions provide information on a threshold value, individually for each one or more analytes, that is an indicator of the likelihood that a patient will demonstrate a sustained response after administration of cell therapy. In some embodiments, the instructions provide information on a threshold value, individually for each one or more analytes, that is an indicator of the likelihood that a patient will demonstrate toxicity after administration of cell therapy.

Brief description of the drawings

[0179] FIG. 1 shows the percentage of patients with laboratory abnormalities and treatment-related adverse events (TEAE) occurring in ≥20% of patients. *: One Grade 5 AE in the form of multiple organ failure unrelated to study treatment and resulting from the development of lymphoma; †: One Grade 5 AE of diffuse alveolar injury, as assessed by the investigator, associated with fludarabine, cyclophosphamide, and CAR T-cell therapy occurring on day 23 in a patient who refused mechanical ventilation for progressive respiratory failure, demonstrating neutropenia on growth factors and broad-spectrum antibiotics and antifungals.

[0180] FIG. 2 is a Kaplan-Meier curve depicting the observed time to onset of CRS and neurotoxicity.

[0181] FIG. 3A and FIG. 3B depicts the objective response rate (ORR) over a 3-month period among subgroups of treated patients.

[0182] FIG. 4A and FIG. 4B shows duration of response (CR/PR, CR or PR) and overall survival in the overall and core patient population.

[0183] FIG. 5A shows the pharmacokinetics of CAR ⁺ T cells in peripheral blood at different time points after treatment at different dose levels. FIG. 5B shows the pharmacokinetics of CAR ⁺ T cells in peripheral blood at different time points after treatment between responders and non-responders. FIG. 5C shows the pharmacokinetics of CAR ⁺ T cells in peripheral blood at different time points after treatment in patients who developed or did not develop neurotoxicity.

[0184] FIG. 6A shows the number of CD3 ⁺ /CAR ⁺ , CD4 ⁺ /CAR ⁺ , CD8 ⁺ /CAR ⁺ T cells in the peripheral blood of a patient with chemorefractory transformed DLBCL, measured at certain time points. FIG. 6B is a pre-treated axial PET-CT image showing intracranial pathology in the right middle cranial fossa and extensive pathology in the subcutaneous tissues in the right posterior auricular region. FIG. 6C is a post-treatment PET-CT image of the pathology in FIG. 2B after treatment with anti-CD19 CAR ⁺ T cells. FIG. 6D is a pre-processed MRI of the brain (T ₁ high resolution weighted image using contrast; axial view) showing a homogeneously increased mass in the right middle cranial fossa. FIG. 6E is a post-treatment MRI image showing almost complete elimination of the increased mass. FIG. 6F is an axial PET-CT image in the right posterior ear region showing tumor recurrence associated with extensive accumulation ^{of 18} F-fluorodeoxyglucose (arrow). FIG. 6G is a PET-CT image showing clearance of a posterior ear tumor after incisional biopsy and CAR ⁺ T cell re-expansion.

[0185] FIG. 7 shows the levels of analytes measured in the serum of patients prior to administration of CAR ⁺ T cells and the correlation with the development of neurotoxicity.

[0186] FIG. 8 shows a graph showing time without progression (months) and best complete response and duration of response, and individual clinical outcomes observed over time in individual patients in the Full and Core NHL cohorts treated with anti-CD19 cell therapy containing CAR-T-expressing CD4 ⁺ and CD8 ⁺ T cells. ^a : Patients achieved BOR at month 1, unless otherwise noted; ^b : Complete elimination of CNS involvement in lymphoma seen in 2 patients; ^c : One patient with biopsy re-expansion after disease progression

[0187] FIG. 9A depicts the median (±quartiles) number of CAR-expressing CD3 ⁺ cells/µl of blood, estimated by flow cytometry using an antibody specific for the truncated receptor (CD3, circle; N=87); or median (±quartiles) integrated CAR transgene copy number/mg genomic DNA, as assessed by quantitative polymerase chain reaction (qPCR) using primers specific for the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) present in the CAR-coding vector (qPCR, square ; N=85) in blood samples from 87 patients injected with CAR-expressing anti-CD19 cells. The cutoff for detection of CAR ⁺ cells in flow cytometry was set to ≥ 25 events in CAR ⁺ gates, and the detection limit for qPCR was ≥ 12.5 copies of transgenic CARs per µg of genomic DNA.

[0188] FIG. 9B depicts the relative numbers of CD4 ⁺ and CD8 ⁺ CAR-expressing cells/µl in blood and bone marrow samples from 67 patients treated with anti-CD19 CAR-expressing cells on day 11 ± 3 days. The line represents a unity line and is not a regression line.

[0189] FIG. 10A and 10B depict the median (±quartiles) area under the curve between days 0 and 28 (AUC _0-28 ; FIG. 10A) and peak serum concentration (AUC _0-28 ; CAR ⁺ cells/µl blood; FIG. 10B) CD4 ⁺ and CD8 ⁺ CAR ⁺ cells in subgroups of patients with de novo diffuse large B-cell lymphoma or transformed from indolent lymphoma (DLBCL, NOS; N=27), transformed follicular lymphoma (tFL; N=10), DLBCL transformed from lymphoma marginal zone or chronic lymphocytic leukemia (tMZL/tCLL; N=4), or mantle cell lymphoma (MCL; N-5) who received CAR-expressing T cells in DL1.

[0190] FIG. 11A and 11B depict the median (±quartiles) area under the curve between days 0 and 28 (AUC _0-28 ; FIG. 11A) and peak serum concentration (AUC _0-28 ; CAR ⁺ cells/µl blood; FIG. 11B) CD3 ⁺ , CD4 ⁺ and CD8 ⁺ CAR ⁺ cells in patients who received CAR ⁺ cells in DL1 or DL2.

[0191] FIG. 12A-12D depict the median (±quartiles) number of CAR-expressing CD4 ⁺ and CD8 ⁺ CAR ⁺ cells/µL of blood over time in patients who developed cytokine release syndrome (any CRS) compared with patients who did not develop CRS (no CRS) (CD4 ⁺ : FIG. 12A; CD8 ⁺ : FIG. 12B), or in patients who developed neurotoxicity (any NT) compared to patients who did not develop NT (no NT) (CD4 ⁺ : FIG 12C; CD8 ⁺ : FIG 12D).

[0192] FIG. 13A and 13B depict the number of peak CD3 ⁺ CAR ⁺ cells/µl (CD3 ⁺ AUC _0-28 ) in patients divided into patient groups who had a best overall response (BOR) of CR, PR or PD or a 3-month sustained response ( M3) CR, PR or PD.

[0193] FIG. 14A depicts pre-lymphodepletion blood analyte levels in serum samples from patients showing high CAR ⁺ cell expansion (CD3 ⁺ AUC _0-28 >500) and patients showing low CAR ⁺ cell expansion (CD3 ⁺ AUC _0-28 <500) .

[0194] FIG. 14B depicts peak blood analyte levels in serum samples from patients showing high CAR ⁺ cell expansion (CD3 ⁺ AUC _0-28 >500) and patients showing low CAR ⁺ cell expansion (CD3 ⁺ AUC _0-28 <500).

[0195] FIG. 15 depicts a plot depicting SPD before lymphodepletion (cm ² ) vs. AUC _0-28 (cells*day/µl) of CD3 ⁺ CAR ⁺ cells for individual patients treated with DL1 or DL2 CAR ⁺ cells.

[0196] FIG. 16A and 16B depict pre-lymphodepletion blood analyte levels in serum samples from patients who developed cytokine release syndrome (CRS grades 1-4) compared to patients who did not develop CRS (CRS grade 0) (FIG. 16A) , or in patients who developed neurotoxicity (NT grade 0) compared to patients who did not develop NT (NT grade 1-4) (FIG. 16B). Units were as follows: Ferritin and D-dimer (µg/l); CRP (mg/l) and cytokines (pg/ml).

[0197] FIG. 17 depicts pre-lymphodepletion evaluation of a patient parameter as sum of product dimensions (SPD; cm ² ) showing tumor mass and lactate dehydrogenase (LDH; U/L) level in patients who developed cytokine release syndrome (any CRS) compared to patients who did not develop CRS (no CRS), or in patients who developed neurotoxicity (any NT), compared to patients who did not develop NT (no NT).

[0198] FIG. 18A is a graph depicting SPD (cm ² ) before lymphodepletion versus LDH levels (U/L) before lymphodepletion in individuals who developed neurotoxicity (NT grades 1-4) or patients who did not develop NT (NT grade 0) (left group), and in individuals who developed CRS (CRS grade 1-4) or patients who did not develop CRS (CRS grade 0) (right group). Dotted lines represent SPD (50 cm ² or higher) or LDH (500 U/L or higher) levels, which are associated with higher CRS or NT. FIG. 18B depicts estimates of the likelihood of developing CRS or NT based on SPD (50 cm ² or greater) or LDH (500 U/L or greater) levels, with 95% confidence intervals (CI). FIG. 18C depicts estimates of the likelihood of developing CRS or NT based on SPD or LDH levels, including probability estimates for below threshold values, with 95% confidence intervals (CI).

[0199] FIG. 19 depicts the tumor mass parameter (SPD) before lymphodepletion and blood analyte levels for patients who had a sustained response at 3 months compared to patients who did not have a response at 3 months. Units were as follows: Ferritin and D-dimer (µg/l); CRP and SAA-1 (mg/l) and cytokines (pg/ml).

[0200] FIG. 20A and 20B depict peak blood analyte levels in serum samples from patients who developed cytokine release syndrome (any CRS) compared to patients who did not develop CRS (no CRS) (FIG. 20A), or in patients who who developed neurotoxicity (any NT) compared to patients who did not develop NT (no NT) (FIG. 20B). The units were as follows: CRP (mg/l), SAA-1 (mg/l) and cytokines (pg/ml).

[0201] FIG. 21A depicts peak blood analyte levels in serum samples from patients who had a better overall response (BOR) in the form of a complete response (CR) or partial response (PR) (N=57) compared to levels in patients who had stable disease (SD). or progressive disease (PD) (N=17). FIG. 21B depicts peak blood analyte levels in serum samples from patients who had a 3-month SD/PD response (N=31) compared to patients who had a 3-month CR/PR response (N=35). The units were as follows: CRP (mg/l), SAA-1 (mg/l) and cytokines (pg/ml).

[0202] FIG. 22 depicts the objective response rate (ORR) at month 3 among subgroups of treated patients with a 95% confidence interval.

[0203] FIG. 23A and 23B depict duration of response (DOR) for full population (FIG. 23A) and core population (FIG. 23B) and FIG. 23C and 23D depict overall survival for the full cohort (FIG. 23C) and core cohort (FIG. 23D), patients who achieved CR, PR, all responders, non-responders, and all treated patients. The median F/U was 6.3 months during the response.

[0204] FIG. 24 shows the percentage of patients experiencing treatment-related adverse events (TEAE) in the FULL DLBCL cohort that occurred in ≥20% of patients. Data for 5 MCL patients treated with product in DL1 with at least 28 days of follow-up were not included. ^b : One Grade 5 AE in the form of septic shock unrelated to CAR ⁺ T cell infusion. ^c : One Grade 5 AE in the form of diffuse alveolar injury, as assessed by the investigator associated with fludarabine, cyclophosphamide, and CAR ⁺ T cells, occurred on day 23 in a patient who refused mechanical ventilation for progressive respiratory failure, demonstrating neutropenia on growth factors and broad-spectrum antibiotics and antifungals. ^d : Laboratory abnormalities.

[0205] FIG. 25 shows the percentage of patients who developed CRS or neurotoxicity over time in the full cohort.

[0206] FIG. 26 shows graphs showing the purity of T cells of T cell compositions enriched in CD4 ⁺ and CD8 ⁺ cells at various stages of the generation process of genetically engineered cell compositions containing CAR T cells, which are described in Example 8. The frequency (% of total leukocytes) is shown. a) CD4 ⁺ and CD8 ⁺ cells in compositions.

[0207] FIG. 27A-27C are graphs showing the concentration (FIG. 27A), viability (FIG. 27B), and frequency of caspase-3 negative (FIG. 27C) CD4 ⁺ and CD8 ⁺ CAR ⁺ T cells in high or low volume therapeutic cell formulations. .

[0208] FIG. 28A shows the number of CD3 ⁺ CAR ⁺ T cells present in CAR T cell formulations for introduction into DL1 and DL2. FIG. 28B shows the number of CD4 ⁺ CAR ⁺ and CD8 ⁺ CAR ⁺ cells and CD4 ⁺ CAR ⁺ TNF-α ⁺ cells and CD8 ⁺ CAR ⁺ TNF-α ⁺ cells present in CAR T cell formulations for introduction into DL1 and DL2.

[0209] FIG. 29 shows the percentage of patients experiencing on-treatment adverse events (TEAE) in the FULL DLBCL cohort that occurred in ≥20% of patients at the time point described in Example 6. Data for 6 MCL patients treated with product in DL1 at least with 28 days of observation were not included. ^b : One Grade 5 AE in the form of septic shock unrelated to CAR ⁺ T cell infusion occurred with disease progression. ^c : One Grade 5 AE in the form of diffuse alveolar injury, as assessed by the investigator, associated with fludarabine, cyclophosphamide, and CAR ⁺ T cells occurred on day 23 in a patient who refused mechanical ventilation for progressive respiratory failure, demonstrating neutropenia on growth factors and broad-spectrum antibiotics and antifungals. ^d : Laboratory abnormalities.

[0210] FIG. 30 depicts the objective response rate (ORR) over six (6) months among subgroups of treated patients with a 95% confidence interval. ^a Includes all patients with DLBCL treated at all dose levels in the core cohort.

[0211] FIG. 31A and 31B depict duration of response (DOR) for full population (FIG. 31A) and core population (FIG. 31B), and FIG. 31C and 31D depict overall survival for the full cohort (FIG. 31C) and core cohort (FIG. 31D), for patients who achieved CR, PR, all responders, non-responders, and all treated patients. NE, not quantifiable.

Detailed description

I. Methods and uses of cell therapy with genetically engineered cells

[0212] The invention relates to methods and uses of genetically engineered cells (e.g., T cells) and/or compositions thereof for the treatment of patients having a disease or condition, which is usually or includes cancer or a tumor, such as leukemia or lymphoma, in particular, non-Hodgkin's lymphoma (NHL). In some aspects, the methods and uses provide or achieve improved response and/or longer responses or efficacy and/or reduced risk of toxicity or other side effects, for example, in certain groups of patients receiving treatment, compared to certain alternative methods. In some embodiments, the methods are advantageous based on the introduction of specific numbers or relative amounts of genetically engineered cells, the introduction of certain ratios of certain cell types, the treatment of a certain population of patients, such as those with a certain risk profile, stage and/or previous treatment history, and /or combinations of these. The invention also relates to methods, including the evaluation of certain parameters, for example, the expression of certain biomarkers or analytes, which may correlate with the development of toxicity, and methods of treatment, for example, interventional therapy, to prevent and/or alleviate toxicity. The invention also relates to methods involving the evaluation of certain parameters, for example, the expression of certain biomarkers or analytes, which can be correlated with an outcome, such as a therapeutic outcome, including a response, such as a complete response (CR) or a partial response (PR), optionally a long-term response. such as a response that lasts for at least 3 months, 6 months or more; or a safety outcome such as the development of toxicity, eg neurotoxicity or CRS, following administration of immunotherapy and/or cell therapy. The invention also relates to methods for assessing the likelihood of a response and/or the likelihood of a risk of toxicity based on the evaluation of parameters such as the expression of biomarkers or analytes. The invention also relates to compositions for use in cell therapy. The invention also relates to articles and kits, for example, for use in the methods of the invention. In some embodiments, the articles and kits optionally contain instructions for use in accordance with the methods of the invention.

[0213] In some embodiments, the methods and uses include administering to a patient cells expressing engineered (recombinant) cell surface receptors in adoptive cell therapy, which are typically chimeric receptors such as chimeric antigen receptors (CARs) that recognize an antigen expressed bound with and/or specific for leukemia or lymphoma and/or the type of cell from which it is derived. Cells are usually administered in compositions formulated for administration; methods typically involve administering one or more doses of cells to a patient, wherein the dose(s) may include a specific number or relative number of cells or genetically engineered cells, and/or a specific ratio or composition of two or more subtypes in the composition, e.g., CD4 vs. CD8 T cells.

[0214] In some embodiments, cells, populations, and compositions are administered to a patient having a particular disease or condition being treated, for example, by adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the methods include treating a patient having a lymphoma or leukemia, such as non-Hodgkin's lymphoma (NHL), with a dose of antigen receptor-expressing cells (eg, CAR-expressing cells).

[0215] In some embodiments, the methods of the invention include treating a specific group or subpopulation of patients, eg, patients identified as being at high risk for disease, eg, high risk for NHL. In some aspects, the methods are used to treat patients who have an aggressive and/or poor prognosis form of B-cell non-Hodgkin's lymphoma (NHL), such as NHL that is relapsed or refractory (R/R) to standard therapy, has a poor prognosis. In some cases, the overall response rate (ORR; also known in some cases as objective response rate) to available therapies, standard methods, or reference therapies for the disease and/or patient population for which the therapy is indicated is less than 40% and/or a complete response (CR; also known in some cases as a complete remission) is less than 20%. In some embodiments, in the case of chemo-refractory DLBCL, the RRR of the reference or available treatment or standard therapy is approximately 26% and the CR level is approximately 8% (Crump et al . Outomes in refractory aggressive diffuse large B-cell lymphoma (DLBCL): Results from the international SCHOLAR study ASCO 2016 [Abstract 7516]). In some aspects, the methods of the invention, compositions, uses, and articles of manufacture achieve improved and superior responses over available therapies.

[0216] In some embodiments, methods, uses, and articles of manufacture include or are used to treat patients, including selection or identification of a specific group or subpopulation of patients, for example, based on certain types of disease, diagnostic criteria, prior treatments, and/or response to prior treatments. In some embodiments, the methods include treating a patient who has relapsed in remission after treatment with, or has become refractory to, one or more prior treatments; or a patient who is relapsed or refractory (R/R) to one or more prior therapies, eg one or more lines of standard therapy. In some embodiments, the methods include treating patients having diffuse large B-cell lymphoma (DLBCL), unspecified (NOS; de novo and transformed from indolent), primary mediastinal B-cell lymphoma (PMBCL), or follicular lymphoma, such as stage follicular lymphoma. 3B (FL3B). In some embodiments, the methods include treating a patient having an Eastern Cooperative Oncology Group Performance Status (ECOG) of 0-1 or 0-2. In some embodiments, the methods are used to treat populations with a poor prognosis, or patients with DLBCL, or patients who generally respond poorly to therapies or certain reference therapies, such as having one or more, for example, two or three, chromosome translocations (such as the so-called "double-hit" or "triple-hit" lymphoma; having translocations of the MYC/8q24 loci, usually in combination with the t(14; 18) (q32; q21)bcl-2 gene and/or BCL6/3q27 chromosomal translocation; see , e.g., Xu et al (2013) Int J Clin Exp Pathol 6(4): 788-794), and/or patients who relapse, optionally relapse within 12 months, after administration of an autologous stem cell transplant (ASCT), and/or patients identified as chemorefractory.

[0217] In some aspects, embodiments of the invention are based on observations that the methods of the invention can be used to achieve high response rates with high duration compared to some of the available cell therapies, without increased risk of toxicity. In some embodiments, the methods of the invention provide prolonged persistence of adoptively transferred cells for cell therapy and/or a low rate of development of toxicity in a patient. In some embodiments, the methods can be used to select for cell therapy treatment patients who are likely or more likely to respond to therapy and/or determine appropriate doses or dosing regimen for a higher response rate and/or longer response while reducing the risk of toxicity. Such methods may provide rational strategies for facilitating the safe and effective clinical use of adoptive cell therapy, such as CAR-T cell therapy.

[0218] In some embodiments, an antigen receptor (eg, CAR) specifically binds to a target antigen associated with a disease or condition, such as associated with NHL. In some embodiments, the antigen associated with the disease or disorder is selected from CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b, or CD30.

[0219] In some embodiments, methods include administering cells or a composition containing cells to a patient, tissue or cell, such as having, at risk, or suspected of having a disease, condition, or disorder. In some embodiments, the patient is an adult patient. In some embodiments, the patient is approximately 30, 40, 50, 60, or 70 years of age or older.

[0220] In some embodiments, the methods include administering the cells to a patient selected or identified as having a particular prognosis or risk for NHL. Non-Hodgkin's lymphoma (NHL) can be a variant disease. Some NHL patients may survive without treatment, while others may require immediate intervention. In some cases, patients with NHL can be classified into groups that can report on the prognosis of the disease and/or the recommended treatment strategy. In some cases, these groups may be "low risk", "intermediate risk", "high risk" and/or "very high risk" and patients may be classified into them depending on many factors, including but not limited to , genetic abnormalities and/or morphological or physical properties. In some embodiments, patients receiving treatment in accordance with the methods and/or articles or compositions are classified or identified based on the risk of NHL. In some embodiments, the patient is a patient at high risk for NHL.

[0221] In some embodiments, the patient has previously been treated with a therapy or therapeutic agent that targets a disease or condition, such as NHL, prior to administration of cells expressing the recombinant receptor. In some embodiments, the patient has previously received treatment with a hematopoietic stem cell transplant (HSCT), such as allogeneic HSCT or autologous HSCT. In some embodiments, the patient had a poor prognosis after treatment with standard therapy and/or did not have a positive outcome after one or more lines of previous therapy. In some embodiments, the patient has been treated or previously received at least or about at least or about 1, 2, 3 or 4 other therapies for the treatment of NHL other than anti-lymphocyte therapy and/or a dose of antigen receptor expressing cells. In some embodiments, the patient has previously been treated with radiation therapy or chemotherapy. In some aspects, the patient is refractory or unresponsive to another therapy or therapeutic agent. In some embodiments, the patient has a persistent or recurrent disease, such as after treatment with another therapy or therapeutic intervention, including chemotherapy or radiation.

[0222] In some embodiments, the patient is a transplant eligible patient, such as an eligible hematopoietic stem cell transplant (HSCT), such as an allogeneic HSCT. In some such embodiments, the patient has not previously received a transplant, despite being eligible, prior to the administration of the engineered cells (eg, CAR-T cells) or composition containing the cells to the patient, as provided in the present invention.

[0223] In some embodiments, the patient is a patient who is not eligible for a transplant, such as not eligible for a hematopoietic stem cell transplant (HSCT), such as an allogeneic HSCT. In some embodiments, genetically engineered cells (eg, CAR-T cells) or a composition containing cells according to embodiments of the invention are administered to such a patient.

[0224] In some embodiments, the methods include administering the cells to a patient selected or identified as having a high risk of NHL. In some embodiments, the patient exhibits one or more cytogenetic abnormalities, such as those associated with a high risk of NHL. In some embodiments, the patient is selected or identified based on the presence of a disease or condition characterized as or defined as aggressive NHL, diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma (PMBCL), T-cell/histocytic- enriched large B cell lymphoma (TCHRBCL), Burkitt's lymphoma, mantle cell lymphoma (MCL) and/or follicular lymphoma (FL). In some embodiments, a patient that may be treated with the methods of the invention includes patients with aggressive NHL, particularly diffuse large B-cell lymphoma (DLBCL), unspecified (NOS; de novo and transformed from indolent), primary mediastinal B -cell lymphoma (PMBCL) or stage 3B follicular lymphoma (FL3B). In some embodiments, the implementation of the patient has a status of unsatisfactory treatment. In some aspects, the population being treated includes patients having an Eastern Cooperative Oncology Group Performance Status (ECOG) that ranges from 0-2. In other aspects of any of the embodiments, patients being treated include patients with ECOG 0-1 or do not include patients with ECOG 2. previous therapies. In some embodiments, the patient does not have DLBCL transformed from marginal zone lymphoma (MZL) and chronic lymphocytic leukemia (CLL; Richter). In some embodiments, a patient with CLL may exhibit Richter syndrome (RS), defined as the transformation of CLL into an aggressive lymphoma, usually diffuse large B-cell lymphoma (DLBCL) (see, for example, Parikh et al. Blood 2014 123:1647-1657 ). In some embodiments, the patient has mantle cell lymphoma (MCL). In some embodiments, the implementation of the patient has signs that correlate with low overall survival. In some embodiments, the patient has never achieved a complete response (CR), has never received an autologous stem cell transplant (ASCT), is refractory to 1 or more second-line therapies, has primary refractory disease, and/or has an ECOG condition score of 2.

[0225] In some embodiments, the patient being treated includes a group of patients with diffuse large B-cell lymphoma (DLBCL), de novo or transformed from indolent lymphoma (NOS), primary mediastinal large B-cell lymphoma (PMBCL), and follicular stage 3B (FL3B) lymphoma after failure of 2 lines of therapy and an ECOG score of 0-2, and the patient may have optionally previously received an allogeneic stem cell transplant (SCT). In some embodiments, the implementation of such a group of patients may be referred to as "full population". In some embodiments, a patient is selected for treatment with adoptive cell therapy if the patient meets the specified criteria. In some embodiments, in the specified group (“full population”), the patient is not selected for treatment or excluded from treatment if the patient has an unsatisfactory status (for example, ECOG 2) and/or has DLBCL transformed from marginal zone lymphoma (MZL) and chronic lymphocytic leukemia (CLL, Richter). Thus, in some embodiments, a patient is selected for treatment if the patient has patients with diffuse large B-cell lymphoma (DLBCL), de novo or transformed from indolent lymphoma (NOS), primary mediastinal large B-cell lymphoma (PMBCL), and stage 3B follicular lymphoma (FL3B) after failure of 2 lines of therapy and with an ECOG score of 0 or 1, and the patient may not necessarily have previously received an allogeneic stem cell transplant (SCT) but does not have DLBCL transformed from marginal zone lymphoma (MZL) and chronic lymphocytic leukemia (CLL, Richter). In some embodiments, such a group of patients may be referred to as a "basic population". In some embodiments, the implementation of the patient receiving treatment are patients in the "basic population".

[0226] In some aspects, embodiments of the invention are based on the observation that certain patient populations, such as "core population" patients who are administered a particular dose of cell therapy, exhibit an overall response rate (ORR) of more than 80%, with a complete response rate (CR ) is greater than 55%, with a high duration, eg, a response that persists for a longer period of time, eg, more than 3 months, with a 3-month RRR greater than 65% and a 3-month CR level of approximately 50%. In particular, observations showed that the 3-month RRR was high in patients with two or three chromosomal translocations ("double-hit" or "triple-hit" lymphoma; having translocations at the MYC/8q24 loci, usually in combination with t(14 18)(q32;q21)bcl-2 genome and/or BCL6/3q27 chromosomal translocation, see e.g. Xu et al (2013) Int J Clin Exp Pathol 6(4): 788-794), primary refractory lymphomas, chemo-refractory DLBCL, and patients who have never previously achieved CR.

[0227] In some aspects, the invention relates to compositions, methods, and uses for administering a specific cell therapy composition, at specific doses, associated with a high response rate and/or high duration of response and low levels and/or frequency of toxicity. In some embodiments, the composition or dose administered is a flat and/or fixed dose, such as a precise flat dose, of cells and/or one or more cells having a particular phenotype, such as a certain number of such cells or a number that is within a certain range. and/or the degree of variability or difference compared to the target number. In some embodiments, the composition or dose administered contains a certain ratio of CD4 ⁺ and CD8 ⁺ cells (for example, a ratio of 1:1 CD4 ⁺ : CD8 ⁺ CAR ⁺ T cells), and / or contains a ratio that is in a certain degree of variability from such a ratio, such as no more than +10%, such as no more than +8%, such as a degree of variability or difference of no more than +10%, such as no more than +8%. In some embodiments, CD4 ⁺ and CD8 ⁺ cells are individually formulated and administered. In some embodiments, the administered cells show consistent activity and/or function, such as cytokine production, apoptosis, and/or growth. In some embodiments, compositions of the invention exhibit highly consistent and defined activity and low inter-cell variability, eg in terms of cell number, cell function and/or cell activity, within the composition or between formulations. In some embodiments, consistency in activity and/or function, such as low inter-drug variability of the compositions, may enhance efficacy and/or safety. In some embodiments, administration of certain compositions results in low product variability and low toxicity, eg, CRS or neurotoxicity, compared to administration of highly heterogeneous cell compositions. In some embodiments, the implementation of a certain concordant composition also shows concordant cell growth. Such consistency may facilitate identification of dose, therapeutic window, dose-response assessment, and identification of patient factors that may correlate with safety or toxicity results.

[0228] In some embodiments, in a certain cohort of patients receiving a single infusion of a certain dose level, a sustained response rate after 6 months of more than 60% can be achieved. In some embodiments, patients in certain cohorts can achieve an overall response rate (ORR, in some cases also known as an objective response rate) of greater than 80%, a complete response (CR) rate of greater than 60%, and/or a high sustained CR at 6 months. In some embodiments, patients receiving a certain dose show improved safety outcomes, for example, more than two-thirds of patients do not show CRS or NT. In some aspects, the level of severe CRS or severe NT is low. In some embodiments, the higher exposure (eg, AUC _0-28 and AUC _0-28 ) observed with a particular specific dose is not associated with increased toxicity, eg, CRS or NT. In some embodiments, specific patient factors, such as certain biomarkers, may be used to predict the risk of toxicity. In some embodiments, embodiments of the invention may be used to achieve a high response rate with a low risk of toxicity.

[0229] In some embodiments, no more than 25%, no more than 20%, no more than 15%, no more than 10%, or no more than 5% of patients treated with compositions, articles, kits, methods and uses of the invention, an agent (eg, tocilizumab and/or dexamethasone) is administered to alleviate, treat, or prevent toxicity both before and after administration of the cell therapy. In some embodiments, the patient is not given any prophylactic treatment until the genetically engineered cells (eg, CAR-T cells) are obtained.

[0230] In some embodiments, embodiments of the invention provide an advantage, such as allowing cell therapy to be performed on an outpatient basis. In some embodiments, administration of a cell therapy, such as a dose of T cells according to embodiments of the invention, may be on an outpatient basis or does not require admission of the patient to a hospital, such as admission to an overnight hospital. In some embodiments, such outpatient administration may provide increased access and reduced costs while maintaining a high long-term response rate with low toxicity. In some aspects, outpatient treatment may be beneficial for patients who are already immunocompromised by previous treatments, such as after lymphodepletion, and are at greater risk of infection while in the hospital or in hospital. In some aspects, outpatient treatments also increase treatment options for patients who may not have access to hospital, inpatient care, or transplant centers, thereby expanding access to treatment. In some embodiments, patients treated on an outpatient basis with the compositions, articles, kits, methods, and uses of the invention remain on an outpatient basis for at least 3 days, or a certain percentage of patients, such as at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% of patients remain on an outpatient basis for at least 3 days. In some aspects, patients remain on an outpatient basis for at least 4 days, 5 days, 6 days, 7 days, 8 days or more. In some embodiments, patients treated with the compositions, articles, kits, methods, and uses of the invention show a reduction in length of hospital stay, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40%, compared to patients treated with other compositions, articles, kits, methods, and uses.

[0231] In some embodiments, the methods, cells, and compositions can provide a patient with a high sustained response rate across a range of patient and/or tumor mass characteristics. In some embodiments, the methods, cells, and compositions can provide a high sustained response rate for a high-risk patient with a poor prognosis, with a reduced risk of adverse effects or toxicity. In some embodiments, the methods and uses provide or achieve higher response rates and/or longer response times or efficacy and/or reduced risk of toxicity or other side effects that may be associated with cell therapy, such as neurotoxicity (NT) or release syndrome. cytokines (CRS). In some aspects, the observations showed a low percentage of severe NT (sNT) or severe CRS (sCRS), and a high proportion of patients without any toxicity, such as NT or CRS.

[0232] 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 patients treated according to the methods of the invention and/or using the articles or compositions of the invention achieve a complete response (CR). In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of patients receiving treatment according to the methods of the invention and/or using articles or compositions according to invention achieve an objective response (OR). In some embodiments, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more of patients receiving treatment according to the methods of the invention and/or using articles or compositions of the invention achieve CR or OR within one month, two months or three months.

[0233] In some embodiments, within three months, four months, five months, six months or more after the initiation of the introduction of cell therapy, at least 60%, at least 70%, at least 80%, at least at least 90%, at least 95% or more of patients treated according to the methods of the invention and/or using articles or compositions of the invention maintain a response such as CR or OR. In some embodiments, such a response, such as a CR or OR, lasts for at least three months, four months, five months, six months, seven months, eight months, or nine months, such as at least or approximately at least at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more of patients treated according to the methods of the invention, or in such patients who achieve a CR in one month, or for three months. In some embodiments, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more of patients receiving treatment according to the methods of the invention and/or using articles or compositions of the invention, or such patients who achieve a CR in one month or three months, remain alive or remain alive without progression for more than or more than about three months, four months, five months, six months, seven months , eight months or nine months.

[0234] In some embodiments, the resulting response observed in such patients when treated in accordance with the methods of the invention and/or using the articles or compositions of the invention is associated with or results in a low risk of any toxicity or a low risk of severe toxicity. in most treated patients. In some embodiments, more than or more than about 30%, 35%, 40%, 50%, 55%, 60% or more of patients treated according to the methods of the invention and/or using the articles or compositions of the invention do not show any degree of CRS or any degree of neurotoxicity (NT). In some embodiments, more than or more than about 50%, 60%, 70%, 80% or more of patients treated according to the methods of the invention and/or using the articles or compositions of the invention do not show severe or grade CRS. 3 or higher. In some embodiments, greater than or greater than about 50%, 60%, 70%, 80% or more of patients treated according to the methods of the invention and/or using articles or compositions of the invention do not exhibit severe or grade neurotoxicity. 3 or higher, such as neurotoxicity grade 4 or 5.

[0235] In some embodiments, at least or about 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of patients receiving treatment according to the method and/ or using articles or compositions of the invention, do not show early onset CRS or neurotoxicity and/or show no onset of CRS earlier than 1 day, 2 days, 3 days, or 4 days after initiation of administration. In some embodiments, at least or about 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of patients treated according to the methods and/or using articles or compositions of the invention do not show the onset of neurotoxicity earlier than 3 days, 4 days, 5 days, six days, or 7 days after initiation of administration. In some aspects, the median onset of neurotoxicity among patients treated according to the methods and/or using the articles or compositions of the invention occurs at or after the median peak or median time to CRS clearance in patients treated according to the method. In some cases, the average onset of neurotoxicity among patients treated according to the method occurs after more than or about 8, 9, 10, or 11 days.

[0236] In some embodiments, such results are observed after administration of from or about 5 x 10 ⁷ to 1.5 x 10 ⁸ , e.g., 5 x 10 ⁷ to 1 x 10 ⁸ total recombinant receptor-expressing T cells, such as dose of T cells, including CD4 ⁺ and CD8 ⁺ T cells, administered in a certain ratio as described here, for example, in or about a ratio of 1: 1, and / or in a precise or flat or constant number of CAR ⁺ T- cells, or a precise or flat, or constant number of a particular type of CAR ⁺ T cell, such as CD4 ⁺ CAR ⁺ T cells and/or CD8 ⁺ CAR ⁺ T cells, and / or the number of any of such cells, which is in a specified degree of variability, for example, no more than + or - (plus or minus, in some cases indicated as ±) 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15% compared to such exact or flat or constant number. In some embodiments, such a flat or constant number of cells is or approximately 2.5 x 10 ⁷ , 5 x 10 7 , 10 x 10 ⁷ , 15 x 10 ⁷ , or 20 x 10 ⁷ , such as total CAR ⁺ T cells or CD8 ⁺ and/or CD4 ⁺ CAR ⁺ T cells. In some embodiments, the number of cells in a dose comprises or consists of or consists essentially of 5 x 10 ⁷ CD4 ⁺ CAR ⁺ T cells (optionally 2.5 x 10 ⁷ CD4 ⁺ CAR ⁺ T cells and 2.5 x 10 ⁷ CD8 ⁺ CAR ⁺ T cells); in some embodiments, this number includes or consists of or consists essentially of 10 x 10 ⁷ CAR ⁺ T cells (optionally 5 x 10 ⁷ CD4 ⁺ CAR ⁺ T cells and 5 x 10 ⁷ CD8 ⁺ CAR ⁺ T cells) . In some aspects, the number of injected cells is within a certain degree of variability of such numbers in the above embodiments, for example, plus or minus (±) 5, 6, 7, 8, 9 or 10%, for example, plus or minus 8%, according to compared to that number(s) of cells. In some aspects, the dose is in a range in which there is a correlation (not necessarily a linear relationship) between the number of such cells (for example, total CAR ⁺ T cells or CD8 ⁺ and/or CD4 ⁺ CAR ⁺ T cells) and one or more results, indicative of the therapeutic response or its duration (for example, the likelihood of achieving remission, complete remission and/or a certain duration of remission) and/or the duration of any of the previous ones. In some aspects, it has been found that a higher dose of administered cells can lead to a stronger response with or without significantly affecting or affecting the level or risk of toxicity (e.g., CRS or neurotoxicity) or the degree of level or risk of toxicity in a patient, e.g., severe CRS. or severe neurotoxicity.

[0237] In some aspects, the methods of the invention may achieve a high or defined response rate (such as a response rate in a population as measured over a period of time after treatment, such as three months or six months), such as an RRR (such as 6 months or 3-month RRR) 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or 80% or 81%, 82 %, 83%, 84% or 85% or more and CR level (such as 6-month or 3-month CR level) 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 71%, 72%, 73% or more, or about 75% or more, which is also long-term, such as for a certain a period of time or at least a certain period of time, for example, lasts for more than 1, 3 or 6 months or more or 9 months or more after the initiation of therapy. In some embodiments, such frequency and duration of response is obtained after only a single administration or dose of such therapy. Treatment of such patients with the methods of the invention and/or using the articles or compositions of the invention, in some embodiments, also results in patients achieving a high response rate without showing a higher rate of development of toxicity such as neurotoxicity or CRS, even at a higher cell dosage. In some embodiments, about or more than 50%, 55%, or 60% of patients who achieve such responses do not develop a degree of toxicity, such as any degree of CRS and/or neurotoxicity.

[0238] Thus, in some embodiments, the methods, articles, and/or compositions of the invention may provide advantages over other available methods or solutions, or treatment approaches, such as adoptive cell therapy. In particular, among the embodiments of the invention can be mentioned those that provide an advantage for patients at risk of NHL, by achieving a long-term response with a high frequency, with a reduced level of toxicity or side effects.

A. Method of treatment

[0239] The invention relates to methods of treatment, including the introduction of genetically engineered cells, or compositions containing genetically engineered cells, such as genetically engineered T cells. The invention also relates to methods and uses of engineered cells (e.g., T cells) and/or compositions thereof, including methods for treating patients having a disease or condition such as leukemia or lymphoma, e.g., non-Hodgkin's lymphoma (NHL), comprising administering genetically engineered cells and/or their compositions. In some embodiments, the methods and uses of the invention may achieve improved response and/or longer responses or efficacy, and/or reduced risk of toxicity or other side effects, e.g., in certain groups of treated patients, compared to some alternative methods. In some aspects, the invention also relates to methods of introducing genetically engineered cells or compositions containing genetically engineered cells, such as genetically engineered T cells, to a patient, such as a patient having a disease or disorder. In some aspects, the invention also relates to the use of genetically engineered cells or compositions containing genetically engineered cells, such as engineered T cells for the treatment of a disease or disorder. In some aspects, the invention also relates to uses of engineered cells or compositions containing engineered cells, such as engineered T cells, for the production of a medicament for the treatment of a disease or disorder. In some aspects, the invention also relates to methods of administering engineered cells or compositions containing engineered cells, such as engineered T cells, for use in the treatment of a disease or disorder, or for administration to a patient having a disease or disorder. In some aspects, the use of engineered cells or compositions containing engineered cells, such as engineered T cells, is in accordance with any of the methods described here.

[0240] Conventional methods for introducing cells for adoptive cell therapy are known and can be used in connection with the methods and compositions of the invention. For example, methods of adoptive T-cell therapy are described, for example, in Publication No. 2003/0170238 US patent applications to Gruenberg et al; US Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.

[0241] The disease or condition being treated can be any in which antigen expression is associated with and/or involved in the etiology of the disease state or disorder, e.g., causes, exacerbates or is otherwise involved in such disease, condition or disorder. Examples of diseases and conditions may include diseases or conditions associated with cancer or cell transformation (eg, cancer), an autoimmune disease or an inflammatory disease, or an infectious disease, eg, caused by a bacterial, viral, or other pathogen. Examples of antigens, including antigens associated with various diseases and conditions that can be treated, are described above. In some embodiments, the chimeric antigen receptor or transgenic TCR specifically binds to an antigen associated with a disease or condition.

[0242] Diseases, conditions, and disorders include tumors, including solid tumors, hematologic malignancies, and melanomas, and including localized and metastatic tumors, infectious diseases such as infection by a virus or other pathogen, e.g., HIV, HCV, HBV, CMV , HPV, and parasitic disease, and autoimmune diseases and inflammatory diseases. In some embodiments, the disease, disorder, or condition is a tumor, cancer, cancer, neoplasm, or other proliferative disease or disorder. Such diseases include, but are not limited to, leukemia, lymphoma, such as acute myeloid (or myelogenous) leukemia (AML), chronic myeloid (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), small lymphocyte lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt's lymphoma, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), anaplastic large cell lymphoma (ALCL), follicular lymphoma, refractory follicular lymphoma, diffuse large B cell lymphoma (DLBCL) and multiple myeloma (MM). In some embodiments, the disease or condition is a B-cell cancer selected from acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin's lymphoma (NHL), and diffuse large B-cell lymphoma (DLBCL). ). In some embodiments, the disease or condition is NHL and the NHL is selected from the group consisting of aggressive NHL, diffuse large B cell lymphoma (DLBCL), NOS (de novo and transformed from indolent), primary mediastinal large B cell lymphoma (PMBCL), T-cell/histocyte-rich large B-cell lymphoma (TCHRBCL), Burkitt's lymphoma, mantle cell lymphoma (MCL) and/or follicular lymphoma (FL), optionally stage 3B follicular lymphoma (FL3B).

[0243] In some embodiments, NHL may be subdivided into stages based on the 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 by Roman numerals I to IV (1-4), and limited stage (I or II) lymphomas affecting an organ outside the lymphatic system (extranodal organ) are indicated by the letter E. Stage I means involvement in one node or a group of adjacent nodes , or a single extranodal lesion without central involvement (IE). Stage 2 means participation in two or more central groups on the same side of the diaphragm or stage I or II central with limited adjacent extranodal involvement (IIE). Stage III means involvement in nodes on both sides of the diaphragm or nodes above the diaphragm involving the spleen. Stage IV means participation in additional extralymphatic involvement consisting of several non-contiguous sites. Also, "massive lymphadenopathy" can be used to describe large tumors in the breast, particularly stage II. The extent of the disease is determined by positron emission tomography (PET) - computed tomography (CT) for specific lymphomas and CT for non-specific histologies.

[0244] In some embodiments, the Eastern Cooperative Oncology Group Performance Status (ECOG) indicator can be used to evaluate or select patients for treatment, for example, patients who have had an unsatisfactory result of previous therapies (see, for example, Oken et al. (1982 ) Am J Clin Oncol 5:649-655). The ECOG Performance Status scale describes the level of functioning of patients in terms of their ability to care for themselves, daily activities and physical abilities (eg walking, working, etc.). In some embodiments, performance status ECOG 0 indicates that the patient is able to carry out normal activities. In some aspects, patients with performance status ECOG 1 show some limitation in physical activity, but the patient is fully outpatient. In some aspects, patients with an ECOG performance status of 2 are more than 50% outpatients. In some cases, a patient with an ECOG performance status of 2 may also be capable of self-care; see, for example, Sørensen et al., (1993) Br J Cancer 67(4) 773-775. Criteria reflecting the performance status of the ECOG are described in Table 1 below:

Table 1. ECOG Performance Status Criteria

Degree ECOG performance status 0 Fully active, able to continue all performance until illness without limitation 1 Limited in physically strenuous activity, but ambulatory and able to perform work of a light or sedentary nature, such as lighthouse work, office work 2 Ambulatory and capable of all self-care, but unable to perform any work activity; up to and approximately more than 50% of waking hours 3 Capable of only limited self-care; bedridden or chair-ridden for more than 50% of waking hours 4 Completely independent; unable to take care of themselves; completely chained to a bed or chair 5 Dead

[0245] In some embodiments, the patient has or has been identified as having double/triple hit lymphoma or double/triple-hit molecular subtype lymphoma. In some embodiments, the lymphoma is a double-hit lymphoma characterized by the presence of gene rearrangements (eg, translocations) of MYC (myelocytomatosis oncogene), BCL2 (B cell lymphoma 2), and/or BCL6 (B cell lymphoma 6). In some embodiments, a gene rearrangement affects the MYC/8q24 locus in combination with another gene rearrangement. For example, another gene permutation includes t(14; 18)(q32; q21) including BCL2. In some embodiments, the gene rearrangements affect the MYC/8q24 locus in combination with BCL6/3q27. In some embodiments, the lymphoma is 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 patient is ECOG 0-1 or does not have or is not suspected or characterized as having DLBCL transformed from MZL or CLL. In some aspects, therapy is indicated for such patients and/or the instructions indicate administration to a patient from such a population. In some embodiments, based on the 2016 WHO criteria (Swerdlow et al., (2016) Blood 127(20):2375-2390), double/triple hit lymphoma can be considered high-grade B-cell lymphoma with MYC and BCL2 permutations and/or BCL6 with DLBCL histology (double/triple hit).

[0246] In some embodiments, the disease or condition is an infectious disease or condition, such as, but not limited to, viral, retroviral, bacterial, and protozoal infections, immunodeficiency, Cytomegalovirus (CMV), Epstein-Barr Virus (EBV), adenovirus, polyomavirus BK. In some embodiments, the disease or condition is an autoimmune disease or inflammatory disease or condition, such as arthritis, e.g., rheumatoid arthritis (RA), type 1 diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, diffuse toxic goiter, Crohn's disease, multiple sclerosis, asthma, and/or transplant related disease or condition.

[0247] In some embodiments, the antigen associated with the disease or disorder is selected from αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), testicular cancer antigen, testicular cancer antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, Chemokine Motif Ligand 1 CC (CCL-1 ), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), mutations in epidermal growth factor receptor type III (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin A2 receptor (EPHa2), estrogen receptor, Fc-like receptor 5 (FCRL5) ; also known as the Fc receptor 5 or FCRH5 homologue), embryonic acetylcholine receptor (embryonic AchR), folate binding protein (FBP), folate alpha receptor, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G protein-coupled 5D receptor (GPCR5D), Her2/neu (erb-B2 receptor tyrosine kinase), Her3 (erb-B3), HeR ⁴ (erb-B4), dimers erbB, human high molecular weight melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte A1 antigen (HLA-A1), human leukocyte A2 antigen (HLA-A2), IL-22 receptor alpha (IL-22Rα), receptor alpha IL-13 2 (IL-13Rα2), receptor containing kinase insertion domain (kdr), kappa light chain, cell adhesion molecule L1 (L1-CAM), CE7 epitope L1-CAM containing leucine-rich repeats of family A member 8 ( LRRC8A), Ley antigen, melanoma associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, mouse cytomegalovirus (CMV), mucin 1 (MUC1), MUC16 , natural killer group 2 member D ligand (NKG2D), melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, preferentially expressed melanoma antigen (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), orphan receptor tyrosine kinase-like 1 (ROR1), survivin, trophoblastic glycoprotein (TPBG, also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase, related protein 1 (TRP1, also known as TYRP1 or gp75), tyrosinase-dependent protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor 1 (WT-1), a pathogen-specific or pathogen-expressed antigen or an antigen associated with a universal label, and/or biotinylated molecules and/or molecules expressed by HIV, HCV, HBV or other pathogens. Antigens targeted by receptors in some embodiments include antigens associated with B cell cancer, such as any of a variety of known B cell markers. In some embodiments, the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b, or CD30.

[0248] In some embodiments, the antigen is or includes a pathogen-specific or pathogen-expressed antigen. In some embodiments, the antigen is a viral antigen (such as a viral antigen from HIV, HCV, HBV, etc.), bacterial antigens, and/or parasitic antigens.

[0249] In some embodiments, cell therapy, such as adoptive T cell therapy, is performed by autologous transfer, in which cells are isolated and/or otherwise obtained from a patient to receive cell therapy, or from a sample obtained from such a patient. Thus, in some aspects, the cells are obtained from a patient, such as a patient in need of treatment, and the cells, after isolation and processing, are administered to the same patient.

[0250] In some embodiments, cell therapy, such as adoptive T-cell therapy, is performed by allogeneic transfer, in which cells are isolated and/or otherwise obtained from a patient other than the patient who is to receive or who ultimately receives cell therapy eg the first patient. In such embodiments, the cells are then administered to another patient, such as a second patient, of the same species. In some embodiments, the first and second patients are genetically identical. In some embodiments, the first and second patients are genetically similar. In some embodiments, the second patient expresses the same HLA class or supertype as the first patient.

[0251] Cells may be administered by any suitable means, e.g., bolus infusion, injection, e.g., intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjunctival injection, subconjunctival injection, subtenon injection, retrobulbar injection, peribulbar injection, or posterior parascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary and intranasal, and if topical treatment is required, intralesional administration. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal or subcutaneous administration. In some embodiments, the administration dose is administered as a single bolus of cells. In some embodiments, it is administered by multiple bolus injections of cells, for example, over no more than 3 days, or by continuous infusion of cells. In some embodiments, administration of a dose of cells or any additional therapies, for example, anti-lymphocyte therapy, interventional therapy, and/or combination therapy, is carried out by outpatient delivery.

[0252] For the prevention or treatment of a disease, a suitable dosage may depend on the type of disease being treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are administered prophylactically or therapeutically, previous therapy, the patient's medical history and response. on cells, and the discretion of the attending physician. Compositions and cells in some embodiments, the implementation is preferably administered to the patient once or as a series of treatments.

[0253] In some embodiments, the cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another or additional therapeutic intervention, such as an antibody or engineered cell or receptor, or an agent, such as a cytotoxic or therapeutic agent. Cells, in some embodiments, are administered in conjunction with one or more additional therapeutic agents or in connection with another therapeutic intervention, simultaneously or sequentially in any order. In some embodiments, the additional therapeutic agent is any of the interventions or agents described herein, such as any of the described interventions or agents that can alleviate the symptoms of toxicity described here, for example, in Section II. In some contexts, cells are co-administered with another therapy at sufficiently short intervals such that the cell population enhances the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, cells are administered up to one or more additional therapeutic agents. In some embodiments, cells are administered after one or more additional therapeutic agents. In some embodiments, the implementation of one or more additional agents include a cytokine, such as IL-2, for example, to improve persistence. In some embodiments, the methods include administering a chemotherapeutic agent.

[0254] In some embodiments, methods include administering a chemotherapeutic agent, eg, a conditioning chemotherapeutic agent, eg, to reduce tumor mass prior to administration.

[0255] Preconditioning patients with immunodepleting (eg, lymphodepleting) therapy may enhance the effects of adoptive cell therapy (ACT) in some aspects.

[0256] Thus, in some embodiments, the methods include administering a preconditioning agent, such as a lymphodepletion agent, or a chemotherapeutic agent, such as cyclophosphamide, fludarabine, or combinations thereof, to a patient prior to initiating cell therapy. For example, a preconditioning agent may be administered to a patient at least 2 days, such as at least 3, 4, 5, 6, or 7 days prior to the initiation of cell therapy. In some embodiments, the patient is administered a preconditioning agent no more than 7 days, such as no more than 6, 5, 4, 3, or 2 days prior to the initiation of cell therapy.

[0257] In some embodiments, the patient is preconditioned with cyclophosphamide at a dose of from or about 20 mg/kg to 100 mg/kg, for example, from or from about 40 mg/kg to 80 mg/kg. In some aspects, the patient is preconditioned with or about 60 mg/kg of 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 three days. In some embodiments, cyclophosphamide is administered once a day for one or two days. In some embodiments, where the lymphodepletion agent comprises cyclophosphamide, the patient is administered cyclophosphamide at a dose of from or about 100 mg/m ² to 500 mg/m ² , for example, from or from about 200 mg/m ² to 400 mg/m ² , or from 250 mg/m ² to 350 mg/m ² inclusive. In some cases, approximately 300 mg/m ² cyclophosphamide is administered to the patient. 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 three days. In some embodiments, the implementation of cyclophosphamide is administered daily, for example, for 1-5 days, for example, for 3-5 days. In some cases, the patient is administered approximately 300 mg/m ² cyclophosphamide daily for 3 days prior to the initiation of cell therapy.

[0258] In some embodiments, where the lymphodepletion agent comprises fludarabine, the patient is administered fludarabine at a dose of from or about 1 mg/m ² to 100 mg/m ² , such as from or from about 10 mg/m ² to 75 mg / m ² , from 15 mg/m ² to 50 mg/m ² , from 20 mg/m ² to 40 mg/m ² or from 24 mg/m ² to 35 mg/m ² , inclusive. In some cases, the patient is administered approximately 30 mg/m ² fludarabine. 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 three days. In some embodiments, fludarabine is administered daily, for example, for 1-5 days, for example, for 3-5 days. In some cases, the patient is administered approximately 30 mg/m ² fludarabine daily for 3 days prior to the initiation of cell therapy.

[0259] In some embodiments, the lymphodepletion agent includes a combination of agents, such as a combination of cyclophosphamide and fludarabine. Thus, the combination of agents may include cyclophosphamide at any dose or schedule as described above and fludarabine at any dose or schedule as described above. For example, in some aspects, the patient is administered 60 mg/kg (~2 g/m ² ) cyclophosphamide and 3 to 5 doses of 25 mg/m ² fludarabine prior to the first or subsequent dose.

[0260] After the introduction of the cells, in some embodiments, the biological activity of the population of genetically engineered cells is measured, for example, by any of the many known methods. Parameters for evaluation include specific binding of engineered or natural T cells or other immune cells to an antigen in vivo, eg by imaging, or ex vivo, eg by flow cytometry or ELISA. In some embodiments, the ability of engineered cells to destroy target cells can be measured using any suitable known methods, such as cytotoxicity tests described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In some embodiments, the biological activity of cells is measured by analyzing the expression and/or secretion of one or more cytokines such as CD107a, IFNγ, IL-2, and TNF. In some aspects, biological activity is measured by assessing the outcome of the disease, such as reduction in tumor mass or burden.

[0261] In some embodiments, the genetically engineered cells are further modified in any number of ways such that their therapeutic or prophylactic efficacy is increased. For example, engineered CARs or TCRs expressed by a population can be conjugated, directly or indirectly via a linker, to a targeting group. The practice of conjugating compounds, eg CAR or TCR, with targeting groups is known. See, for example, Wadwa et al., J. Drug Targeting 3: 1 1 1 (1995), and US Patent 5,087,616. In some embodiments, cells are administered as part of a combination treatment, for example, simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor, or an agent, such as a cytotoxic or therapeutic agent. Cells, in some embodiments, are administered in conjunction with one or more additional therapeutic agents or in connection with another therapeutic intervention, simultaneously or sequentially in any order. In some contexts, cells are co-administered with another therapy at sufficiently short intervals such that the cell population enhances the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, cells are administered up to one or more additional therapeutic agents. In some embodiments, cells are administered after one or more additional therapeutic agents. In some embodiments, one or more additional agents include a cytokine such as IL-2, for example, to improve persistence.

B. Dosing

[0262] In some embodiments, a dose of cells is administered to patients in accordance with the methods of the invention and/or using articles or compositions of the invention. In some embodiments, the choice of size or timing of doses is determined depending on the specific disease or condition of the patient. In some cases, the choice of the size or timing of the introduction of doses for a particular disease in view of the description presented can be determined empirically.

[0263] In some embodiments, the cell dosage includes from or from about 2 x 10 ⁵ cells/kg to or up to about 2 x 10 ⁶ cells/kg, for example, from or from about 4 x 10 ⁵ cells/kg to or up to about 1 x 10 ⁶ cells/kg, or from or from about 6 x 10 ⁵ cells/kg, and up to or up to about 8 x 10 ⁵ cells/kg. In some embodiments, the cell dose includes no more than 2 x 10 ⁵ cells (e.g., antigen-expressing, such as CAR-expressing cells) per kilogram of patient body weight (cells/kg), e.g., no more than or about 3 x 10 ⁵ cells/kg, no more than or approximately 4 x 10 ⁵ cells/kg, no more than or approximately 5 x 10 ⁵ cells/kg, no more than or approximately 6 x 10 ⁵ cells/kg, no more than or approximately 7 x 10 ⁵ cells/kg, not more than or approximately 8 x 10 ⁵ cells/kg, not more than or approximately 9 x 10 ⁵ cells/kg, not more than or approximately 1 x 10 ⁶ cells/kg, or not more than or approximately 2 x 10 ⁶ cells/kg. In some embodiments, the cell dosage comprises at least or at least about or up to or up to about 2 x 10 ⁵ cells (e.g., antigen-expressing, such as CAR-expressing cells) per kilogram of patient body weight (cells/kg), for example, at least or at least about or up to or up to about 3 x 10 ⁵ cells/kg, at least or at least about or up to or up to about 4 x 10 ⁵ cells/kg, at least or at least at least about or up to or up to about 5 x 10 ⁵ cells/kg, at least or at least about or up to or up to about 6 x 10 ⁵ cells/kg, at least or at least about or up to or up to about 7 x 10 ⁵ cells/kg, at least or at least about or up to or up to about 8 x 10 ⁵ cells/kg, at least or at least about or up to or up to about 9 x 10 ⁵ cells/kg, at at least or at least about or up to or up to about 1 x 10 ⁶ cells/kg, or at least or at least about or up to or up to about 2 x 10 ⁶ cells/kg.

[0264] In some embodiments, cells or a distinct population of cellular subtypes are administered to a patient in the range of from about one million to about 100 billion cells and/or as many cells per kilogram of body weight as, for example, from 1 million to about 50 billion cells ( e.g., approximately 5 million cells, approximately 25 million cells, approximately 500 million cells, approximately 1 billion cells, approximately 5 billion cells, approximately 20 billion cells, approximately 30 billion cells, approximately 40 billion cells, or a range of any two of the preceding values ), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells , about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or the range of any two of the preceding values), and in some cases from about 100 million cells to about 50 billion cells (e.g., approximately 120 million cells, approximately 250 million cells, approximately 350 million cells, approximately 450 million cells, approximately 650 million cells, approximately 800 million cells, approximately 900 million cells, approximately 3 billion cells, approximately 30 billion cells, approximately 45 billion cells) or any value in between these ranges and/or per kilogram of body weight. Dosages may vary depending on the characteristics of the disease or disorder and/or the patient and/or other treatments.

[0265] In some embodiments, the cell dose is a flat cell dose or a fixed cell dose such that the cell dose is not tied to or based on the patient's surface area or body weight.

[0266] In some embodiments, the dose of genetically engineered cells comprises from or about 1 x 10 ⁵ to 5 x 10 ⁸ total CAR-expressing T cells, 1 x 10 ⁵ to 2.5 x 10 ⁸ total CAR-expressing T cells, 1 x 10 ⁵ to 1 x 10 ⁸ total CAR-expressing T cells, 1 x 10 ⁵ to 5 x 10 ⁷ total CAR-expressing T cells, 1 x 10 ⁵ to 2.5 x 10 ⁷ total CAR-expressing T cells, 1 x 10 ⁵ to 1 x 10 ⁷ total CAR-expressing T cells, 1 x 10 ⁵ to 5 x 10 ⁶ total CAR-expressing T cells, 1 x 10 ⁵ to 2.5 x 10 ⁶ total CAR expressing T cells, 1 x 10 ⁵ to 1 x 10 ⁶ total CAR expressing T cells, 1 x 10 ⁶ to 5 x 10 ⁸ total CAR expressing T cells cells, 1 x 10 ⁶ to 2.5 x 10 ⁸ total CAR-expressing T cells, 1 x 10 ⁶ to 1 x 10 ⁸ total CAR-expressing T cells, 1 x 10 ⁶ to 5 x 10 ⁷ total CAR-expressing T cells, 1 x 10 ⁶ to 2.5 x 10 ⁷ total CAR-expressing T cells, 1 x 10 ⁶ to 1 x 10 ⁷ total CAR-expressing T cells, 1 x 10 ⁶ to 5 x 10 ⁶ total CAR expressing T cells, 1 x 10 ⁶ to 2.5 x 10 ⁶ total CAR expressing T cells, 2.5 x 10 ⁶ to 5 x 10 ⁸ total CAR expressing T cells, 2.5 x 10 ⁶ to 2.5 x 10 ⁸ total CAR-expressing T cells, 2.5 x 10 ⁶ to 1 x 10 ⁸ total CAR-expressing T cells, from 2.5 x 10 ⁶ to 5 x 10 ⁷ total CAR-expressing T cells, 2.5 x 10 ⁶ to 2.5 x 10 ⁷ total CAR-expressing T cells, 2.5 x 10 ⁶ to 1 x 10 ⁷ total CAR-expressing T cells, 2.5 x 10 ⁶ to 5 x 10 ⁶ total CAR-expressing T cells, 5 x 10 ⁶ to 5 x 10 ⁸ total CAR-expressing T cells, 5 x 10 ⁶ to 2.5 x 10 ⁸ total CAR expressing T cells, 5 x 10 ⁶ to 1 x 10 ⁸ total CAR expressing T cells, 5 x 10 ⁶ to 5 x 10 ⁷ total CAR expressing T cells cells, 5 x 10 ⁶ to 2.5 x 10 ⁷ total CAR-expressing T cells, 5 x 10 ⁶ to 1 x 10 ⁷ total CAR-expressing T cells, 1 x 10 ⁷ to 5 x 10 ⁸ total CAR-expressing T cells, 1 x 10 ⁷ to 2.5 x 10 ⁸ total CAR-expressing T cells, 1 x 10 ⁷ to 1 x 10 ⁸ total CAR-expressing T cells, 1 x 10 ⁷ to 5 x 10 ⁷ total CAR expressing T cells, 1 x 10 ⁷ to 2.5 x 10 ⁷ total CAR expressing T cells, 2.5 x 10 ⁷ to 5 x 10 ⁸ total CAR expressing T cells, 2.5 x 10 ⁷ to 2.5 x 10 ⁸ total CAR expressing T cells, 2.5 x 10 ⁷ to 1 x 10 ⁸ total CAR expressing T cells, from 2.5 x 10 ⁷ to 5 x 10 ⁷ total CAR expressing T cells, 5 x 10 ⁷ to 5 x 10 ⁸ total CAR expressing T cells, 5 x 10 ⁷ to 2.5 x 10 ⁸ total CAR expressing T cells, 5 x 10 ⁷ to 1 x 10 ⁸ total CAR expressing T cells, 1 x 10 ⁸ to 5 x 10 ⁸ total CAR expressing T cells, 1 x 10 ⁸ to 2.5 x 10 ⁸ total CAR expressing T cells or 2.5 x 10 ⁸ to 5 x 10 ⁸ total CAR expressing T cells.

[0267] In some embodiments, the dose of genetically engineered cells includes at least or at least about 1 x 10 ⁵ CAR-expressing cells, at least or at least about 2.5 x 10 ⁵ CAR-expressing cells, according to at least or at least about 5 x 10 ⁵ CAR expressing cells, at least or at least about 1 x 10 ⁶ CAR expressing cells, at least or at least about 2.5 x 10 ⁶ CAR expressing cells, at least or at least about 5 x 10 ⁶ CAR-expressing cells, at least or at least about 1 x 10 ⁷ CAR-expressing cells, at least or at least about 2.5 x 10 ⁷ CAR-expressing cells, at least or at least about 5 x 10 ⁷ CAR-expressing cells, at least or at least about 1 x 10 ⁸ CAR-expressing cells, at least or at least about 2.5 x 10 ⁸ CAR-expressing cells, or at least or at least about 5 x 10 ⁸ CAR-expressing cells.

[0268] In some embodiments, cell therapy comprises administering a dose comprising a number of cells from or from about 1 x 10 ⁵ to 5 x 10 ⁸ total recombinant receptor-expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMC), from or from about 5 x 10 ⁵ to 1 x 10 ⁷ total recombinant receptor-expressing cells, total T-cells, or total peripheral blood mononuclear cells (PBMCs) or from or from about 1 x 10 ⁶ to 1 x 10 ⁷ total recombinant receptor-expressing cells , total T cells, or total peripheral blood mononuclear cells (PBMC), inclusive. In some embodiments, cell therapy comprises administering a dose of cells comprising at least or at least about 1 x ¹⁰⁵ total recombinant receptor expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMCs), e.g., at least at least or at least about 1 x 10 ⁶ , at least or at least about 1 x 10 ⁷ , at least or at least about 1 x 10 ⁸ such cells. In some embodiments, this number refers to the total number of CD3 ⁺ or CD8 ⁺ , in some cases also expressing the recombinant receptor (eg, CAR ⁺ ) cells. In some embodiments, the cell therapy comprises administering a dose comprising a number of cells from or from about 1 x 10 ⁵ to 5 x 10 ⁸ CD3 ⁺ or CD8 ⁺ total T cells, or CD3 ⁺ or CD8 ⁺ recombinant receptor-expressing cells, from or from about 5 x 10 ⁵ to 1 x 10 ⁷ CD3 ⁺ or CD8 ⁺ total T cells or CD3 ⁺ or CD8 ⁺ recombinant receptor-expressing cells, or from or about 1 x 10 ⁶ to 1 x 10 ⁷ CD3 ⁺ or CD8 ⁺ total T -cells or CD3 ⁺ or CD8 ⁺ expressing recombinant receptor cells, inclusive. In some embodiments, cell therapy comprises administering a dose comprising a cell count from or from about 1 x 10 ⁵ to 5 x 10 ⁸ total CD3 ⁺ /CAR ⁺ or CD8 ⁺ /CAR ⁺ cells, from or from about 5 x 10 ⁵ to 1 x 10 ⁷ total CD3 ⁺ /CAR ⁺ or CD8 ⁺ /CAR ⁺ cells, or from or from approximately 1 x 10 ⁶ to 1 x 10 ⁷ total CD3 ⁺ /CAR ⁺ or CD8 ⁺ /CAR ⁺ cells, inclusive.

[0269] In some embodiments, the dose of T cells includes: 5 x 10 ⁷ or about 5 x 10 ⁷ expressing the recombinant receptor T cells or 2.5 x 10 ⁷ or up to about 2.5 x 10 ⁷ expressing the recombinant CD8 ⁺ receptor T cells. In some embodiments, the dose of T cells includes: 1 x 10 ⁸ or about 1 x 10 ⁸ recombinant receptor-expressing T cells, or 5 x 10 ⁷ or up to about 5 x 10 ⁷ recombinant receptor-expressing CD8 ⁺ T cells. In some embodiments, the dose of T cells includes: 1.5 x 10 ⁸ or about 1.5 x 10 ⁸ expressing the recombinant receptor T cells or 0.75 x 10 ⁸ or up to about 0.75 x 10 ⁸ expressing the recombinant CD8 receptor ⁺ T cells.

[0270] In some embodiments, the T cell dose includes CD4 ⁺ T cells, CD8 ⁺ T cells, or CD4 ⁺ and CD8 ⁺ T cells.

[0271] In some embodiments, for example, where the patient is human, the CD8 ⁺ T cell dose, including in a dose that includes CD4 ⁺ and CD8 ⁺ T cells, includes from about 1 x 10 ⁶ to 5 x 10 ⁸ total expressing recombinant receptor (e.g., CAR) CD8 ⁺ cells, e.g., in the range of about 5 x 10 ⁶ to 1 x 10 ⁸ such cells, 1 x 10 ⁷ , 2.5 x 10 ⁷ , 5 x 10 ⁷ , 7.5 x 10 ⁷ , 1 x 10 ⁸ , or 5 x 10 ⁸ such total cells, or a range between any two of the preceding values. In some embodiments, multiple doses are administered to the patient, and each of the doses or the total dose may be any of the preceding values. In some embodiments, the cell dosage comprises administering from or about 1 x 10 ⁷ to 0.75 x 10 ⁸ total recombinant CD8 ⁺ receptor expressing T cells, 1 x 10 ⁷ to 2.5 x 10 ⁷ total recombinant CD8 receptor expressing ⁺ T cells, from or from about 1 x 10 ⁷ to 0.75 x 10 ⁸ total recombinant CD8 ⁺ receptor expressing T cells, inclusive. In some embodiments, the cell dosage comprises administering 1 x 10 ⁷ , 2.5 x 10 ⁷ , 5 x 10 ⁷ 7.5 x 10 ⁷ , 1 x 10 ⁸ , or 5 x 10 ⁸ , or approximately that amount of total recombinant CD8 ⁺ receptor-expressing T cells.

[0272] In some embodiments, for example, where the patient is a human, the dose comprises less than about 2 x 10 ⁸ 1 x 10 ⁸ total recombinant receptor (e.g., CAR) expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs) , for example, in the range from about 1 x 10 ⁶ to 2 x 10 ⁸ or from 1 x 10 ⁶ to 1 x 10 ⁸ such cells, for example, 2 x 10 ⁶ , 5 x 10 ⁶ , 1 x 10 ⁷ , 5 x 10 ⁷ , 1 x 10 ⁸ , or 1.5 x 10 ⁸ such total cells, or a range between any two of the preceding values. In some embodiments where the patient is a human, the dose comprises from about 1 x 10 ⁶ to 3 x 10 ⁸ total recombinant receptor (e.g., CAR) expressing cells, e.g., in the range of about 1 x 10 ⁷ to 2 x 10 ⁸ such cells, for example, 1 x 10 ⁷ , 5 x 10 ⁷ , 1 x 10 ⁸ or 1.5 x 10 ⁸ such total cells, or a range between any two of the preceding values. In some embodiments, multiple doses are administered to the patient, and each of the doses or the total dose may be any of the preceding values. In some embodiments, the cell dosage comprises administering from or about 1 x 10 ⁵ to 5 x 10 ⁸ total recombinant receptor expressing T cells or total T cells, 1 x 10 ⁵ to 1.5 x 10 ⁸ total recombinant receptor expressing T cells or total T cells, 1 x 10 ⁵ to 1 x 10 ⁸ total recombinant receptor-expressing T cells or total T cells, from or about 5 x 10 ⁵ to 1 x 10 ⁷ total recombinant T receptor expressing α-cells or total T cells, or from or from about 1 x 10 ⁶ to 1 x 10 ⁷ total recombinant receptor-expressing T cells or total T cells, inclusive.

[0273] In some embodiments, the T cell dose includes CD4 ⁺ T cells, CD8 ⁺ T cells, or CD4 ⁺ and CD8 ⁺ T cells.

[0274] In some embodiments, for example, where the patient is human, the CD8 ⁺ T cell dose, including in a dose that includes CD4 ⁺ and CD8 ⁺ T cells, comprises from about 1 x 10 ⁶ to 1 x 10 ⁸ total expressing recombinant receptor (e.g., CAR) CD8 ⁺ cells, e.g., in the range of about 5 x 10 ⁶ to 1 x 10 ⁸ such cells, 1 x 10 ⁷ , 2.5 x 10 ⁷ , 5 x 10 ⁷ , 7.5 x 10 ⁷ or 1 x 10 ⁸ such total cells, or a range between any two of the preceding values. In some embodiments, multiple doses are administered to the patient, and each of the doses or the total dose may be any of the preceding values. In some embodiments, the cell dosage comprises administering from or about 1 x 10 ⁷ to 0.75 x 10 ⁸ total recombinant CD8 ⁺ receptor expressing T cells, 1 x 10 ⁷ to 2.5 x 10 ⁷ total recombinant CD8 receptor expressing ⁺ T cells, from or from about 1 x 10 ⁷ to 0.75x108x 10 ⁸ total expressing recombinant CD8 ⁺ receptor T cells, inclusive. In some embodiments, the cell dosage comprises administering 1x10 ⁷ , 2.5x10 ⁷ , 5x10 ⁷ 7.5x10 ⁷ , or 1x10 ⁸ or approximately that number of total recombinant CD8 ⁺ receptor-expressing T cells.

[0275] In some embodiments, a dose of cells, e.g., expressing a recombinant T cell receptor, is administered to a patient 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.

[0276] In the context of adoptive cell therapy, the administration of a given "dose" encompasses the administration of a given number or number of cells in the form of a single composition and/or a single continuous administration, for example, in the form of a single injection or continuous infusion, and also encompasses the administration of a given number or number of cells in the form of divided doses or in the form of multiple compositions supplied in multiple individual compositions or infusions, over a set period of time, such as no more than 3 days. Thus, in some contexts, a dose is a single or continuous administration of a specific number of cells given or initiated at a single point in time. In some contexts, however, the dose is administered in multiple injections or infusions over a period of no more than three days, such as once a day for three days or two days, or multiple infusions over a period of one day.

[0277] Thus, in some aspects, the dose cells are administered in a single pharmaceutical composition. In some embodiments, dose cells are administered in a plurality of compositions collectively containing dose cells.

[0278] In some embodiments, the term “split dose” refers to a dose divided so that it is administered over more than one day. This type of dosing is covered by the methods of the invention and is considered a single dose.

[0279] Thus, the dose of cells can be administered as a divided dose, for example, a divided dose administered over time. For example, in some embodiments, the dose may be administered to the patient over 2 days or over 3 days. Examples of split administration methods include 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% administered 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 divided dose is not spread over more than 3 days.

[0280] In some embodiments, dose cells can be administered by administering multiple compositions, or solutions, such as the first and second, optionally more, each containing some dose cells. In some aspects, multiple compositions, each containing different cell populations and/or subtypes, are administered separately or independently, optionally over a period of time. For example, cell populations or subtypes may include CD8 ⁺ and CD4 ⁺ T cells, respectively, and/or CD8 ⁺ - and CD4 ⁺ -rich populations, respectively, for example, CD4 ⁺ and/or CD8 ⁺ T cells, each individually includes cells genetically modified to express the recombinant receptor. In some embodiments, administering a 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 a different dose of CD4 ⁺ T cells and CD8 ⁺ T cells.

[0281] In some embodiments, administering a composition or dose, for example, administering multiple cell compositions, comprises separately administering the cell compositions. In some aspects, the separate introductions are carried out simultaneously or sequentially, in any order. In some embodiments, the dose comprises a first composition and a second composition, and the first composition and second composition are administered at 0 to 12 hour intervals, 0 to 6 hour intervals, or 0 to 2 hour intervals. In some embodiments, the initiation of the administration of the first composition and the initiation of the administration of the second composition are carried out with an interval of no more than 2 hours, no more than 1 hour, or no more than 30 minutes, no more than 15 minutes, no more than 10 minutes, or no more than 5 minutes. In some embodiments, the initiation and/or completion of the administration of the first composition and the completion and/or initiation of the administration of the second composition is carried out with an interval of no more than 2 hours, no more than 1 hour, or no more than 30 minutes, no more than 15 minutes, no more than 10 minutes or not more than 5 minutes.

[0282] In some compositions, the first composition, for example, the first dose composition, includes CD4 ⁺ T cells. In some compositions, the first composition, for example, the first dose composition, includes CD8 ⁺ T cells. In some embodiments, the first composition is administered prior to the second composition.

[0283] In some embodiments, the dose or cell composition comprises a specific or target ratio of CD4 ⁺ cells expressing the recombinant receptor to CD8 ⁺ cells expressing the recombinant receptor and/or CD4+ cells to CD8+ cells, the ratio optionally being about 1: 1 or from about 1:3 to about 3:1, for example, about 1:1. In some aspects, administering a composition or dose at a target or desired ratio of different cell populations (such as a CD4 ⁺ :CD8 ⁺ ratio or a CAR ⁺ CD4 ⁺ :CAR ⁺ CD8 ⁺ ratio, e.g., 1:1) comprises administering a cell composition containing one of populations, and then the introduction of a separate composition of cells, including another population, where the introduction is carried out in or approximately in the target or desired ratio. In some aspects, the introduction of a dose or composition of cells in a certain respect leads to improved growth, persistence and/or antitumor activity of T-cell therapy.

[0284] In some embodiments, the patient receives multiple doses, such as two or more doses, or multiple consecutive doses of cells. In some embodiments, two doses are administered to the patient. In some embodiments, the patient receives a subsequent dose, for example, a second dose, which is administered approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days after the first dose. In some embodiments, multiple subsequent doses are administered after the first dose, such that an additional dose or doses are administered after the subsequent dose is administered. In some aspects, the number of cells administered to the patient in an additional dose is the same as or similar to the first dose and/or the subsequent dose. In some embodiments, the additional dose or doses are greater than the previous doses.

[0285] In some aspects, the size of the first and / or subsequent dose is determined based on one or more criteria, such as the response of the patient to prior treatment, for example, chemotherapy, the severity of the disease in the patient, such as tumor burden, mass, size or degree, degree or type of metastasis, stage and/or likelihood or incidence of toxicity in the patient, eg, CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or host immune response to injected cells and/or recombinant receptors.

[0286] In some aspects, the time between the first dose and the next dose is about 9 to about 35 days, about 14 to about 28 days, or 15 to 27 days. In some embodiments, the next dose is administered at a time point greater than about 14 days after and less than about 28 days after the first dose. In some aspects, the time between the first and subsequent dose is approximately 21 days. In some embodiments, an additional dose or doses, such as subsequent doses, is administered after the subsequent dose is administered. In some aspects, the additional sequential dose or doses are administered at least about 14 and less than about 28 days after the previous dose. In some embodiments, an additional dose is administered less than about 14 days after the previous dose, such as 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.

[0287] In some embodiments, the dose of cells, e.g., recombinant receptor-expressing cells, comprises two doses (e.g., a double dose), including a first dose of T cells and a subsequent dose of T cells, wherein one or both of the first dose and the second dose involves administering a divided dose of T cells.

[0288] In some embodiments, the implementation of the dose of cells is usually large enough to be effective in reducing the severity of the disease.

[0289] In some embodiments, cells are administered at the desired dosage, in some aspects including the desired dose or number of cells or cell type(s) and/or the desired ratio of cell types. Thus, the dosage of cells in some embodiments is based on the total number of cells (or number per kg of body weight) and the desired ratio of individual populations or subtypes, such as the ratio of CD4 ⁺ to CD8 ⁺ . In some embodiments, cell dosage is based on the desired total number (or number per kg of body weight) of cells in particular populations or particular cell types. In some embodiments, the implementation of the dosage is based on a combination of such features, such as the desired number of total cells, the desired ratio and the desired total number of cells in individual populations.

[0290] In some embodiments, populations or subtypes of cells, such as CD8 ⁺ and CD4 ⁺ T cells, are administered at or within a tolerable difference from the desired dose of total cells, such as the desired dose of T cells. In some aspects, the desired dose is the desired number of cells, or the desired number of cells per unit body weight of the patient to whom the cells are administered, eg, cells/kg. In some aspects, the desired dose is at or above the minimum number of cells or the minimum number of cells per unit body weight. In some aspects, among total cells administered at the desired dose, distinct populations or subtypes are present at or near a desired output ratio (such as the ratio of CD4 ⁺ to CD8 ⁺ ), for example, in a certain tolerated difference or error in that ratio.

[0291] In some embodiments, the cells are administered at or in a tolerable variation of a desired dose of one or more distinct cell populations or subtypes, such as a desired dose of CD4 ⁺ cells and/or a desired dose of CD8 ⁺ cells. In some aspects, the desired dose is the desired number of cells of a subtype or population, or the desired number of such cells per unit body weight of the patient to whom these cells are administered, eg, cells/kg. In some aspects, the desired dose is at or above the minimum cell population or subtype, or the minimum cell population or subtype per unit body weight.

[0292] Thus, in some embodiments, the dosage is based on the desired fixed dose of total cells and the desired ratio, and/or based on the desired fixed dose of one or more, e.g. each, individual subtypes or subpopulations. Thus, in some embodiments, the dosage is based on the desired fixed or minimum dose of T cells and the desired ratio of CD4 ⁺ to CD8 ⁺ cells, and/or is based on the desired fixed or minimum dose of CD4 ⁺ and/or CD8 ⁺ cells.

[0293] In some embodiments, cells are administered within or within the tolerable range of the desired output ratio of multiple cell populations or subtypes, such as CD4 ⁺ and CD8 ⁺ cells or subtypes. In some aspects, the desired ratio may be a specific ratio, or may be a range of ratios. For example, in some embodiments, the desired ratio (e.g., ratio of CD4 ⁺ to CD8 ⁺ cells) is from or from about 5:1 to or up to about 5:1 (or greater than about 1:5 and less than about 5: 1), or from or from about 1:3 to or up to about 3:1 (or more than about 1:3 and less than about 3:1), for example, from or from about 2:1 to or up to about 1:5 (or greater than about 1:5 and less than about 2:1, e.g., a ratio of or approximately a ratio of 5:1, 4.5:1, 4:1, 3.5:1, 3:1 , 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3 :1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1 :1.6, 1:1.7, 1:1.8, 1:1.9: 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4 .5 or 1:5 In some aspects, the tolerable difference is about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30 %, about 35%, about 40%, about 45%, about 50% of the desired ratio, including anything in between these ranges.

[0294] In some embodiments, the numbers and/or concentrations of cells refer to the number of recombinant receptor (eg, CAR) expressing cells. In other embodiments, cell numbers and/or concentrations refer to the number or concentration of all cells, T cells, or peripheral blood mononuclear cells (PBMCs) administered.

[0295] In some aspects, the dose size is determined based on one or more criteria, such as the patient's response to prior treatment, such as chemotherapy, the severity of the disease in the patient, such as tumor burden, mass, size or grade, grade or type of metastasis, stage and/or the likelihood or frequency of a patient developing toxicity, eg, CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or host immune response to injected cells and/or recombinant receptors.

[0296] In some embodiments, the methods also include administering one or more additional doses of cells expressing a chimeric antigen receptor (CAR) and/or antilymphocyte therapy, and/or one or more steps of the methods are repeated. In some embodiments, one or more additional doses are the same as the initial dose. In some embodiments, one or more additional doses differ from the initial dose, for example, are higher, such as 2x, 3x, 4x, 5x, 6x, 7x, 8x , 9-fold or 10-fold or even higher than the initial dose, or lower, such as, for example, higher, such as 2-fold, 3-fold, 4-fold, 5-fold, 6-fold , 7-fold, 8-fold, 9-fold or 10-fold or even lower than the initial dose. In some embodiments, the administration of one or more additional doses is determined based on the patient's response to initial treatment or any prior treatment, the severity of the disease in the patient, such as tumor burden, mass, size or grade, grade or type of metastasis, stage and/or likelihood, or the frequency of the patient developing toxicity, eg, CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or host immune response to injected cells and/or recombinant receptors.

C. Response, efficiency and survival

[0297] In some embodiments, the implementation effectively treats the patient, despite the fact that the patient is resistant to other therapy. In some embodiments, at least 30%, at least 35%, at least 40%, or at least 50% of patients treated according to the method achieve complete remission (CR); and/or at least about 40%, at least about 50%, at least about 60%, or at least about 70% of patients treated according to the method achieve an objective response (OR). In some embodiments, at least or about at least 50% of patients, at least or about at least 60% of patients, at least or about at least 70% of patients, at least or about at least 80% patients or at least or about at least 90% of patients treated according to the method achieve CR and/or achieve an objective response (OR). In some embodiments, criteria evaluated for effective treatment include overall response rate (ORR; also known in some cases as objective response rate), complete response (CR; also known as complete response in some cases), duration of response (DOR), progression-free survival (PFS) and/or overall survival (OS).

[0298] In some embodiments, at least 40% or at least 50% of patients treated according to the methods achieve complete remission (CR; also known in some cases as a complete response), demonstrate progression-free survival (PFS), and/ or overall survival (OS) of more than or about 3 months, 6 months or 12 months or more than 13 months or about 14 months; on average, patients treated according to the method show an average PFS or OS of more than or about 6 months, 12 months or 18 months; and/or the patient shows PFS or OS after therapy for at least or about 6, 12, 18 or more months or longer.

[0299] In some aspects, response rates in patients, such as patients with NHL, are based on the Lugano criteria. (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, any of the clinical, hematological and/or molecular methods are used in evaluating the response. In some aspects, the response assessed using the Lugano criteria includes the use of positron emission tomography (PET)-computed tomography (CT) and/or CT, as appropriate. PET-CT evaluations may additionally include the use of fluorodeoxyglucose (FDG) for lymphomas with increased FDG accumulation. In some aspects, where PET-CT is used to assess response in histologies with increased FDG accumulation, a 5-point scale may be used. In some respects, the 5-point scale includes the following criteria: 1, no accumulation above background; 2, accumulation ≤ mediastinum; 3, accumulation > mediastinum, but ≤ liver; 4, accumulation is moderate > liver; 5, the accumulation is markedly higher than the liver and/or new lesions; X, new areas of accumulation, probably not associated with lymphoma.

[0300] In some aspects, a complete response, as described using the Lugano criteria, includes a complete metabolic response and a complete radiographic response at various measurable sites. In some aspects, these sites include lymph nodes and extralymphatic sites, in which CR is described as a score of 1, 2, or 3 with or without residual mass on a 5-point scale when PET-CT is used. In some aspects, in Waldeyer's ring or extranodal sites with high physiological accumulation or with activation in the spleen or bone marrow (for example, with chemotherapy or myeloid colony stimulating factors), the accumulation may be greater than in the normal mediastinum and/or liver. Under this circumstance, a complete metabolic response can be inferred if the accumulation in the sites of initial involvement is not greater than in the surrounding normal tissue, even if the tissue has a high physiological accumulation. In some aspects, response is assessed in lymph nodes by CT, with CR being described as if no extralymphatic disease sites and target nodes/central masses should regress to ≤ 1.5 cm in the longest transverse diameter of the lesion (LDi). Further evaluation sites include the bone marrow, in which PET-CT evaluation should show no evidence of disease with increased FDG accumulation in the bone marrow, and CT evaluation should show normal morphology, which, if found to be indeterminate, should be regarded as IHC- negative. Further areas may include an assessment of organ enlargement, which should regress to normal. In some aspects, unmeasured lesions and new lesions are evaluated, which in the case of CR should be absent (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).

[0301] In some aspects, partial response (PR; also known in some cases as partial remission), as described using the Lugano criteria, includes a partial metabolic and/or radiological response at various measurable sites. In some aspects, these sites include lymph nodes and extralymphatic sites, in which the PR is described as a score of 4 or 5 with reduced accumulation compared to the initial and residual mass(s) of any size when PET-CT is used. In the interim, such test results may indicate a responding disease. At the end of treatment, these test results may indicate residual disease. In some aspects, the response is assessed in the lymph nodes using CT, and PR is described as a decrease of ≥50% in SPD to 6 target measurable nodes and extranodal sites. If the lesion is too small to be measured by CT, 5 mm x 5 mm is assigned as the default value; if the lesion is no longer visible, the value is 0 mm x 0 mm; for a knot > 5 mm x 5 mm but less than normal, the actual measurements are used for the calculation. Other sites to assess include bone marrow, with PET-CT assessment showing residual accumulation higher than normal bone marrow but reduced from baseline (diffuse accumulation consistent with reactive changes from chemotherapy). In some aspects, if there are persistent focal changes in the bone marrow in the context of a nodal response, consideration should be given to further evaluation using MRI or biopsy or interval scanning. In some aspects, other sites may include assessment of organ enlargement, where the spleen must regress >50% in length beyond normal. In some aspects, unmeasured lesions and new lesions are evaluated, which in the case of PR should be absent/normal, regress but not increase. Non-response/stable disease (SD) or progressive disease (PD) can also be measured by assessment based on PET-CT and/or CT. (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) .

[0302] In some respects, progression-free survival (PFS) is described as the length of time during and after treatment for a disease, such as cancer, during which the patient lives with the disease, but it does not worsen. In some aspects, an objective response (OR) is described as a measurable response. In some aspects, the objective response rate (ORR; also known in some cases as the overall response rate) is described as the proportion of patients who achieve CR or PR. In some aspects, overall survival (OS) is described as the length of time either from the date of diagnosis or from the start of treatment for a disease, such as cancer, during which a patient diagnosed with the disease is still alive. In some aspects, event-free survival (EFS) is described as the length of time after the end of cancer treatment during which the patient does not experience certain complications or events that the treatment was intended to prevent or delay. These events may include a recurrence of the cancer or the onset of certain symptoms, such as bone pain due to cancer spreading to the bone, or death.

[0303] In some embodiments, the measure of duration of response (DOR) includes the time from documentation of tumor response to disease progression. In some embodiments, the parameter for evaluating response may include a sustained response, such as a response that persists after a period of time from the initiation of therapy. In some embodiments, a sustained response is referred to as a response duration of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months after initiation of therapy. In some embodiments, the response lasts for more than 3 months or more than 6 months.

[0304] In some aspects, the RECIST criteria are used to determine an objective carcinogenic effect; in some aspects, in solid tumors. (Eisenhauer et al., European Journal of Cancer 45 (2009) 228-247). In some aspects, the RECIST criteria are used to determine the objective carcinogenic effect for target organ damage. In some respects, a complete response, as defined using the RECIST criteria, is described as the resolution of all target organ lesions, and any pathological lymph nodes (either target or non-target) should have short axis reduction to < 10 mm. In other aspects, a partial response, as defined using the RECIST criterion, is described as at least a 30% reduction in the sum of target organ lesion diameters, taking as a reference value the sum of the initial diameters. In other aspects, progressive disease (PD) is described as at least a 20% increase in the sum of target organ lesion diameters, using as a reference value the smallest amount over the course of the study (this includes the initial amount if it is the smallest over the course of the study) . In addition to a relative increase of 20%, the sum must also show an absolute increase of at least 5 mm (in some aspects, the appearance of one or more new lesions is also considered a progression). In other aspects, stable disease (SD) is described as showing neither a sufficient decrease to acquire PR status, nor a sufficient increase to acquire PD status, taking as a reference value the smallest sums of diameters throughout the study.

[0305] In some aspects, administration in accordance with the methods of the invention and/or using the articles or compositions of the invention generally reduces or prevents the increase or severity of a disease or condition in a patient. For example, if the disease or condition is a tumor, the methods typically reduce the size, tumor mass, metastasis, percentage of blasts in the bone marrow or molecularly detectable cancer, and/or improve prognosis or survival or other symptom associated with tumor burden.

[0306] The severity of a disease may encompass the total number of disease cells in a patient or in a patient's organ, tissue, or bodily fluid, such as an organ or tissue of a tumor or other location, for example, that is indicative of metastasis. For example, tumor cells can be detected and/or quantified in blood or bone marrow in the context of certain hematological malignancies. The severity of the disease may include, in some embodiments, the mass of the tumor, the number or extent of metastases, and/or the percentage of blast cells present in the bone marrow.

[0307] In some embodiments, the patient has leukemia. The severity of the disease can be determined by assessing residual leukemia in the blood or bone marrow.

[0308] Some aspects of response rates in patients, such as patients with CLL, are based on the response criteria defined at the International Workshop on Chronic Lymphocytic Leukemia (IWCLL) (Hallek, et al., Blood 2008, Jun 15; 111(12): 5446-5456). In some aspects, these criteria are described as follows: complete remission (CR; also known in some cases as a complete response), which in some aspects requires the absence of clonal peripheral blood lymphocytes on immunophenotyping, the absence of lymphadenopathy, the absence of hepatomegaly or splenomegaly, the absence of symptoms indicative of generalization of the process and satisfactory blood tests; complete remission with incomplete bone marrow recovery (CRi), in some aspects described as CR above, but without normal blood tests; partial remission (PR; also known in some cases as a partial response), which in some aspects is described as ≥ 50% drop in lymphocyte count, ≥ 50% reduction in lymphadenopathy, or ≥ 50% reduction in liver or spleen, together with improvement in the amount of peripheral blood; progressive disease (PD), in some aspects described as ≥ 50% increase in lymphocyte count to > 5 x 10 ⁹ /l, ≥ 50% increase in lymphadenopathy, ≥ 50% increase in liver or spleen size, Richter's transformation, or new cytopenias due to CLL; and stable disease, in some aspects described as not meeting the criteria for CR, CRi, PR, or PD.

[0309] In some embodiments, patients show a CR or OR if, within 1 month after a dose of cells, the patient's lymph nodes are less than or about 20 mm in size, less than or about 10 mm in size, or less than or about 10 mm in size. .

[0310] In some embodiments, the CLL index clone is not detected in the patient's bone marrow (or in the bone marrow of more than 50%, 60%, 70%, 80%, 90%, or more of patients treated according to the methods). In some embodiments, the CLL index clone is evaluated by deep IgH sequencing. In some embodiments, an index clone is not detected at a time point that is either about or at least about or about 1, 2, 3, 4, 5, 6, 12, 18, or 24 months after cell injection.

[0311] In some embodiments, the patient shows a morphological disease if the number of blasts in the bone marrow is greater than or equal to 5%, for example, as in the case of detection by light microscopy, for example, the number of blasts in the bone marrow is greater than or equal to 10%, the number of blasts in the bone more than or equal to 20%, the number of blasts in the bone marrow is more than or equal to 30%, the number of blasts in the bone marrow is more than or equal to 40%, the number of blasts in the bone marrow is more than or equal to 50%. In some embodiments, the patient shows complete or clinical remission if there are less than 5% of blasts in the bone marrow.

[0312] In some embodiments, the patient has leukemia. The severity of the disease can be determined by assessing residual leukemia in the blood or bone marrow.

[0313] In some embodiments, the patient shows a morphological disease if the number of blasts in the bone marrow is greater than or equal to 5%, for example, as in the case of detection by light microscopy, for example, the number of blasts in the bone marrow is greater than or equal to 10%, the number of blasts in the bone more than or equal to 20%, the number of blasts in the bone marrow is more than or equal to 30%, the number of blasts in the bone marrow is more than or equal to 40%, the number of blasts in the bone marrow is more than or equal to 50%. In some embodiments, the patient shows complete or clinical remission if there are less than 5% of blasts in the bone marrow.

[0314] In some embodiments, the patient may show complete remission, but a small proportion of morphologically undetectable (by light microscopy) residual leukemic cells are present. A patient is said to show minimal residual disease (MRD) if the patient shows less than 5% of blasts in the bone marrow and shows molecularly detectable cancer. In some embodiments, a molecularly detectable cancer can be assessed using any of a variety of molecular techniques that provide sensitive detection of a small number of cells. In some aspects, such methods include PCR tests that can identify unique Ig/T-cell receptor gene permutations or fusion transcripts produced by chromosome translocations. In some embodiments, flow cytometry can be used to identify cancer cells based on leukemia-specific immunophenotypes. In some embodiments, cancer molecular diagnostics can detect only 1 leukemic cell per 100,000 normal cells. In some embodiments, the patient shows an MRD that is detectable at the molecular level if at least or more than 1 leukemia cell in 100,000 is detectable, for example, by flow cytometry or PCR. In some embodiments, the severity of the patient's disease at the molecular level is not detected or MRD ^- so that in some cases, no leukemic cells can be detected in the patient using PCR or flow cytometric methods.

[0315] In some embodiments, a leukemia index clone, e.g., CLL, is not detected in the bone marrow of a patient (or in the bone marrow of more than 50%, 60%, 70%, 80%, 90% or more of patients treated according to the methods ). In some embodiments, a leukemia index clone, eg, CLL, is assessed by deep IGH sequencing. In some embodiments, an index clone is not detected at a time point that is either about or at least about or about 1, 2, 3, 4, 5, 6, 12, 18, or 24 months after cell injection.

[0316] In some aspects, MRD is detected by flow cytometry. Flow cytometry can be used to monitor bone marrow and peripheral blood samples for cancer cells. In certain aspects, flow cytometry is used to detect or monitor the presence of cancer cells in the bone marrow. In some aspects, multivariable immunological detection by flow cytometry is used to detect cancer cells (see, for example, Coustan-Smith et al., (1998) Lancet 351:550-554). In some aspects, multiparametric immunological mass cytometry detection is used to detect cancer cells. In some examples, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 parameters may be used for detection of cancer cells. The antigens used for detection are selected depending on the cancer being diagnosed (Foon and Todd (1986) Blood 68:1-31).

[0317] In some examples, bone marrow is obtained from bone marrow aspirates or bone marrow biopsies, and lymphocytes are isolated for analysis. Fluorochrome-conjugated monoclonal and/or polyclonal antibodies (eg, fluorescein isothiocyanate (FITC), phycoerythrin, peridinin chlorophyll protein, or biotin) can be used to detect epitopes such as terminal deoxynucleotidyl transferase (TdT), CD3, CD10, CD11c, CD13 , CD14, CD33, CD19, CD20, CD21, CD22, CD23, CD34, CD45, CD56, CD79b, IgM and/or KORSA3544, on isolated lymphocytes. Labeled cells can be detected using flow cytometry, such as multiparameter flow cytometry, or mass cytometry, for the detection of multiple epitopes.

[0318] Lymphoid cells can be identified and grouped based on a scatter plot of light and then grouped secondarily based on the identification of a population of cells expressing immunophenotypic features of interest. Examples of epitopes are presented in Table 2 below. Another immunological classification of leukemias and lymphomas is given by Foon and Todd (Blood (1986) 68(1): 1-31). In some aspects, MRD flow cytometric evaluation can be performed by quantifying live lymphocytes having one or more CLL immunophenotypes (e.g., low forward/lateral scatter; CD3 ^neg ; CD5 ⁺ ; CD14 ^neg ; CD19 ⁺ ; CD23 ⁺ ; CD45 ⁺ ; CD56 ^neg ).

Table 2. Example of immunophenotype and cytogenetic characteristics Disease Immunophenotype Cytogenetics Chronic lymphocytic leukemia (CLL) Pan B+; CD5+; CD23+; CD79b/CD22 weak; FMC7-; sIg weak Trisomy 12
del(13)(q14,3)
del 11q22-q23
del 17p13 (p53)
t(11;14)(q13;q32) BCL1/IgH rearrangement
t(14;19)(q32;q13)
IgH deletion (14q32)
del(6q)
+8q24
+3
+18
del 6q21 Small cell lymphocytic lymphoma (SLL) Pan B+; CD5+; CD23+; CD10-; sIgM+ unclear del(6)(q21-23) Lymphoplasmic lymphoma Pan B+; CD5-; CD10-; cyIgM+ t(9;14)(p13;q32) PAX5/IgH Lymphoma from cells of the central follicle Pan B+; CD10+/-; CD5-; sIg+ t(14;18)(q32;q21) / BCL2 Rearr Diffuse large cell lymphoma CD19+; CD22+; CD10-/+; SIg+ t(14;18) and p53 mutations
t(3;V)(q27;V)/ BCL6 Rearr
c-MYC Rearr variants Burkitt's lymphoma Pan B+; TdT-; CD10+; CD5-; sIgM+ t(8;14)(q24;q32) or options/c-MYC R earr Burkitt-like lymphoma Pan B+; TdT-; CD10-/+CD5-; sIg+ t(8;14) or options
t(8;14)+ t(14;18) mantle cell lymphoma Pan-B+; CD5+; CD23-; CD10-/+; sIgM+ bright t(11;14)(q13;q32) / BCL1 Rearr B-cell marginal zone lymphoma (MZBCL) pan-B+; CD5-/+; CD10-; CD23-; CD11c+/-; cyIg+ (40% cells), sIgM+ bright; sIgD- t(11;18)(q21;q21) / PI2/MLT fused: low-grade extranodal MALT-type lymphoma; sluggish disease
t(1;14)(p21;q32): MALT-type extranodal lymphoma
del(7)(q22-31): Spleen MZBCL
/+3q: MZBCL nodal, extranodal and spleen +: positive in >90% of cases
+/-: positive in more than 50% of cases
-/+: positive in less than 50% of cases
-: positive in <10% of cases
Pan-B markers: e.g. CD19, CD20, CD79a
sIG: surface immunoglobulins
cyIg: cytoplasmic immunoglobulins

[0319] In some aspects, deep sequencing of the immunoglobulin heavy chain (IGH) locus of harvested B cells can be used to detect minimal residual disease (MRD). The clonal presence of a particular IgG permutation can provide a marker for detecting the presence of B-cell cancers such as CLL or NHL and/or the residual presence of cancer cells from these tumors. In some aspects, cells, such as a population containing or suspected of containing B cells, are obtained and isolated from the blood. In some aspects, cells are obtained and isolated from bone marrow, such as from bone marrow aspirates or bone marrow biopsies, and/or from other biological samples. In some aspects, polymerase chain reaction (PCR) amplification of complementarity determining region 3 (CDR3) is performed using primers for highly conserved sequences in the V and J regions of the gene locus, which can be used to identify clonal cell populations in order to evaluate minimal residual disease. Other methods for detecting clonal populations, such as single cell sequencing approaches, including those providing information on the number of cells of a particular lineage, and/or expression of a particular variable chain, such as a heavy chain variable region or its binding site, such as a clonal population, can be used. In some aspects, IGH DNA is amplified using degenerate primers or primers that recognize variable chain regions common to different cell clones, such as those that recognize the consensus V and degenerate consensus J regions of the IGH sequence. An example of a variable region sequence is ACACGGCCTCGTGTATTACTGT (SEQ ID NO: 57). An example of a degenerate J consensus sequence is ACCTGAGGAGACGGTGACC (SEQ ID NO: 58).

[0320] The PCR product or sequencing result is, in some aspects, specific for the rearranged allelic segment and serves as a clonal marker for MRD detection. After PCR amplification of the CDR3 region, PCR products can be sequenced to obtain patient-specific oligonucleotides designed as allele-specific PCR probes for sensitive MRD detection after treatment of B-cell malignancies with CAR-T cell therapy, e.g., CD19 CAR-T- cell therapy. In instances where the PCR product is not generated using consensus primers, V-region family-specific primers for frame region 1 can be used instead.

[0321] In some aspects, the persistence of PCR-detectable tumor cells, such as B-cell cancer cells such as NHL or CLL, such as detectable IGH sequences corresponding to malignant or clonal IGH sequences, after treatment is associated with an increased risk of relapse. In some aspects, patients who are negative for IGH malignant sequences after treatment (in some aspects, even in the context of other criteria indicating progressive disease or only a partial response, such as persistent lymph node enlargement or other criteria that may in some contexts be associated with disease or lack of a complete response) can be considered to have an increased likelihood of PFS or entered into a CR or prolonged CR or prolonged survival compared to patients with persistent IGH malignant sequences. In some embodiments, such prognostic and staging determinations are particularly important for treatments in which clearance of malignant cells is observed in a short period of time after the administration of therapy, for example, compared with the resolution of other clinical symptoms, such as lymph node size or other staging criteria. For example, in some such aspects, the absence of detectable IGH or minimal residual disease in a sample, such as bone marrow, may be a preferred reading of response, or probability of response, or duration, over other available staging or predictive approaches. In some aspects, MRD results, such as IGH deep sequencing information, may provide information on further intervention or lack thereof. For example, according to the methods and other embodiments of the invention, in some contexts, a patient who is considered negative for malignant IGH may, in some aspects, receive no additional treatment or dose of therapy, or a lower or reduced dose may be administered to the patient. On the other hand, a patient showing MRD through IGH deep sequencing may be found or determined to receive additional treatment, such as therapy initially administered at a similar or higher dose, or another treatment agent. In some aspects, the disease or condition persists after the first dose and/or the first dose is not sufficient to eliminate the disease or condition in the patient.

[0322] In some embodiments, the method reduces the severity of the disease or condition, for example, the number of tumor cells, tumor size, duration of patient survival or event-free survival, to a greater extent and/or for a longer period of time compared to the reduction that would be observed in a comparable manner with an alternative dosing regimen, such as one in which the patient takes one or more alternative therapeutic agents and/or one in which the patient does not receive a dose of cells and/or a lymphodepletion agent in accordance with the methods of the invention, and /or using articles or compositions according to the invention. In some embodiments, the severity of a disease or condition in a patient is detected, assessed, or measured. The severity of a disease can be detected in some aspects by detecting the total number of disease cells or cells associated with the disease, such as tumor cells, in a patient, or in a patient's organ, tissue, or body fluid, such as blood or serum. In some aspects, a patient's survival, time interval survival, degree of survival, presence or duration of event-free survival, asymptomatic survival, or relapse-free survival are assessed. In some embodiments, any symptom of a disease or condition is evaluated. In some embodiments, the extent of the disease or the severity of the condition is determined.

[0323] In some embodiments, the event-free survival or overall survival of the patient is improved in ways compared to other ways, for example, ways in which the patient takes one or more alternative therapeutic agents and/or in a way in which the patient does not receive a dose cells and/or a lymphodepletion agent according to the methods of the invention and/or using articles or compositions of the invention. For example, in some embodiments, the rate or probability of event-free survival for patients treated with the methods 6 months after administration is greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, more than about 80%, more than about 90%, or more than about 95%. In some aspects, overall survival 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 patient receiving treatment with the methods demonstrates event-free survival, relapse-free survival, or survival of 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 enhanced, for example, the time to progression is greater than or about 6 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.

[0324] In some embodiments, after treatment with the method, the likelihood of relapse is reduced compared to other methods, for example, methods in which the patient takes one or more alternative therapeutic agents, and / or a method in which the patient does not receive a dose of cells and / or a lymphodepletion agent according to the methods of the invention and/or using the articles or compositions of the invention. For example, in some embodiments, the likelihood of recurrence at 6 months after the first dose 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%.

[0325] In some cases, the pharmacokinetics of administered cells, eg, adoptively transferred cells, is determined to assess the availability, eg, bioavailability, of the administered cells. Methods for determining the pharmacokinetics of adoptively tolerated cells may include sampling peripheral blood from patients receiving engineered cells and determining the number or ratio of engineered cells in peripheral blood. Cell selection and/or isolation approaches may include the use of chimeric antigen receptor (CAR) specific antibodies (e.g., Brentjens et al., Sci. Transl. Med. 2013 Mar; 5(177): 177ra38) Protein L (Zheng et al ., J. Transl. Med. 2012 Feb; 10:29), epitope tags such as Strep-Tag sequences introduced directly into active sites in CAR, whereby Strep-Tag binding reagents are used to directly assess CAR (Liu et al., (2016) Nature Biotechnology, 34:430; International Patent Publication No. WO2015095895) and monoclonal antibodies that specifically bind to a CAR polypeptide (see International Patent Publication No. WO2014190273). External marker genes can in some cases be used in connection with genetically engineered cell therapies to allow detection or selection of cells and, in some cases, also promote cell suicide. Truncated epidermal growth factor receptor (EGFRt) in some cases can be co-expressed with a transgene of interest (CAR or TCR) in transduced cells (see, for example, US Patent No. 8,802,374). EGFRt may contain an epitope recognized by the cetuximab antibody (Erbitux®) or other therapeutic anti-EGFR antibody, or a binding molecule that can be used to identify or select cells engineered with an EGFRt construct and another recombinant receptor, such as a chimeric antigen receptor (CAR), and/or to eliminate or separate cells expressing the receptor. See US Patent No. 8,802,374 and Liu et al., Nature Biotech. April 2016; 34(4): 430-434.

[0326] In some embodiments, the number of CAR ⁺ T cells in a biological sample obtained from a patient, such as blood, can be determined in the period of time after the introduction of cell therapy, for example, to determine the pharmacokinetics of cells. In some embodiments, the number of CAR ⁺ T cells, optionally CAR ⁺ CD8 ⁺ T cells and/or CAR ⁺ CD4 ⁺ T cells detected in the blood of the patient or in the majority of patients treated with the method, is greater than 1 cell per μl, more than 5 cells per µl or more than 10 cells per µl.

D. Toxicity

[0327] In some embodiments, the methods of the invention are designed for or include features that result in a lower frequency and/or lower degree of toxicity, toxic outcome, or symptom causing a toxicity profile, factor, or property, such as a symptom or outcome associated with or indicative of cytokine release syndrome (CRS) or neurotoxicity, e.g. compared to administration of an alternative cell therapy, such as an alternative CAR ⁺ T cell formulation, and/or alternative cell administration, e.g., administration of cells not administered in a particular ratio.

[0328] In some embodiments, the methods of the invention do not result in a high frequency or likelihood of toxicity or toxic results, or reduce the frequency or likelihood of toxicity or toxic results, such as neurotoxicity (NT), cytokine release syndrome (CRS), compared to some other cell therapies. In some embodiments, the methods do not result in or increase the risk of severe NT (sNT), severe CRS (sCRS), macrophage activation syndrome, tumor lysis syndrome, fever of at least or about 38 degrees Celsius for three or more days and a plasma CRP level of at least up to or about 20 mg/dl. In some embodiments, more than or more than about 30%, 35%, 40%, 50%, 55%, 60% or more of patients treated according to the methods of the invention do not show any degree of CRS or any degree of neurotoxicity. In some embodiments, no more than 50% of treated patients (e.g., at least 60%, at least 70%, at least 80%, at least 90% or more of treated patients) exhibit cytokine release syndrome ( CRS) greater than Grade 2 and/or neurotoxicity greater than Grade 2. In some embodiments, at least 50% of patients receiving treatment according to the method (e.g., at least 60%, at least 70%, at least at least 80%, at least 90% or more of treated patients) do not show a severe toxic outcome (eg, severe CRS or severe neurotoxicity), such as grade 3 neurotoxicity or higher, and/or do not exhibit or exhibit severe CRS within a certain period of time after treatment, for example, within a week, two weeks or one month after the introduction of cells. In some embodiments, the parameters evaluated to determine specific toxicity include adverse events (AEs), dose-limiting toxicity (DLTs), CRS, and NT.

[0329] The introduction of adoptive T cell therapy, such as treatment with T cells expressing chimeric antigen receptors, can cause toxic effects or outcomes such as cytokine release syndrome and neurotoxicity. In some examples, such effects or results are consistent with high levels of circulating cytokines, which may underlie the observed toxicity.

[0330] In some aspects, the toxic outcome is or is associated with or is indicative of cytokine release syndrome (CRS) or severe CRS (sCRS). CRS, eg sCRS, can occur in some cases after adoptive T-cell therapy and administration of other biological products to patients. See Davila et al., Sci Transl Med 6, 224ra25 (2014); Brentjens et al., Sci. Transl. Med. 5, 177ra38 (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.

[0331] Typically, CRS is caused by an excessive systemic immune response mediated, for example, by T cells, B cells, NK cells, monocytes, and/or macrophages. Such cells can release large amounts of inflammatory mediators such as cytokines and chemokines. Cytokines can induce an acute inflammatory response and/or cause endothelial organ damage, which can lead to capillary permeability, heart failure, or death. Severe life-threatening CRS can lead to pulmonary infiltration and lung injury, kidney failure, or disseminated intravascular coagulation. Other severe life-threatening toxicity may include cardiac toxicity, respiratory distress syndrome, neurological toxicity, and/or liver failure.

[0332] CRS can be treated with anti-inflammatory therapy, such as anti-IL-6 therapy, for example, an anti-IL-6 antibody, for example, tocilizumab, or antibiotics or other substances, as described. The results, signs and symptoms of CRS are known and include those described here. In some embodiments, in which a particular dosage or administration regimen has or does not have an effect on a given CRS-associated outcome, sign, or symptom, the specific outcomes, signs, and symptoms and/or their amounts or degrees can be determined.

[0333] In the context of administration 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 infusion of CAR T cells. The level and timing of CRS onset may be related to initial 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 can be rapidly induced in CRS are IL-1β, IL-6, IL-8 and IL-10.

[0334] Examples of CRS-related outcomes include fever, shivering, chills, hypotension, dyspnea, acute respiratory distress syndrome (ARDS), encephalopathy, ALT/AST elevation, renal failure, cardiovascular disorders, hypoxia, neurological disorders and death. Neurological complications include delirium, seizure-like activity, confusion, difficulty finding words, aphasia, and/or lethargy. Other CRS-related outcomes include fatigue, nausea, headache, convulsions, tachycardia, myalgias, rash, acute vascular permeability syndrome, worsening liver function, and kidney failure. In some aspects, CRS is associated with an increase in one or more factors such as serum ferritin, d-dimer, aminotransferases, lactate dehydrogenase, and triglycerides, or with hypofibrinogenemia or hepatosplenomegaly. Other examples of signs or symptoms associated with CRS include hemodynamic instability, febrile neutropenia, increased serum C-reactive protein (CRP), changes in coagulation parameters (eg, international normalization ratio (INR), prothrombin index (PTI), and/or fibrinogen) , changes in heart and other organ function, and/or absolute neutrophil count (ANC).

[0335] In some embodiments, CRS-related findings include one or more of the following: persistent fever, e.g., fever with fever, e.g., greater than or about 38 degrees Celsius, for two or more, e.g., three or more, such as four or more days, or for at least three consecutive days; an increase in temperature to more than or about 38 degrees Celsius; an increase in cytokines, such as a maximum fold change, e.g., at least or about 75, compared to pre-treatment levels, of at least two cytokines (e.g., at least two from the group consisting of interferon (IFNγ), GM-CSF , IL-6, IL-10, Flt-3L, fractalkine, and IL-5 and/or tumor necrosis factor alpha (TNFα)), or a maximum fold change, e.g., at least or about 250, of at least one of these cytokines; and/or at least one clinical sign of toxicity such as hypotension (eg, as measured by at least one intravenous vasoactive vasopressor agent); hypoxia (eg, plasma oxygen levels (PO ₂ ) less than or about 90%); and/or one or more neurological disorders (including mental status changes, stupor, and seizures).

[0336] Examples of CRS-associated results include increased or high serum levels of one or more factors, including cytokines and chemokines and other factors associated with CRS. Examples of results further include increases in the synthesis or secretion of one or more of these factors. Such synthesis or secretion can be carried out by a T cell or a cell interacting with a T cell, such as an innate immune system cell or a B cell.

[0337] In some embodiments, CRS-associated serum factors or CRS-associated outcomes include inflammatory cytokines and/or chemokines, including interferon (IFN-γ), TNF-a, IL-1β, 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 (TNFα), IL-6 and IL-10, IL-1β, IL-8, IL-2, MIP-1, Flt-3L, fractalkine and/or IL-5. In some embodiments, the factor or result comprises a C-reactive protein (CRP). In addition to being an early and easily measurable risk factor for CRS, CRP is also a marker of cell growth. In some embodiments, patients measured for high levels of CRP, such as ≥ 15 mg/dl, have CRS. In some embodiments, patients measured for high levels of CRP do not have CRS. In some embodiments, the measurement of CRS includes the measurement of CRP and another factor that is indicative of CRS.

[0338] In some embodiments, one or more inflammatory cytokines or chemokines are monitored before, during, or after CAR treatment. In some aspects, one or more cytokines or chemokines include IFN-γ, TNF-α, IL-2, IL-1β, IL-6, IL-7, IL-8, IL-10, IL-12, sIL-2Rα, granulocyte-macrophage colony stimulating factor (GM-CSF) or macrophage inflammatory protein (MIP). In some embodiments, IFN-γ, TNF-α, and IL-6 are monitored.

[0339] Criteria for CRS have been developed that appear to correlate with the onset of CRS to predict which patients are more likely to be at risk of developing sCRS (see Davilla et al. Science translational medicine. 2014;6(224): 224ra25). Factors include fever, hypoxia, hypotension, neurological changes, increased serum levels of inflammatory cytokines such as a series of seven cytokines (IFNγ, IL-5, IL-6, IL-10, Flt-3L, fractalkine, and GM-CSF) whose the treatment-induced increase may correlate well with both pre-treatment tumor burden and sCRS symptoms. Other guidelines for the diagnosis and management of CRS are known (see, for example, Lee et al, Blood. 2014;124(2):188-95). In some embodiments, criteria reflecting the degree of CRS are presented in Table 3 below.

Table 3: Examples of CRS evaluation criteria Degree Description of symptoms 1
Weak Not life-threatening, requires only symptomatic treatment such as antipyretics and antiemetics (eg, fever, nausea, fatigue, headache, myalgia, malaise) 2
Moderate Requires and responds to moderate intervention:
Oxygen demand <40%, or
Hypotension responsive to fluids or a low dose of a single vasopressor, or
Grade 2 organotoxicity (CTCAE v4.0) 3
heavy Demands and responds to aggressive intervention:
Oxygen demand ≥ 40%, or
Hypotension requiring a higher dose of a single vasopressor agent (eg, norepinephrine ≥ 20 mg/kg/min, dopamine ≥ 10 mg/kg/min, phenylephrine ≥ 200 mg/kg/min, or epinephrine ≥ 10 mg/kg/min), or
Hypotension requiring multiple vasopressors (eg, vasopressin + one of the above agents, or a combination of vasopressors equivalent to ≥ 20 mg/kg/min norepinephrine), or
Grade 3 organotoxicity or Grade 4 transaminitis (CTCAE v4.0) 4
life-threatening Dangerous for life:
Requires a ventilator, or
Grade 4 organotoxicity (excluding transaminitis) 5
Lethal Death

[0340] In some embodiments, a patient is considered to develop "severe CRS"("sCRS") in response to or following administration of a cell therapy or dose of cells if, following administration, the patient exhibits: (1) a temperature increase of at least 38 degrees Celsius for at least three days; (2) an increase in cytokines, comprising either (a) a maximum fold change of at least 75 for at least two of the following group of seven cytokines compared to the level immediately after administration: interferon (IFNγ), GM-CSF, IL-6, IL-10, Flt-3L, fractalkine and IL-5 and/or (b) a maximum fold change of at least 250 for at least one of the following group of seven cytokines compared to the level immediately after administration: interferon (IFNγ), GM-CSF, IL-6, IL-10, Flt-3L, fractalkine and IL-5; and (c) at least one clinical sign of toxicity such as hypotension (requiring at least one intravenous vasoactive vasopressor agent) or hypoxia (PO ₂ <90%), or one or more neurological disorders (including mental status changes, and/or seizures). In some embodiments, severe CRS includes grade 3 or greater CRS, such as those set forth in Table 3.

[0341] In some embodiments, findings associated with severe CRS or CRS grade 3 or greater, such as grade 4 or greater, include one or more of the following: persistent fever, e.g., a fever with a specified temperature, e.g., greater than or approximately 38 degrees Celsius for two or more, such as three or more, such as four or more days, or for at least three consecutive days; an increase in temperature to more than or about 38 degrees Celsius; an increase in cytokines, such as a maximum fold change, e.g., at least or about 75, compared to pre-treatment levels of at least two cytokines (e.g., at least two from the group consisting of interferon (IFNγ), GM-CSF, IL -6, IL-10, Flt-3L, fractalkine, and IL-5 and/or tumor necrosis factor alpha (TNFα)), or a maximum fold change, eg, at least or about 250, of at least one of such cytokines; and/or at least one clinical sign of toxicity such as hypotension (eg, as measured by at least one intravenous vasoactive vasopressor agent); hypoxia (eg, plasma oxygen levels (PO ₂ ) less than or about 90%); and/or one or more neurological disorders (including mental status changes, stupor, and seizures). In some embodiments, severe CRS includes CRS requiring monitoring or treatment in an intensive care unit (ICU).

[0342] In some embodiments, CRS, such as severe CRS, encompasses a combination of (1) persistent fever (a rise in temperature of at least 38 degrees Celsius for at least three days) and (2) a serum CRP level of at least or approximately 20 mg/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 dosage of the vasopressor agents is increased on the second or subsequent administration.

[0343] In some embodiments, severe CRS or CRS grade 3 encompasses an increase in alanine aminotransferase, an increase in aspartate aminotransferase, chills, febrile neutropenia, headache, left ventricular dysfunction, encephalopathy, hydrocephalus, and/or tremor.

[0344] A method for measuring or detecting various results can be defined.

[0345] In some aspects, the toxic outcome is or is associated with neurotoxicity. In some embodiments, symptoms associated with the clinical risk of neurotoxicity include confusion, delirium, aphasia, expressive aphasia, stupor, myoclonus, lethargy, altered mental status, convulsions, seizure-like activity, seizures (not necessarily confirmed by an electroencephalogram [EEG]), elevated levels of beta amyloid (Aβ), elevated levels of glutamate, and elevated levels of oxygen radicals. In some embodiments, neurotoxicity is classified based on severity (e.g., using a scale of 1-5 (see, e.g., Guido Cavaletti & Paola Marmiroli Nature Reviews Neurology 6, 657-666 (December 2010); National Cancer Institute-Common Toxicity Criteria version 4.03 (NCI-CTCAE v4.03).

[0346] In some cases, neurological symptoms may be the earliest symptoms of sCRS. In some embodiments, neurological symptoms are observed to begin 5-7 days after the cell therapy infusion. In some embodiments, the implementation of the duration of neurological changes can be from 3 to 19 days. In some cases, recovery of neurological changes occurs after other symptoms of sCRS are resolved. In some embodiments, the time or extent of resolution of neurological changes is not accelerated by treatment with anti-IL-6 and/or steroid(s).

[0347] In some embodiments, a patient is considered to develop "severe neurotoxicity" in response to or following administration of a cell therapy or dose of cells if, following administration, the patient exhibits symptoms that limit self-care (e.g., bathing, bandaging and bandaging, feeding, using the toilet, taking medication) from among: 1) symptoms of peripheral motor neuropathy, including inflammation or degeneration of the peripheral motor nerves; 2) symptoms of peripheral sensory neuropathy, including inflammation or degeneration of peripheral sensory nerves, dysesthesias such as distortion of sensory perception resulting in abnormal and unpleasant sensations, neuralgia such as intense pain along a nerve or group of nerves, and/or paresthesias such as functional impairment of sensory neurons resulting in abnormal skin sensations of tingling, numbness, pressure, cold, and heat in the absence of a stimulus. In some embodiments, severe neurotoxicity includes grade 3 or greater neurotoxicity, such as set forth in Table 4.

Table 4: Examples of criteria for evaluating neurotoxicity Degree Description of symptoms 1
Asymptomatic or weak Mild or asymptomatic symptoms 2
Moderate Presence of symptoms that limit instrumental activities of daily living (ADL), such as cooking, shopping for food or clothing, using the phone, handling money 3
heavy Presence of symptoms that limit ADL self-care, such as bathing, bandaging and bandaging, self-feeding, toileting, medication 4
life-threatening Symptoms that are life-threatening requiring urgent intervention 5
lethal Death

[0348] In some embodiments, the methods reduce symptoms associated with CRS or neurotoxicity compared to other methods. In some aspects, the methods of the invention reduce symptoms, outcomes, or factors associated with CRS, including symptoms, outcomes, or factors associated with severe CRS or CRS grade 3 or higher, compared to other methods. For example, patients receiving treatment according to the methods of the invention may have no detectable CRS symptoms, outcomes, or factors and/or have reduced CRS symptoms, outcomes, or factors, e.g., severe CRS or CRS grade 3 or higher, such as any described, e.g. formulated in Table 3. In some embodiments, patients receiving treatment according to the methods of the invention may have reduced symptoms of neurotoxicity, such as weakness or numbness in the limbs, decreased memory, vision and/or intelligence, obsessive and/or compulsive behavior, not controllable, hallucinations, headache, cognitive and behavioral problems, including motor dysregulation, cognitive impairment and autonomic nervous system dysfunction and sexual dysfunction, compared with patients receiving other treatments. In some embodiments, patients receiving treatment according to the methods of the invention may have reduced symptoms associated with peripheral motor neuropathy, peripheral sensory neuropathy, dysesthesia, neuralgia, or paresthesia.

[0349] In some embodiments, the methods reduce outcomes associated with neurotoxicity, including damage to the nervous system and/or brain, such as neuronal death. In some aspects, the methods reduce levels of factors associated with neurotoxicity, such as beta amyloid (Aβ), glutamate, and oxygen radicals.

[0350] In some embodiments, the result of toxicity is dose-limiting toxicity (DLT). In some embodiments, the toxic result is dose-limiting toxicity. In some embodiments, the toxic result is the absence of dose-limiting toxicity. In some embodiments, Dose-Limiting Toxicity (DLT) is defined as Grade 3 or higher toxicity as assessed by any known or published guidelines for evaluating specific toxicity, such as any of those described above and including the Common Terminology Criteria for Adverse Events (CTCAE) version 4 .0 published by the National Cancer Institute (NCI).

[0351] In some embodiments, a low incidence, risk, or likelihood of developing toxicity, e.g., CRS or neurotoxicity, or severe CRS or neurotoxicity, e.g., CRS or stage 3 or higher neurotoxicity, observed with a dose of T cells in accordance with the methods of the invention and/or using the articles or compositions of the invention allows for the administration of cell therapy on an outpatient basis. In some embodiments, administration of cell therapy, e.g., a dose of T cells (e.g., CAR ⁺ T cells), in accordance with the methods of the invention and/or using articles or compositions of the invention, is on an outpatient basis or does not require admission of the patient to hospital, such as admission to a hospital requiring an overnight stay.

[0352] In some aspects, patients who are administered cell therapy, for example, a dose of T cells (e.g., CAR ⁺ T cells) in accordance with the methods of the invention and / or using articles or compositions of the invention, including patients receiving treatment on an outpatient basis, do not administer an intervention to treat any toxicity prior to or with a cell dose unless or until the patient exhibits a sign or symptom of toxicity such as neurotoxicity or CRS. Examples of agents for treating, delaying, reducing or alleviating toxicity are described in Section II.

[0353] In some embodiments, if a patient receiving cell therapy, e.g., a dose of T cells (e.g., CAR ⁺ T cells), including patients receiving treatment on an outpatient basis, shows an increase in temperature, the patient is prescribed or given treatment to lower the temperature. In some embodiments, the patient's temperature rise is characterized as the patient's body temperature, which is (or measured as such) a temperature at or above a temperature of a certain threshold or level. In some aspects, the threshold temperature is a temperature that is associated with at least low-grade fever, at least moderate fever, and/or at least severe fever. In some embodiments, the threshold temperature is a specific temperature or range. For example, the threshold temperature may be or approximately, or at least approximately, or approximately approximately 38, 39, 40, 41, or 42 degrees Celsius, and/or may be in the range from or from about 38 degrees Celsius to or to approximately 39 degrees Celsius, in the range of or from approximately 39 degrees Celsius to or to approximately 40 degrees Celsius, in the range of or from approximately 40 degrees Celsius to or to approximately 41 degrees, or in the range of or from approximately 41 degrees Celsius to or to approximately 42 degrees Celsius.

[0354] In some embodiments, the treatment designed to reduce fever includes treatment with an antipyretic agent. The antipyretic may include any agent, e.g., a compound, composition, or ingredient that reduces fever, such as any number of agents known to have antipyretic effects, such as NSAIDs (such as ibuprofen, naproxen, ketoprofen, and nimesulide), salicylates, such as aspirin, choline salicylate, magnesium salicylate and sodium salicylate, paracetamol, acetaminophen, metamizole, nabumetone, phenaxone, antipyrine, antipyretics. In some embodiments, the antipyretic is acetaminophen. In some embodiments, the implementation of acetaminophen can be administered at a dose of 12.5 mg/kg orally or intravenously at a frequency of up to once every four hours. In some embodiments, the antipyretic is or includes ibuprofen or aspirin.

[0355] In some embodiments, if the fever is a prolonged fever, the patient is administered an alternative treatment for the treatment of toxicity, such as any described in Section II below. For patients receiving treatment on an outpatient basis, the patient is instructed to return to the hospital if the patient has and/or is diagnosed with or has a prolonged fever. In some embodiments, the patient has, and/or is diagnosed with or is believed to have, a prolonged fever if he or she exhibits a fever at or above an appropriate threshold temperature, and where the fever or the patient's body temperature does not decrease, or does not decrease by or more than a specified amount (e.g., more than 1°C, and does not typically fluctuate by about or more than about 0.5°C, 0.4°C, 0.3°C, or 0, 2°C) after said treatment, such as a treatment designed to reduce fever, such as treatment with an antipyretic, eg NSAID or salicylates, eg ibuprofen, acetaminophen or aspirin. For example, a patient is considered to have a prolonged fever if he or she shows or is diagnosed with a fever of at least or about 38 or 39 degrees Celsius that does not decrease or decrease by more than or about 0.5 °C, 0.4 °C, 0.3°C or 0.2 °C, or by or approximately 1%, 2%, 3%, 4%, or 5% within 6 hours within 8 hours or within 12 hours, or within 24 hours, even after treatment with an antipyretic such as acetaminophen. In some embodiments, the dosage of the antipyretic is the dosage normally effective for such a patient to reduce fever or to reduce a particular type of fever, such as fever associated with a bacterial or viral infection, such as a localized or systemic infection.

[0356] In some embodiments, a patient has and/or is determined or believed to have a prolonged fever if he or she shows a temperature rise to or above an appropriate threshold temperature, and where the patient's temperature rise or body temperature does not fluctuate approximately or more than approximately 1 °C, and usually do not fluctuate approximately or more than approximately 0.5 °C, 0.4 °C, 0.3 °C or 0.2 °C. This lack of fluctuation above or by a certain amount is usually measured over a set period of time (for example, over a 24-hour, 12-hour, 8-hour, 6-hour, 3-hour, or 1-hour period of time, which can be measured from the first fever symptom or first temperature above a designated threshold). For example, in some embodiments, a patient is considered to exhibit, or is determined to exhibit, a prolonged temperature rise if he or she exhibits a temperature rise of at least or about or at least to or about 38 or 39 degrees Celsius. , which does not fluctuate by more than or approximately 0.5°C, 0.4°C, 0.3°C, or 0.2°C within 6 hours, within 8 hours, or within 12 hours, or within 24 hours.

[0357] In some embodiments, the temperature rise is a prolonged temperature rise; in some aspects, the patient is receiving treatment at the point in time at which a prolonged fever was detected for the patient, such as one, two, three, four, five, six or less hours from such detection or the first such detection after initial therapy with potential to stimulate toxicity, such as cell therapy, such as a dose of T cells, for example, CAR ⁺ T cells.

[0358] In some embodiments, one or more interventions or agents for the treatment of toxicity, such as toxicity-targeted therapies, are administered at or immediately after which the patient is determined or confirmed (e.g., first determined or confirmed), a long-term an increase in temperature, for example, when measured according to any of the above embodiments. In some embodiments, one or more toxicity-targeting therapies are administered within a specified time period following such confirmation or determination, such as within 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, or 8 hours.

E. Biomarkers, analytes or parameters

[0359] Among the methods of the invention are methods for assessing the risk of developing toxicity associated with cell therapy in a patient, including the assessment or detection of biomarkers (for example, analytes) or parameters associated with toxicity, for example, neurotoxicity, such as severe neurotoxicity, and/ or CRS, such as severe CRS. Also among the methods of the invention are methods for assessing the likelihood of response to cell therapy in a patient, including the assessment or detection of biomarkers (for example, analytes) or parameters associated with the outcome of the response, such as objective response (OR), including complete response (CR) and partial response (PR). In some embodiments, the associated response outcome includes a sustained response, such as a response that lasts for 3 months, 6 months, 9 months, 12 months or more after the initial response.

[0360] In some embodiments, the methods include evaluating or detecting the presence or absence of one or a group of biomarkers (e.g., analytes) and/or parameters (e.g., concentration, amount, level, or activity) associated with one or a group of biomarkers (e.g., analytes). ). In some instances, the methods may include comparing one or more parameters to a specific reference value, such as a cut-off value (also referred to herein as a “threshold”), e.g., associated with a risk of developing toxicity or associated with a particular response, such as OR, CR, or PR. , or a prolonged response, such as a response that lasts for 3 months, 6 months, 9 months, 12 months, or more after the initial response. In some embodiments, the methods also include selecting patients for cell therapy treatment based on an assessment of the presence or absence of a biomarker and/or comparing the biomarkers to a biomarker reference or threshold value. In some embodiments, the methods also include administering an agent or therapy that can treat, prevent, delay, and/or reduce the development of toxicity, such as by assessing the presence or absence of a biomarker and/or comparing the biomarkers to a biomarker reference or threshold value.

[0361] In some embodiments, the methods include assessing the likelihood of a patient's response or the risk of developing toxicity after administration of cell therapy. In some embodiments, the methods include assessing the level, amount, or concentration of one or more analytes in a biological sample, wherein the biological sample is from a patient who is a candidate for cell therapy treatment, said cell therapy optionally comprising a dose or composition of genetically engineered cells expressing recombinant receptor; and the biological sample is obtained from the patient prior to the application of cell therapy, and/or said biological sample does not include the recombinant receptor and/or said genetically engineered cells. In some aspects, the methods include comparing, individually, the level, amount, or concentration of an analyte in a sample to a cut-off value, thereby determining the risk of developing toxicity after administration of cell therapy. In some aspects, comparisons can be used to determine the likelihood of a patient's response or the risk of developing toxicity after administration of cell therapy.

[0362] In some embodiments, the methods also include selecting patients for treatment with a cell therapy, such as a specific dose of cell therapy, including administering a specific dose of cell therapy, such as those described herein, for example, in Section I.A and I.B, based on an assessment of the presence or absence of biomarker and/or comparison of biomarkers with a reference value or threshold value of a biomarker. In some embodiments, the methods also include selecting patients for treatment with an additional agent, such as an agent or other treatment capable of treating, preventing, delaying, attenuating, or reducing the development or risk of developing toxicity, based on an assessment of the presence or absence of a biomarker and/or comparisons of biomarkers with a reference value or threshold value of a biomarker.

[0363] In some embodiments, the parameter is or includes features, factors, a characteristic of a patient, and/or a disease or condition. In some embodiments, the parameter is a tumor burden related parameter, such as a measurement of tumor mass. In some aspects, the methods also include further monitoring the patient for possible symptoms of toxicity based on the risk of toxicity determined by assessing the presence or absence of a biomarker and/or comparing the biomarkers to a biomarker reference or threshold value. In some aspects, a biological sample, such as a blood sample or tissue sample from a patient, may be obtained to detect the presence or absence of a biomarker (e.g., analyte), such as to detect or measure a parameter (e.g., concentration, amount, level, or activity) of the biomarker and/or assessing the presence of a biomarker, for analysis, correlation and/or detection of certain results and/or toxicity. In some embodiments, certain physiological or biological parameters associated with a biomarker, including the expression of biomarkers, and/or clinical and laboratory parameters, can be assessed in a biological sample, such as blood, from patients before or after administration of cell therapy. In some embodiments, expression biomarkers or analytes and/or clinical and laboratory parameters may be assessed in a biological sample, such as blood, from patients prior to administration of cell therapy (pre-circulation). In some embodiments, expression biomarkers or analytes and/or clinical and laboratory parameters can be assessed in a biological sample, such as blood, from patients after administration of cell therapy (follow-up treatment). In some embodiments, the concentration, amount, level, or activity of biomarkers (eg, analytes) and/or clinical and laboratory parameters can be assessed at one or more time points before or after administration of the cell therapy. In some embodiments, the peak concentration, amount, level, or activity of biomarkers (eg, analytes) and/or clinical and laboratory parameters over a specified period of time may also be determined.

[0364] In some embodiments, a biomarker or analyte is an objectively measurable characteristic or molecule expressed by or in a biological sample, including cells, that may be indicative of or associated with a particular condition or phenomenon, such as a biological process, therapeutic outcome, phenotype cells or disease status. In some aspects, the biomarker or analyte, or parameters associated with the biomarker or analyte, can be measured or detected. For example, the presence or absence of expression of a biomarker or analyte can be detected. In some aspects, parameters such as the concentration, amount, level, or activity of a biomarker or analyte can be measured or detected. In some embodiments, the presence, absence, expression, concentration, amount, level, and/or activity of a biomarker may be associated with, correlate with, be indicative of, and/or predictive of certain conditions, such as certain therapeutic outcomes or patient conditions. In some aspects, the presence, absence, expression, concentration, amount, level, and/or activity of a biomarker or analyte, such as any described herein, can be used to assess the likelihood of a particular outcome or condition, such as a particular therapeutic outcome, including a response outcome or a toxicity outcome. . In some embodiments, examples of biomarkers include cytokines, cell surface molecules, chemokines, receptors, soluble receptors, soluble serum proteins, and/or cleavage products. In some embodiments, biomarkers or analytes may also include certain features, factors, characteristics of the patient and/or disease or condition, or factors indicative of the patient's condition and/or disease or condition of the patient (including disease severity), and/or clinical or laboratory parameters. .

[0365] In some embodiments, biomarkers may be used alone or in combination with other biomarkers, such as in a panel of biomarkers. In some embodiments, expression of certain biomarkers may correlate with certain outcomes or toxicity, such as the development of neurotoxicity. In some embodiments, biomarkers (e.g., analytes), including their parameters that can be assessed, include lactate dehydrogenase (LDH), ferritin, C-reactive protein (CRP), Interleukin-6 (IL-6), IL-7, IL-8, IL-10, IL-15, IL-16, tumor necrosis factor alpha (TNF-α), interferon alfa-2 (IFN-α2), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein 1 alpha (MIP-1α), macrophage inflammatory protein 1 beta (MIP-1β), eotaxin, granulocyte colony stimulating factor (G-CSF), IL-1 receptor alpha (IL-1Rα), IL-1β, IFN-γ inducible protein 10 (IP-10), perforin and D-dimer (fibrin breakdown product). In some embodiments, biomarkers (eg, analytes), including their parameters, include LDH, ferritin, CRP, IL-6, IL-8, IL-10, TNF-α, IFN-α2, MCP-1, and MIP-1β. In some embodiments, the biomarkers (eg, analytes), including their parameters, include ferritin, CRP, D-dimer, IL-6, IL-15, TNF-α, and MIP-1α. In some embodiments, the biomarkers (eg, analytes), including their parameters, include ferritin, CRP, IL-10, IL-15, IL-16, TNF-α, or MIP-1β.

[0366] In some embodiments, the methods include detecting the presence or absence of one or more biomarkers, such as a parameter (e.g., concentration, amount, level, or activity) associated with one or more biomarkers, wherein the one or more biomarkers are selected from Lactate dehydrogenase (LDH), ferritin, C-reactive protein (CRP), Interleukin-6 (IL-6), IL-7, IL-8, IL-10, IL-15, IL-16, tumor necrosis factor alpha (TNF- α), interferon alfa 2 (IFN-α2), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein 1 alpha (MIP-1α), macrophage inflammatory protein 1 beta (MIP-1β), eotaxin, granulocyte colony stimulating factor (G-CSF), IL-1 receptor alpha (IL-1Rα), IL-1β, IFN-γ-inducible Protein 10 (IP-10), perforin and D-dimer (fibrin breakdown product).

[0367] In some embodiments, the estimated parameter is or includes features, factors, characteristics of the patient and/or disease or condition and/or expression of biomarkers. In some embodiments, the parameter is or includes one or more factors indicative of the status of the patient and/or the disease or condition of the patient. In some embodiments, the implementation of the parameter is indicative of the tumor mass. In some embodiments, the tumor mass indicative factor is a volumetric parameter of the tumor(s). In some embodiments, the volume parameter is the size of the lesion(s), such as tumor size, tumor diameter, tumor volume, tumor mass, tumor burden, tumor-associated edema, tumor-associated necrosis, and/or the number or extent of metastases. In some embodiments, the implementation of the tumor volume parameter is a two-dimensional size. For example, in some embodiments, the lesion(s) is calculated as the product of the longest diameter and the longest perpendicular diameter of all measurable tumors. In some cases, the tumor volume parameter is a one-dimensional size. In some cases, the size of measurable lesions is estimated as the longest diameter. In some embodiments, sum product diameters (SPD), longest tumor diameters (LD), sum longest tumor diameters (SLD), necrosis, tumor volume, necrosis volume, necrosis to tumor ratio (NTR), peritumoral edema (PTE) are measured and edema to tumor ratio (ETR).

[0368] Examples of methods for measuring and evaluating tumor mass include those described in, for example, Carceller et al., Pediatr Blood Cancer. (2016) 63(8):1400-1406 and Eisenhauer et al., Eur J Cancer. (2009) 45(2):228-247. In some embodiments, the volumetric parameter is the sum of product diameters (SPD), measured by determining the sum of the products of the largest perpendicular diameters of all measurable tumors. In some aspects, the tumor or lesion is measured in a single longest diameter (LD) dimension and/or by determining the sum of the longest tumor diameters (SLD) of all measurable lesions. In some embodiments, the tumor volume parameter is a volumetric quantification of tumor necrosis, such as necrosis volume and/or necrosis to tumor ratio (NTR), see Monsky et al., Anticancer Res. (2012) 32(11): 4951-4961. In some aspects, the tumor volume parameter is a volumetric quantification of tumor-associated edema, such as peritumor edema (PTE) and/or edema to tumor ratio (ETR). In some embodiments, the measurement may be performed using imaging techniques such as computed tomography (CT), positron emission tomography (PET), and/or magnetic resonance imaging (MRI) of the patient.

[0369] In some embodiments, a tumor volume parameter is determined in a screening procedure, such as a routine evaluation or blood draw, to confirm and/or identify a condition or disease in a patient.

[0370] In some embodiments, the presence or absence and/or parameter of one or more biomarkers (eg, analytes) is assessed in a biological sample. In some aspects, the biological sample is a physical fluid or tissue. In some such embodiments, the biological sample, such as a bodily fluid, is or contains whole blood, serum, or plasma.

[0371] In some embodiments, the presence or absence and/or parameter of one or more biomarkers (e.g., analytes) is assessed prior to administration of cell therapy (e.g., pre-infusion), e.g., received up to 2 days, up to 7 days, up to 14 days , up to 21 days, up to 28 days, up to 35 days or up to 40 days before the initiation of the introduction of genetically engineered cells. In some embodiments, the biological sample is obtained from a patient prior to the administration of cell therapy (e.g., a pre-infusion), e.g., obtained up to 2 days, up to 7 days, up to 14 days, up to 21 days, up to 28 days, up to 35 days, or up to 40 days before initiating the introduction of genetically engineered cells.

[0372] In some embodiments, the biological sample is an apheresis or leukapheresis sample. In some embodiments, the presence or absence and/or parameter of one or more biomarkers (eg, analytes) is assessed, or a biological sample is obtained after the administration of cell therapy. In some embodiments, the reagents may be used prior to administration of cell therapy or after administration of cell therapy, for diagnostic purposes to identify patients and/or evaluate treatments and/or toxicity.

[0373] In some embodiments, measuring the value of one or more biomarkers includes performing an in vitro test. In some aspects, the in vitro test is an immunological test, an aptamer-based test, a histological or cytological test, or an mRNA expression level test. In some embodiments, values of one or more biomarkers are measured by enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, flow cytometry test, surface plasmon resonance (SPR), chemiluminescence test, immunochromatographic assay, inhibition test, or test avidity. In some cases, the value of at least one of one or more biomarkers is determined using a binding reagent that specifically binds to at least one biomarker. In some aspects, the binding reagent is an antibody, or an antigen-binding fragment thereof, an aptamer, or a nucleic acid probe.

[0374] In some embodiments, measuring the value of one or more biomarkers (e.g., analytes) includes contacting a reagent capable of directly or indirectly detecting an analyte with a biological sample and determining the presence or absence, level, amount, or concentration of the analyte in the biological sample. In some embodiments, one or more biomarkers (e.g., analyte) is lactate dehydrogenase (LDH), ferritin, CRP, IL-6, IL-7, IL-8, IL-10, IL-15, IL-16, TNF- alpha, IFN-gamma, MCP-1, MIP-1beta, eotaxin, G-CSF, IL-1Ralpha, IL-1Rbeta, IP-10, perforin, and D-dimer (fibrin breakdown product). In some embodiments, one or more biomarkers (eg, analyte) is LDH, ferritin, CRP, IL-6, IL-8, IL-10, TNF-alpha, IFN-alpha2, MCP-1, and MIP-1beta. In some embodiments, one or more biomarkers (eg, analyte) is or includes LDH.

[0375] In some aspects, the reagent is a binding molecule that specifically binds to the analyte. For example, in some embodiments, the reagent is an antibody or an antigen-binding fragment thereof. In some embodiments, the reagent is or includes a substrate or an analyte binding partner.

[0376] In some embodiments, the presence, absence, or parameter (eg, level, amount, concentration, and/or other value) of LDH is detected or determined in a sample. Various methods for detecting or determining LDH are known. For example, a test that measures the conversion of LDH lactate to pyruvate via the reduction of NAD+ to NADH can be used to detect LDH in a sample. In some embodiments, the sample is contacted with lactate in the presence of the NAD coenzyme, which, as a measure of LDH in the sample, results in NADH, which is then oxidized in the presence of an electron transfer agent. In some embodiments, NADH interacts with a probe or dye precursor that can be detected by measuring absorbance in the visible light range. In some examples, diaphorase uses NADH to reduce the tetrazolium salt (INT) to a red formazan product and the product is measured. Therefore, in some embodiments, the amount of color product formed is directly proportional to the LDH activity in the sample.

[0377] In some embodiments, the methods include comparing, individually, the level, amount, or concentration of an analyte in a sample with a threshold value, thereby determining the risk of developing toxicity after administration of cell therapy, or thereby determining the likelihood that the patient will achieve a response to cell therapy. therapy. In some aspects, exemplary thresholds may be determined based on means or medians and values in the range or standard deviation from the means or medians of the level, amount, or concentration of an analyte in a biological sample obtained from a population of patients prior to receiving cell therapy, each of the patients groups continued to show a specific outcome, such as a specific therapeutic outcome, either including response reporting or including no response reporting; or either developing toxicity or not developing toxicity. In some embodiments, certain aspects of determining thresholds include those described below in Sections I.E.1 and I.E.2.

1. Examples of biomarkers, analytes, or parameters associated with response outcomes

[0378] In some embodiments, the analyte or biomarker is associated with, correlates with, is indicative of, and/or predictive of a particular outcome, such as a particular response outcome, such as an objective response (OR), a complete response (CR), or a partial response (PR). ) or a prolonged response such as an OR or CR or PR that lasts 3, 6, 9 months or more. In some embodiments, reduced or decreased levels or increased levels of one or more of these biomarkers (e.g., analytes), e.g., compared to a reference value or threshold value, may be associated with a response, such as OR, CR, or PR, or any results. response described here, for example in Section I.C, optionally a long-term response, such as a response that lasts for at least 3 months, 6 months or more.

[0379] In some embodiments, the analyte or biomarker is associated with, correlates with, is indicative of, and/or predictive of a specific outcome, such as a specific response or sustained response, in a patient who is being administered cell therapy, such as therapy with a composition containing genetically modified cells. In some embodiments, the presence, expression, level, amount, or concentration of one or more analytes in a biological sample obtained from a patient prior to administration of cell therapy may be associated with, correlate with, be indicative of, and/or predictive of a particular outcome, such as the result of a specific response or a sustained response. In some embodiments, the presence, expression, level, amount, or concentration of certain biomarkers may be correlated with the outcome of a particular response or sustained response. In some embodiments, the response result may be any of the response results described herein, for example, in Section I.C.

[0380] In some embodiments, the methods include comparing, individually, the level, amount, or concentration of an analyte in a sample to a threshold value, thereby determining the likelihood that the patient will achieve a response to cell therapy. In some embodiments, the methods include selecting a patient for whom a probability of response to treatment is determined based on the results of determining the probability that the patient will achieve a response to cell therapy by comparing, individually, the level, amount, or concentration of an analyte in a sample with a threshold value. In some embodiments, the methods also include administering cell therapy to the patient selected for treatment. In some embodiments, if the patient is determined to be unlikely to achieve a response or sustained response, the method further comprises administering an additional therapeutic agent to the patient.

[0381] In some embodiments, biomarkers (eg, analytes) include outcome-related response and/or long-term response. In some embodiments, the biomarkers (e.g., analytes), including their parameters, include LDH, ferritin, CRP, D-dimer, serum amyloid A1 (SAA-1), IL-6, IL-10, IL-15, IL-16 , TNF-α, IFN-γ, MIP-1α, and C-X-C motif chemokine 10 (CXCL10).

[0382] In some aspects, examples of analytes or biomarkers that can be assessed or analyzed in relation to the assessment of the likelihood of response after the introduction of cell therapy include one or more analytes selected from ferritin, LDH, CXCL10, G-CSF and IL-10. In some embodiments, for any of the above analytes or biomarkers, a patient is likely to respond if the level, amount, or concentration of one or more analytes is below a threshold, and the patient is unlikely to respond if the level, amount, or concentration of one or more more analytes will be above the threshold. In some embodiments, the response is or includes an objective response. In some embodiments, the objective response is or includes a complete response (CR) or a partial response (PR). In some aspects, reduced levels of ferritin, LDH, CXCL10, G-CSF, and IL-10 in a biological sample from a patient obtained prior to the administration of cell therapy (pre-treatment) may be associated with the achievement of an objective response, including a complete response (CR) or partial response (PR).

[0383] In some embodiments, the threshold value is within 25%, 20%, 15%, 10%, or 5% and/or within a standard deviation below the median or mean level, amount or concentration of ferritin, LDH, CXCL10, G- CSF or IL-10 in a biological sample obtained from a group of patients prior to receiving cell therapy, with each of the patients in the group continuing to achieve a response after administration of the composition of cells expressing the recombinant receptor for the treatment of the same disease or condition. In some embodiments, the threshold is within 25%, 20%, 15%, 10%, or 5% and/or within a standard deviation above the median or mean level, amount or concentration of ferritin, LDH, CXCL10, G-CSF, or IL -10 in a biological sample obtained from a group of patients prior to receiving cell therapy, wherein each of the patients in the group continued to show stable disease (SD) and/or progressive disease (PD) after administration of the composition of cells expressing the recombinant receptor to treat the same disease, or states.

[0384] In some aspects, examples of analytes or biomarkers that can be assessed or analyzed in relation to assessing the likelihood of a long-term response after the introduction of cell therapy include one or more analytes selected from LDH, ferritin, CRP, D-dimer, SAA-1, IL -6, IL-10, IL-15, IL-16, TNF-α, IFN-γ, MIP-1α, CXCL-10, IL-8, MCP-1 and MIP-1β. In some embodiments, for any of the above analytes or biomarkers, the patient is likely to achieve a sustained response if the level, amount, or concentration of one or more analytes is below a threshold, and the patient is unlikely to achieve a sustained response if the level, amount, or concentration of one or more analytes will be above the threshold. In some embodiments, a sustained response is or includes a complete response (CR) or a partial response (PR) that lasts for or more than 3 months, 4 months, 5 months, or 6 months. In some embodiments, the long-term response is or includes a CR or PR that lasts for at least 3 months. In some aspects, decreased levels of LDH, ferritin, CRP, D-dimer, SAA-1, IL-6, IL-10, IL-15, IL-16, TNF-α, IFN-γ, MIP-1α, CXCL-10 , IL-8, MCP-1 and MIP-1β in a biological sample from a patient obtained before the introduction of cell therapy (pre-treatment) may be associated with achieving a long-term response, such as a CR or PR that lasts for at least 3 months.

[0385] In some embodiments, the threshold is within 25%, 20%, 15%, 11%, or 5% and/or within a standard deviation below the median or mean level, amount or concentration of LDH, ferritin, CRP, D- dimer, SAA-1, IL-6, IL-10, IL-15, IL-16, TNF-α, IFN-γ, MIP-1α, CXCL-10, IL-8, MCP-1 or MIP-1β in a biological sample obtained from a group of patients prior to receiving cell therapy, wherein each of the patients in the group continued to achieve sustained response after administration of the recombinant receptor-expressing cell composition for the treatment of the same disease or condition.

[0386] In some embodiments, the threshold is within 25%, 20%, 15%, 11%, or 5% and/or within a standard deviation above the median or mean level, amount or concentration of LDH, ferritin, CRP, D- dimer, SAA-1, IL-6, IL-10, IL-15, IL-16, TNF-α, IFN-γ, MIP-1α, CXCL-10, IL-8, MCP-1 or MIP-1β in a biological sample obtained from a group of patients prior to receiving cell therapy, and each of the patients of the group did not achieve a long-term response after administration of the composition of cells expressing the recombinant receptor for the treatment of the same disease or condition.

[0387] In some embodiments, the response is a long-term response, such as a CR or PR, that lasts for at least 3 months.

[0388] In some embodiments, the threshold value for LDH is or is about or below or about below 600 U/L, 500 U/L, 400 U/L, 300 U/L, or 200 U/L.

[0389] In some embodiments, an exemplary ferritin cut-off value is or is about or below or about below 1000 mg/L, 900 mg/L, 800 mg/L, 700 mg/L, 600 mg/L, 500 mg/L , 400 mg/l, 300 mg/l or 200 mg/l.

[0390] In some embodiments, an exemplary threshold for CRP is or is about or is below or about below 20 mg/L, 19 mg/L, 18 mg/L, 17 mg/L, 16 mg/L, 15 mg/L , 14 mg/l, 13 mg/l, 12 mg/l, 11 mg/l, 10 mg/l, 9 mg/l, 8 mg/l, 7 mg/l, 6 mg/l or 5 mg/l .

[0391] In some embodiments, an exemplary threshold for D-dimer is or is about or below or about below 1000 mg/L, 900 mg/L, 800 mg/L, 700 mg/L, 600 mg/L, 500 mg /l, 400 mg/l, 300 mg/l or 200 mg/l.

[0392] In some embodiments, an exemplary threshold for SAA-1 is or is about or below or about below 100 mg/L, 90 mg/L, 80 mg/L, 70 mg/L, 60 mg/L, 50 mg /l, 40 mg/l, 30 mg/l or 20 mg/l.

[0393] In some embodiments, an exemplary threshold for IL-6 is or is about or less than or less than about 6 pg/mL, 5 pg/mL, 4 pg/mL, 3 pg/mL, or 2 pg/mL.

[0394] In some embodiments, an exemplary threshold for IL-10 is or is about or below or about below 2 pg/mL, 1 pg/mL, 0.9 pg/mL, 0.8 pg/mL, 0.7 pg/ml, 0.6 pg/ml or 0.5 pg/ml.

[0395] In some embodiments, an exemplary threshold for IL-15 is or is about or less than or less than about 7 pg/mL, 6 pg/mL, 5 pg/mL, 4 pg/mL, or 3 pg/mL.

[0396] In some embodiments, an exemplary threshold for IL-16 is or is about or less than or less than 1000 pg/mL, 900 pg/mL, 800 pg/mL, 700 pg/mL, or 600 pg/mL.

[0397] In some embodiments, an exemplary threshold for TNF-α is or is about or less than or less than about 10 pg/mL, 9 pg/mL, 8 pg/mL, 7 pg/mL, or 6 pg/mL.

[0398] In some embodiments, an exemplary threshold for IFN-γ is or is about or below or about below 30 pg/mL, 20 pg/mL, 10 pg/mL, 9 pg/mL, 8 pg/mL, or 7 pg /ml;

[0399] In some embodiments, an exemplary threshold for MIP-1α is or is about or below or about 40 pg/mL, 30 pg/mL, or 20 pg/mL; and/or

[0400] In some embodiments, an exemplary threshold for CXCL-10 is or is about or below or about below 1500 pg/mL, 1000 pg/mL, 900 pg/mL, 800 pg/mL, 700 pg/mL, 600 pg /ml or 500 pg/ml.

[0401] In some aspects, examples of analytes or biomarkers that can be assessed or analyzed in relation to assessing the likelihood of a long-term response after the introduction of cell therapy include one or more analytes selected from ferritin, CRP, LDH, CXCL10, IL-8, IL- 10, IL-15, MCP-1, MIP-1β and TNF-α. In some embodiments, for any of the above analytes or biomarkers, the patient is likely to achieve a sustained response if the level, amount, or concentration of one or more analytes is below a threshold, and the patient is unlikely to achieve a sustained response if the level, amount, or concentration one or more analytes will be above the threshold value. In some embodiments, a sustained response is or includes a complete response (CR) or a partial response (PR) that lasts for or more than 3 months, 4 months, 5 months, or 6 months. In some embodiments, the long-term response is or includes a CR or PR that lasts for at least 3 months. In some aspects, reduced levels of ferritin, CRP, LDH, CXCL10, IL-8, IL-10, IL-15, MCP-1, MIP-1β and TNF-α in a biological sample from a patient obtained before the introduction of cell therapy (pretreatment ) may be associated with achieving a long-term response such as CR or PR that lasts for at least 3 months.

[0402] In some embodiments, the threshold is within 25%, 20%, 15%, 11%, or 5% and/or within a standard deviation below the median or mean level, amount or concentration of ferritin, CRP, LDH, CXCL10, IL-8, IL-10, IL-15, MCP-1, MIP-1β or TNF-α in a biological sample obtained from a group of patients before receiving cell therapy, and each of the patients in the group continued to achieve a long-term response after administration of the cell composition, expressing the recombinant receptor to treat the same disease or condition.

[0403] In some embodiments, the threshold is within 25%, 20%, 15%, 11%, or 5% and/or within a standard deviation above the median or mean level, amount or concentration of ferritin, CRP, LDH, CXCL10, IL-8, IL-10, IL-15, MCP-1, MIP-1β or TNF-α in a biological sample obtained from a group of patients before receiving cell therapy, and each of the patients in the group did not achieve a long-term response after administration of the cell composition, expressing the recombinant receptor to treat the same disease or condition.

[0404] In some aspects, examples of analytes or biomarkers that can be assessed or analyzed in relation to assessing the likelihood of a long-term response after the introduction of cell therapy include one or more analytes selected from hemoglobin and protein. In some embodiments, for any of the above analytes or biomarkers, the patient is likely to achieve a sustained response if the level, amount, or concentration of one or more analytes is above a threshold, and the patient is unlikely to achieve a sustained response if the level, amount, or concentration one or more analytes will be below the threshold value. In some embodiments, a sustained response is or includes a complete response (CR) or a partial response (PR) that lasts for or more than 3 months, 4 months, 5 months, or 6 months. In some embodiments, the long-term response is or includes a CR or PR that lasts for at least 3 months. In some aspects, elevated levels of hemoglobin and protein in a biological sample from a patient obtained prior to the administration of cell therapy (pre-treatment) may be associated with the achievement of a long-term response, such as a CR or PR, that lasts for at least 3 months.

[0405] In some embodiments, the threshold value is within 25%, 20%, 15%, 11%, or 5% and/or within a standard deviation above the median or mean level, amount or concentration of hemoglobin or protein in the biological sample obtained from a group of patients to receiving cell therapy, with each of the patients in the group continuing to achieve a sustained response after administration of the recombinant receptor-expressing cell composition for the treatment of the same disease or condition.

[0406] In some embodiments, the threshold value is within 25%, 20%, 15%, 11%, or 5% and/or within a standard deviation below the median or mean level, amount or concentration of hemoglobin or protein in the biological sample obtained from a group of patients to receiving cellular therapy, each of the patients of the group did not achieve a long-term response after administration of a composition of cells expressing the recombinant receptor for the treatment of the same disease or condition.

2. Examples of biomarkers, analytes or parameters associated with toxicity outcomes

[0407] In some embodiments, the analyte or biomarker is associated with, correlates with, is indicative of, and/or predictive of a particular outcome, such as the development of toxicity, in a patient who is administered cell therapy, such as therapy with a composition containing genetically engineered cells . In some embodiments, the presence, expression, level, amount, or concentration of one or more analytes in a biological sample obtained from a patient prior to administration of cell therapy may be associated with, correlate with, be indicative of, and/or predictive of a particular outcome, such as the development of toxicity, such as any of the toxicity results described here, for example, in Section I.D. In some embodiments, the presence, expression, level, amount, or concentration of certain biomarkers may correlate with certain toxicity outcomes, such as the development of NT or CRS. In some embodiments, the toxicity is a toxicity potentially associated with cell therapy, such as any described here, for example, in Section I.D. In some embodiments, the toxicity is neurotoxicity (NT) or cytokine release syndrome (CRS). In some embodiments, the toxicity is severe NT or severe CRS. In some embodiments, the toxicity is NT grade 2 or higher or CRS grade 2 or higher. In some embodiments, the toxicity is NT grade 3 or higher or CRS grade 3 or higher.

[0408] In some embodiments, the methods include comparing, individually, the level, amount, or concentration of an analyte in a sample to a cut-off value, thereby determining the risk of toxicity following administration of cell therapy. In some embodiments, the methods include identifying a patient who is at risk of developing toxicity after administration of a cell therapy based on a comparison, individually, of an analyte level, amount, or concentration in a sample with a threshold value. In some embodiments, the methods also include, following or based on the results of the assessment, administering to the patient a cell therapy, and optionally, a treatment agent or other agent capable of treating, preventing, delaying, attenuating, or reducing the development or risk of developing toxicity. In some embodiments, the methods also include monitoring the patient for symptoms of toxicity if the patient is receiving cell therapy and is identified as being at risk of developing toxicity.

[0409] In some embodiments, if a patient is identified as being at risk of developing toxicity, one or more of the following steps may be performed by administering to the patient: (a) (1) a treatment agent or other agent capable of treating, preventing, delaying, attenuation or reduction of the development or risk of developing toxicity, and (2) cell therapy, and the introduction of the agent must be carried out (i) before, (ii) within one, two or three days, (iii) simultaneously with and / or (iv) at the first rise in temperature after the initiation of the introduction of cell therapy to the patient; and/or (b) administering the cell therapy to the patient at a reduced dose or at a dose not associated with a risk of developing toxicity or severe toxicity, or not associated with a risk of developing toxicity or severe toxicity in the majority of patients and/or in the majority of patients with the disease or a condition that the patient has or is suspected of having following the administration of cell therapy; and/or (c) administering the cell therapy to the patient on an inpatient basis and/or with hospital admission for one or more days, optionally the cell therapy must otherwise be administered to patients on an outpatient basis or without hospital admission for one or more days. more days.

[0410] In some embodiments, the biomarkers or analytes, including their parameters that can be assessed, include lactate dehydrogenase (LDH), ferritin, C-reactive protein (CRP), Interleukin-6 (IL-6), IL-7, IL -8, IL-10, IL-15, IL-16, tumor necrosis factor alpha (TNF-α), interferon alfa 2 (IFN-α2), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein 1 alpha (MIP-1α), macrophage inflammatory protein 1 beta (MIP-1β), Eotaxin, granulocyte colony stimulating factor (G-CSF), IL-1 receptor alpha (IL-1Rα), IL-1β, IFN-γ inducible protein 10 (IP-10), perforin and D-dimer (fibrin breakdown product). In some embodiments, biomarkers (eg, analytes), including their parameters, include LDH, ferritin, CRP, IL-6, IL-8, IL-10, TNF-α, IFN-α2, MCP-1, and MIP-1β. In some embodiments, the biomarkers (eg, analytes), including their parameters, include ferritin, CRP, D-dimer, IL-6, IL-15, TNF-α, and MIP-1α. In some embodiments, the biomarkers (eg, analytes), including their parameters, include ferritin, CRP, IL-10, IL-15, IL-16, TNF-α, or MIP-1β. In some embodiments, elevated levels or elevated levels of one or more of these biomarkers (e.g., biomarkers), e.g., compared to a reference value or threshold value, may be associated with the development of neurotoxicity, e.g. 4 or 5. In some embodiments, elevated levels or increased levels of one or more of these biomarkers (e.g., analytes), e.g., compared to a reference value or threshold value, may be associated with the development of neurotoxicity, e.g., severe neurotoxicity or grade 3 neurotoxicity. or higher or degree 4 or 5.

[0411] In some aspects, examples of analytes or biomarkers that can be assessed or analyzed in relation to the assessment of the risk of developing toxicity after the introduction of cell therapy include one or more analytes selected from LDH, Ferritin, C-reactive protein (CRP), IL- 6, IL-8, IL-10, TNF-α, IFN-α2, MCP-1 and MIP-1β. In some embodiments, for any of the above analytes or biomarkers, the patient is at risk of developing toxicity if the level, amount, or concentration of one or more analytes is above a threshold, and the patient is at low risk of developing toxicity if the level, amount, or concentration of one or more analytes below the threshold. In some embodiments, the toxicity is neurotoxicity. In some aspects, elevated levels of LDH, Ferritin, C-reactive protein (CRP), IL-6, IL-8, IL-10, TNF-α, IFN-α2, MCP-1, and MIP-1β, in a biological sample from a patient obtained prior to the introduction of cell therapy (pre-treatment) may be associated with a higher risk of neurotoxicity.

[0412] In some embodiments, the threshold is within 25%, 20%, 15%, 30%, or 5% and/or within a standard deviation above the median or mean level, amount or concentration of LDH, Ferritin, C-reactive protein (CRP), IL-6, IL-8, IL-10, TNF-α, IFN-α2, MCP-1 or MIP-1β in a biological sample obtained from a group of patients prior to receiving cell therapy, each of the patients in the group still does not develop any toxicity after receiving a composition of cells expressing the recombinant receptor for the treatment of the same disease or condition.

[0413] In some embodiments, the threshold is within 25%, 20%, 15%, 30%, or 5% and/or within a standard deviation below the median or mean level, amount or concentration of LDH, Ferritin, C-reactive protein (CRP), IL-6, IL-8, IL-10, TNF-α, IFN-α2, MCP-1 or MIP-1β in a biological sample obtained from a group of patients prior to receiving cell therapy, each of the patients in the group continued to develop toxicity after receiving a composition of cells expressing the recombinant receptor for the treatment of the same disease or condition.

[0414] In some embodiments, the toxicity is neurotoxicity.

[0415] In some embodiments, an exemplary LDH threshold is or is at or above or above about 300 U/L, 400 U/L, 500 U/L, 600 U/L, or 700 U/L.

[0416] In some embodiments, an exemplary Ferritin threshold is or is at or above or above about 500 ng/mL, 600 ng/mL, 700 ng/mL, 800 ng/mL, 900 ng/mL, 1000 ng/mL or 1500 ng/ml.

[0417] In some embodiments, an exemplary threshold for CRP is or is about or is above or is above about 20 mg/L, 30 mg/L, 40 mg/L, 50 mg/L, 60 mg/L, 70 mg/L or 80 mg/l.

[0418] In some embodiments, an exemplary threshold for IL-6 is or is about or greater than or greater than about 5 pg/mL, 6 pg/mL, 7 pg/mL, 8 pg/mL, 9 pg/mL, 10 pg /ml, 20 pg/ml or 30 pg/ml.

[0419] In some embodiments, an exemplary threshold for IL-8 is or is about or greater than or greater than about 8 pg/mL, 9 pg/mL, 10 pg/mL, 20 pg/mL, or 30 pg/mL.

[0420] In some embodiments, an exemplary threshold for IL-10 is or is about or greater than or greater than about 20 pg/mL, 30 pg/mL, 40 pg/mL, 50 pg/mL, 60 pg/mL, or 70 pg /ml

[0421] In some embodiments, an exemplary threshold for TNF-α is or is about or greater than or greater than about 20 pg/mL or 30 pg/mL.

[0422] In some embodiments, an exemplary threshold for IFN-α2 is or is about or greater than or greater than about 40 pg/mL, 50 pg/mL, 60 pg/mL, 70 pg/mL, or 80 pg/mL.

[0423] In some embodiments, an example of a threshold for MCP-1; and/or is or approximately is or greater than or greater than approximately 200 pg/ml or 300 pg/ml.

[0424] In some embodiments, an exemplary cutoff for MIP-1β is or is about or greater than or greater than about 40 pg/mL, 50 pg/mL, 60 pg/mL, 70 pg/mL, or 80 pg/mL.

[0425] In some aspects, examples of analytes or biomarkers that can be assessed or analyzed in relation to the assessment of the risk of developing toxicity after the introduction of cell therapy include one or more analytes selected from IL-8, IL-10 and CXCL10. In some embodiments, for any of the above analytes or biomarkers, the patient is at risk of developing toxicity if the level, amount, or concentration of one or more analytes is above a threshold, and the patient is at low risk of developing toxicity if the level, amount, or concentration of one or more analytes is below threshold value. In some embodiments, the toxicity is neurotoxicity. In some embodiments, the toxicity is severe neurotoxicity or grade 3 neurotoxicity or higher. In some aspects, elevated levels of IL-8, IL-10, and CXCL10 in a biological sample from a patient obtained prior to the administration of cell therapy (pre-treatment) may be associated with a higher risk of developing neurotoxicity or severe neurotoxicity, or grade 3 or higher neurotoxicity. .

[0426] In some embodiments, the threshold is within 25%, 20%, 15%, 30%, or 5% and/or within a standard deviation above the median or mean level, amount or concentration of IL-8, IL-10, or CXCL10 in a biological sample obtained from a group of patients before receiving cell therapy, and each of the patients of the group still does not develop any toxicity after receiving the composition of cells expressing the recombinant receptor for the treatment of the same disease or condition.

[0427] In some embodiments, the threshold is within 25%, 20%, 15%, 30%, or 5% and/or within a standard deviation below the median or mean level, amount or concentration of IL-8, IL-10, or CXCL10 in a biological sample obtained from a group of patients prior to receiving cell therapy, with each of the patients in the group continuing to develop toxicity after receiving a composition of cells expressing the recombinant receptor for the treatment of the same disease or condition.

[0428] In some aspects, examples of analytes or biomarkers, or tumor mass volumetric parameter, that can be assessed or analyzed in relation to the assessment of the risk of developing toxicity after the introduction of cell therapy includes one or more analytes or a tumor mass volumetric measure selected from the number of product diameters ( SPD), LDH, Ferritin, C-reactive protein (CRP), D-dimer (fibrin breakdown product), IL-6, IL-10, IL-15, IL-16 TNF-α, MIP-1α and MIP-1β . In some embodiments, for any of the above analytes or biomarkers or tumor mass volume parameter, the patient is at risk of developing toxicity if the level, amount, or concentration of one or more analytes or tumor mass volume parameter is above a threshold value, and the patient is at low risk of developing toxicity if the level, amount, or concentration of one or more analytes, or the volumetric parameter of the tumor mass, is below a threshold value. In some embodiments, the toxicity is neurotoxicity. In some aspects, elevated levels or measurement of diameter products (SPD), LDH, Ferritin, C-reactive protein (CRP), D-dimer (fibrin breakdown product), IL-6, IL-10, IL-15, IL-16 TNF-α, MIP-1α and MIP-1β in a biological sample from a patient obtained before the introduction of cell therapy (pre-treatment) may be associated with a higher risk of developing neurotoxicity (NT) or cytokine release syndrome (CRS).

[0429] In some embodiments, one or more analytes or tumor mass volume parameter selected from LDH, SPD, IL-10, IL-15, IL-16, TNF-α, and MIP-1β, and the toxicity is neurotoxicity. In some embodiments, one or more analytes or tumor mass volume parameter selected from LDH, SPD, CRP, d-dimer, IL-6, IL-15, TNF-α, and MIP-1α, and toxicity is CRS. In some aspects, elevated levels or magnitude of LDH, SPD, IL-10, IL-15, IL-16, TNF-α and MIP-1β in a biological sample from a patient obtained prior to the administration of cell therapy (pretreatment) may be associated with higher risk of developing neurotoxicity (NT). In some aspects, elevated levels or magnitude of LDH, SPD, CRP, d-dimer, IL-6, IL-15, TNF-α, and MIP-1α in a biological sample from a patient obtained prior to the administration of cell therapy (pretreatment) may be associated with a higher risk of developing cytokine release syndrome (CRS).

[0430] In some embodiments, the threshold is within 25%, 20%, 15%, 32%, or 5% and/or within a standard deviation above the median or mean level, amount or concentration of LDH, Ferritin, C-reactive protein (CRP), D-dimer (fibrin degradation product), IL-6, IL-10, IL-15, IL-16 TNF-α, MIP-1α, or MIP-1β, or median or mean volumetric tumor mass parameter of the number of products diameters (SPD) in a biological sample obtained from a group of patients before receiving cell therapy, and each of the patients of the group still does not develop any toxicity after receiving a composition of cells expressing the recombinant receptor for the treatment of the same disease or condition.

[0431] In some embodiments, the threshold is within 25%, 20%, 15%, 32%, or 5% and/or within a standard deviation below the median or mean level, amount or concentration of LDH, Ferritin, C-reactive protein (CRP), D-dimer (fibrin degradation product), IL-6, IL-10, IL-15, IL-16 TNF-α, MIP-1α, or MIP-1β, or median or mean volumetric tumor mass parameter of the number of products diameters (SPD) in a biological sample obtained from a group of patients before receiving cell therapy, and each of the patients of the group continued to develop toxicity after receiving a composition of cells expressing the recombinant receptor for the treatment of the same disease or condition.

[0432] In some embodiments, the toxicity is neurotoxicity and an exemplary LDH threshold is or is at or is above or is above about 300 U/L, 400 U/L, 500 U/L, or 600 U/L.

[0433] In some embodiments, the toxicity is neurotoxicity, and an example of a threshold value for SPD is or is about or is greater than or greater than about 30 cm ² , 40 cm ² , 50 cm ² , 60 cm ² , 70 cm ² , 80 cm ^{2 ,} or 90 ^cm2 .

[0434] In some embodiments, the toxicity is neurotoxicity, and an exemplary cut-off value for IL-10 is or is about or greater than or greater than about 0.8 pg/mL, 0.9 pg/mL, 1 pg/mL, 2 pg/ ml, 3 pg/ml or 4 pg/ml.

[0435] In some embodiments, the toxicity is neurotoxicity, and an exemplary cut-off value for IL-15 is or about or greater than or greater than about 3 pg/mL, 4 pg/mL, 5 pg/mL, 6 pg/mL, or 7 pg /ml

[0436] In some embodiments, the toxicity is neurotoxicity, and an exemplary cut-off value for IL-16 is or is about or greater than or greater than about 600 pg/mL, 700 pg/mL, 800 pg/mL, 900 pg/mL, or 1000 pg /ml

[0437] In some embodiments, the toxicity is neurotoxicity, and an example cut-off value for TNF-α is or is about or greater than or greater than about 6 pg/mL, 7 pg/mL, 8 pg/mL, 9 pg/mL, or 10 pg /ml

[0438] In some embodiments, the toxicity is neurotoxicity, and an exemplary cutoff for MIP-1β is or is at or is above or is above about 70 pg/mL, 80 pg/mL, 90 pg/mL, or 100 pg/mL.

[0439] In some embodiments, the toxicity is CRS and an exemplary LDH threshold is or is at or is above or is above about 300 U/L, 400 U/L, 500 U/L, or 600 U/L.

[0440] In some embodiments, the toxicity is CRS and the cut-off value for SPD is or is at or about 20 cm ² , 30 cm ² , 40 cm ² , or 50 cm ² .

[0441] In some embodiments, the toxicity is CRS and an exemplary cut-off for ferritin is or is at or is above or is above about 300 ng/mL, 400 ng/mL, 500 ng/mL, 600 ng/mL, 700 ng/mL , 800 ng/ml, 900 ng/ml or 1000 ng/ml.

[0442] In some embodiments, the toxicity is CRS, and an exemplary threshold for CRP is or is at or is above or is above about 20 mg/L, 30 mg/L, or 40 mg/L.

[0443] In some embodiments, the toxicity is CRS and an exemplary threshold for d-dimer is or is at or is above or is above about 300 pg/mL, 400 pg/mL, 500 pg/mL, 600 pg/mL, 700 pg /ml, 800 pg/ml, 900 pg/ml or 1000 pg/ml.

[0444] In some embodiments, the toxicity is CRS and an exemplary threshold for IL-6 is or is about or is above or is above about 2 pg/mL, 3 pg/mL, 4 pg/mL, 5 pg/mL, 6 pg /ml, 7 pg/ml, 8 pg/ml or 9 pg/ml.

[0445] In some embodiments, the toxicity is CRS and an exemplary threshold for IL-15 is or is at or is above or is above about 3 pg/mL, 4 pg/mL, 5 pg/mL, 6 pg/mL, 7 pg /ml, 8 pg/ml, 9 pg/ml or 10 pg/ml.

[0446] In some embodiments, the toxicity is CRS and an exemplary threshold for TNF-α is or is about or is above or is above about 7 pg/mL, 8 pg/mL, 9 pg/mL, 10 pg/mL, or 15 pg /ml

[0447] In some embodiments, the toxicity is CRS and an exemplary threshold for MIP-1α is or is at or is above or is above about 20 pg/mL, 30 pg/mL, or 40 pg/mL.

[0448] In some embodiments, the implementation of the biomarker is LDH, and in some cases, the development of toxicity, such as CRS or NT, correlates with an LDH value that is above a threshold value. In some embodiments, the inflammatory marker is LDH and the threshold is or is about 300 units per liter, is or is about 400 units per liter, is or is about 500 units per liter, or is or is about 600 units per liter.

[0449] In some embodiments, if the level, amount, or concentration of a biomarker (e.g., analyte) in a sample is equal to or greater than a threshold value for an analyte, substance, or other treatment capable of treating, preventing, delaying, attenuating, or reducing the development or risk the development of toxicity, administered to the patient before, within one, two or three days, simultaneously with and/or at the first increase in temperature after the initiation of the introduction of cell therapy to the patient. Examples of agents or interventions for use in connection with the methods of the invention to treat, prevent, delay, reduce, or mitigate the risk of developing toxicity are described in Section II.

[0450] In some cases, if the level, amount of concentration of the biomarker in the sample is equal to or above the threshold value, ketogenic therapy is administered to the patient at a reduced dose or at a dose not associated with a risk of developing toxicity or severe toxicity, or not associated with a risk of developing toxicity or severe toxicity in the majority of patients and/or in the majority of patients having a disease or condition that the patient has or is suspected of having, after the introduction of cell therapy. In some cases, if the level, amount of concentration of the biomarker in the sample is equal to or above the threshold value, cell therapy is administered to the patient in hospital mode and / or with admission to the hospital for one or more days, and optionally, cell therapy must otherwise be administered to patients on an outpatient basis or without hospital admission for one or more days.

[0451] In some embodiments, if the level, amount, or concentration of a biomarker (eg, analyte) is below a threshold for the analyte, cell therapy is administered to the patient, optionally at a non-reduced dose. In some cases, cell therapy is not necessarily administered on an outpatient basis or without hospital admission for one or more days. In some embodiments, if the level, amount, or concentration of the analyte is below a threshold, administering the cell therapy does not include administering, prior to or simultaneously with the use of the cell therapy and/or prior to the development of a sign of a symptom of toxicity other than fever, a treatment agent or agent capable of treating, preventing, delaying or reducing the development of toxicity; and/or the administration of the cell therapy must or may be administered to the patient on an outpatient basis and/or without admission of the patient to the hospital overnight or for one or more consecutive days and/or without admission of the patient to the hospital for one or more days.

[0452] In some aspects of the methods of the invention, a patient is determined to be at risk of developing toxicity (e.g., neurotoxicity, such as severe neurotoxicity or grade 3 or higher neurotoxicity, such as grade 4 or 5 neurotoxicity, and/or CRS, such as severe CRS or CRS grade 3 or higher) by comparing a parameter (e.g. concentration, amount, level or activity) of a biomarker (e.g. analyte) or, individually, each of the biomarkers (e.g. analytes) with a reference value, such as a threshold value, of the corresponding parameter for biomarker or each biomarker. In some embodiments, the comparison indicates whether the patient is or is at risk of developing toxicity, e.g., neurotoxicity, such as severe neurotoxicity or Grade 3 or higher neurotoxicity, e.g., Grade 4 or 5 neurotoxicity, and/or CRS, such as severe CRS or CRS grade 3 or higher, and/or indicates the level of risk for developing said toxicity. In some embodiments, a reference value is a value that is a cut-off or cut-off at which there is a good predictive value (e.g., accuracy, sensitivity, and/or specificity) that such toxicity will occur or is likely to occur, individually or in combination with one or more biomarkers in the group. In some cases, such a reference value, such as a cut-off value, may be predetermined or known prior to implementation of the method, for example, from a plurality of patients previously treated with cell therapy and assessed to correlate a biomarker parameter or, individually, each of the biomarkers in a group with the presence of a toxic result (eg, the presence of neurotoxicity, such as severe neurotoxicity or grade 3 or higher neurotoxicity, eg, grade 4 or 5 neurotoxicity and/or CRS, such as severe CRS or grade 3 or higher CRS).

[0453] In some embodiments, a biomarker parameter (e.g., LDH, ferritin, CRP, IL-6, IL-8, IL-10, TNF-α, IFN-α2, MCP-1, and MIP-1β) that is higher or greater than the reference value, for example, the cut-off value, of the relevant parameter is associated with a positive prediction of the risk of toxicity (individually or in combination with the assessment of other biomarkers in the group). In some embodiments, a biomarker parameter that is equal to or lower than a reference value, such as a threshold value, of the respective parameter is associated with a negative predictive risk of toxicity (individually or in combination with assessment of other biomarkers in the group).

[0454] In some embodiments, a threshold value is determined based on the level, amount, concentration, or other value of a biomarker (eg, analyte) in a sample that is positive for the biomarker. In some aspects, the threshold is within 25%, 20%, 15%, 10%, or 5% of the mean level, amount, or concentration, or value, and/or within the standard deviation of the mean level, amount, or concentration, or value, analyte, or parameter. in a biological sample obtained from a group of patients prior to obtaining a therapeutic composition of cells expressing the recombinant receptor, and each of the patients in the group continued to develop toxicity, for example, neurotoxicity, such as severe neurotoxicity or grade 3 or higher neurotoxicity, for example, grade 4 or 5 neurotoxicity and/or CRS, such as severe CRS or CRS grade 3 or higher, after receiving a composition of cells expressing the recombinant receptor for the treatment of the same disease or condition.

[0455] In some embodiments of any of the methods of the invention, the biomarker (e.g., analyte) correlates with and/or is predictive of the risk of developing severe neurotoxicity, such as severe neurotoxicity or grade 3 or higher neurotoxicity, e.g., grade 4 or 5 neurotoxicity and /or severe CRS or CRS grade 3 or higher. In some embodiments, the threshold is within 25%, 20%, 15%, 10%, or 5% of the mean level, amount, or concentration, or amount, and/or within the standard deviation of the mean level, amount, or concentration, or amount, of the analyte or parameter in a biological sample obtained from a group of patients prior to obtaining a therapeutic composition of cells expressing the recombinant receptor, and each of the patients in the group continued to develop severe neurotoxicity or grade 3 or higher neurotoxicity, for example, grade 4 or 5 neurotoxicity and / or severe CRS or CRS grade 3 or higher after obtaining a composition of cells expressing the recombinant receptor for the treatment of the same disease or condition.

[0456] In some embodiments, the implementation of the volumetric parameter is the SPD, and in some cases, the development of toxicity, for example, CRS or NT, correlates with an SPD value that is above a threshold value. In some embodiments, the volume parameter is SPD and the threshold is or is about 30 cm ² , is or is about 40 cm ² , is or is about 50 cm ² , is or is about 60 cm ² , or is or is about 70 cm ² . In some embodiments, the volumetric parameter is SPD and the threshold is or is about 30 cm ² , is or about 40 cm ² , is or about 50 cm ² , is or about 60 cm ² , or is or about 70 cm ² .

[0457] In some embodiments, a parameter, including tumor volume measurements, is associated with response to cell therapy and/or risk of developing toxicity, eg, CRS or neurotoxicity (NT).

[0458] In some embodiments, the volumetric parameter is SPD and the threshold is or is about 30 ^cm2 , is or is about 40 ^cm2 , is or is about 50 ^cm2 , is or is about 60 ^cm2 , or is or is about 70 cm ² . In some embodiments, the implementation of the volume parameter is SPD, and the threshold value is or approximately is 50 cm ² .

[0459] In some embodiments, the analyte is LDH and the cutoff is or is about 300 units per liter (U/L), is or is about 400 U/L, is or is about 500 U/L, or is or about is 600 U / l. In some embodiments, the analyte is LDH and the cut-off is, or is about, 500 U/L.

[0460] In some embodiments, the parameter or biomarker is LDH. In some embodiments, the biomarker is LDH and the threshold is 500 U/L or higher. In some embodiments, the parameter or biomarker is SPD. In some embodiments, the parameter is SPD and the threshold is, or is about, 50 cm ² or greater. In some embodiments, the biomarker or parameters are SPD and LDH and the cutoffs are SPD 50 cm ² or greater and LDH 500 U/l or greater. In some embodiments, biomarkers or parameters are associated with an increased risk of developing CRS or NT.

[0461] In some embodiments, a measurement of a parameter or marker that is above a threshold, such as SPD 50 cm ² or greater and LDH 500 U/L or greater, is associated with approximately 2-, 3-, 4-, 5-, 6 -, 7-, 8-, 9-, 10-fold or more increased risk of developing CRS or NT, such as CRS or NT of any degree. In some embodiments, a measurement of a parameter or marker that is below a threshold, such as SPD below 500 cm ² and LDH below 500 U/L, is associated with approximately 2-, 3-, 4-, 5-, 6- , 7-, 8-, 9-, 10-fold or more reduced risk of developing CRS or NT, such as CRS or NT of any grade.

[0462] In some embodiments, biomarkers (e.g., analytes) include cells associated with increased pharmacokinetic parameters (PK), e.g., increased maximum cell serum concentration (AUC _0-28 ) or increased exposure (e.g., area under the curve (AUC) ). In some embodiments, biomarkers (eg, analytes), including parameters thereof, include IL-7, IL-15, MIP-1α, and TNF-α.

[0463] In some embodiments, the implementation of the parameter is a parameter related to tumor burden, for example, a measurement of tumor mass. In some aspects, the methods also include further monitoring the patient for possible symptoms of toxicity based on the risk of toxicity, specifically assessing the presence or absence of a biomarker, and/or comparing the biomarkers to a biomarker reference or threshold value.

II. AGENTS OR INTERVENTIONS THAT TREAT OR RELIEF THE SYMPTOMS OF TOXICITY

[0464] In some embodiments, the methods and articles of the invention may be used in connection with, or include or employ, one or more agents or agents to treat, prevent, delay, or reduce the development of toxicity. In some examples, an agent or other treatment capable of treating, preventing, delaying, or reducing the development of toxicity is administered prior to and/or simultaneously with administration of a therapeutic cell composition comprising genetically engineered cells.

[0465] In some embodiments, the agent, e.g., a toxicity-targeting agent or treatment capable of treating, preventing, delaying, or reducing the development of toxicity, is a steroid, antagonist, or inhibitor of a cytokine receptor, such as the IL-6 receptor, CD122 (IL-2Rbeta receptor) or CCR2, or an inhibitor of a cytokine such as IL-6, MCP-1, IL-10, IFN-γ, IL-8 or IL-18. In some embodiments, the agent is a cytokine and/or cytokine receptor agonist, such as TGF-β. In some embodiments, the agent, such as an agonist, antagonist, or inhibitor, is an antibody or antigen-binding fragment, small molecule, protein or peptide, or nucleic acid.

[0466] In some embodiments, a liquid bolus may be used as an intervention, such as for the treatment of hypotension associated with CRS. In some embodiments, target hematocrit levels are >24%. In some embodiments, the intervention includes the use of absorbent resin technology with blood or plasma filtration. In some cases, the intervention includes dialysis, plasmapheresis, or similar technologies. In some embodiments, vasopressors or acetaminophen may be used.

[0467] In some embodiments, the agent may be administered sequentially, intermittently, or at the same time as, or in the same composition as, the therapy, such as cells for adoptive cell therapy. For example, the agent may be administered before, during, simultaneously with, or after the administration of cell therapy and/or immunotherapy.

[0468] In some embodiments, the agent is administered at a point in time as described herein and in accordance with the methods of the invention and/or using articles or compositions of the invention. In some embodiments, the toxicity targeting agent is administered at a time point, such as less than or no more than 3, 4, 5, 6, 7, 8, 9, or 10 days after initiation of cell therapy and/or immunotherapy. In some embodiments, the toxicity targeting agent is administered on or within about 1 day, 2 days, or 3 days after initiation of cell therapy and/or immunotherapy administration.

[0469] In some embodiments, an agent, e.g., a toxicity-targeting agent, is administered to a patient after initiation of administration of immunotherapy and/or cell therapy at a point in time at which the patient does not show grade 2 or higher CRS or grade 2 or higher neurotoxicity. In some aspects, the toxicity targeting agent is administered following the initiation of administration of immunotherapy and/or cell therapy at a time point at which the patient is not showing severe CRS or severe neurotoxicity. Thus, between the initiation of administration of immunotherapy and/or cell therapy and the agent targeting toxicity, a patient is a patient not showing grade 2 or higher CRS, such as severe CRS, and/or not showing grade 2 or higher neurotoxicity, such as severe CRS. neurotoxicity.

[0470] Non-limiting examples of interventions to treat or alleviate toxicity, such as severe CRS (sCRS), are described in Table 5. In some embodiments, the intervention comprises tocilizumab or other toxicity-targeting agent as described, which may be at a time point in in which there is a prolonged or persistent increase in temperature to more than or about 38°C or more than or more than about 39°C in a patient. In some embodiments, the patient has had a fever for more than 10 hours, more than 12 hours, more than 16 hours, or more than 24 hours prior to the intervention.

Table 5. Examples of interventions Symptoms associated with CRS Suggested Intervention Temperature rise ≥ 38.3°C Acetaminophen (12.5 mg/kg) PO/IV up to every four hours Persistent fever ≥ 39°C for 10 hours that does not respond to acetaminophen Tocilizumab (8-12 mg/kg) IV Persistent fever ≥ 39°C after tocilizumab Dexamethasone 5–10 mg IV/PO up to every 6–12 hours for prolonged fever Recurrence of symptoms 48 hours after initial dose of tocilizumab Tocilizumab (8-12 mg/kg) IV hypotension Liquid bolus, target hematocrit > 24% Persistent/recurrent hypotension after initial liquid bolus (within 6 hours) Tocilizumab (8-12 mg/kg) IV Use of low dose hypotension pressors for more than 12 hours Dexamethasone 5-10 mg IV/PO up to every 6 hours followed by pressors Initiating a higher dose of pressors or adding a second pressor to treat hypotension Dexamethasone 5-10 mg IV/PO up to every 6 hours followed by pressors Initiation of oxygen addition Tocilizumab (8-12 mg/kg) IV Strengthening IVL with the possibility of intubation Dexamethasone 5-10 mg IV/PO up to every 6 hours followed by pressors Recurrent/persistent symptoms for which tocilizumab was given ≥ 48 hours after initial dose Tocilizumab (8-12 mg/kg) IV

[0471] In some instances, an agent or therapy or intervention, such as an agent targeting toxicity, is administered alone or administered as part of a composition or formulation, such as a pharmaceutical composition or formulation as described herein. Thus, the agent alone or as part of a pharmaceutical composition may be administered intravenously or orally, or by any other acceptable route of administration known in the art, or as described herein.

[0472] In some embodiments, the dosage of the agent or the frequency of administration of the agent in a dosage regimen is reduced compared to the dosage of the agent or its frequency in a method in which the patient is treated with the agent after CRS or grade 2 neurotoxicity or higher has developed or been diagnosed, e.g. , after severe CRS, eg, grade 3 or higher, or after severe neurotoxicity, eg, grade 3 or higher (eg, after physical signs or symptoms of CRS or grade 3 or higher neurotoxicity). In some embodiments, the dosage of the agent or the frequency of administration of the agent in the dosage regimen is reduced compared to the dosage of the agent or its frequency in the method in which the patient is treated for CRS or neurotoxicity after more than 3 days, 4 days, 5 days, 6 days, 1 week , 2 weeks three weeks or more after the introduction of cell therapy and/or immunotherapy. In some embodiments, the dosage is reduced by more than or more than about 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- multiple, 9-fold, 10-fold or more. In some embodiments, the dosage is reduced by more than or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In some embodiments, the frequency of dosing is reduced, for example, the number of daily doses is reduced or the number of days of dosing is reduced.

A. Steroid

[0473] In some embodiments, the agent, e.g., a toxicity-targeting agent, that treats and/or that prevents, delays, or reduces the development or risk of developing toxicity to immunotherapy and/or cell therapy, is a steroid, e.g., a corticosteroid. Corticosteroids typically include glucocorticoids and mineralocorticoids.

[0474] Any corticosteroid, such as a glucocorticoid, can be used in the methods of the invention. In some embodiments, glucocorticoids include synthetic and non-synthetic glucocorticoids. Examples of glucocorticoids include, but are not limited to: alklomethasone, algestone, beclomethasone (eg, beclomethasone dipropionate), betamethasone (eg, betamethasone 17-valerate, betamethasone sodium acetate, betamethasone sodium phosphate, betamethasone valerate), budesonide, clobetasoles (eg, clobetasol propionate ), clobetasones, clocortolones (eg, clocortolone pivalate), cloprednols, corticosterones, cortisones and hydrocortisones (eg, hydrocortisone acetate), cortivasols, deflazacorts, desonides, deoxymethasones, dexamethasone (eg, dexamethasone 21-phosphate, dexamethasone acetate, dexamethasone sodium phosphate) , diflorazones (eg, diflorazone diacetate), diflucortolones, difluprednates, enoxolones, fluazocorts, flucloronides, fludrocortisones (eg, fludrocortisone acetate), flumethasones (eg, flumethasone pivalate), flunisolides, fluocinolones (eg, fluocinolone acetonide), fluocinonides, fluocortins , fluorocortolones , fluorometholones (eg, fluorometholone acetate), fluperolones (eg, fluperolone acetate), fluprednidenes, fluprednisolones, flurandrenolides, fluticasones (eg, fluticasone propionate), formocortals, halcinonides, halobetasoles, halomethasone, halopredones, hydrocortamates, hydrocortisones (eg, hydrocortisones zones 21- butyrate, hydrocortisone aceponate, hydrocortisone acetate, hydrocortisone buteprate, hydrocortisone butyrate, hydrocortisone cypionate, hydrocortisone hemisuccinate, hydrocortisone probutate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, hydrocortisone valerate), loteprednol etabonate, mazipredones, medrisone, meprednisone, methylprednisolones (methylprednisolone aceponate, methylprednisolone acetate, methylprednisolone hemisuccinate, methylprednisolone sodium succinate), mometasone (eg, mometasone furoate), paramethasone (eg, paramethasone acetate), prednisolone, prednisolone (eg, prednisolone 25-diethylaminoacetate, prednisolone sodium phosphate, prednisolone 21-hemisuccinate, prednisolone acetate; prednisolone farnesylate, prednisolone hemisuccinate, prednisolone-21(beta-D-glucuronide), prednisolone metasulfobenzoate, prednisolone steaglate, prednisolone tebutate, prednisolone tetrahydrophthalate), prednisone, prednivals, prednilidenes, rimexolones, thixocortols, triamcinolones (e.g. triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide, triamcinolone acetonide 21-palmitate, triamcinolone diacetate). These glucocorticoids and their salts are discussed in detail, for example, in Remington's Pharmaceutical Sciences, A. Osol, ed., Mack Pub. Co., Easton, Pa. (16th ed. 1980).

[0475] In some examples, the glucocorticoid is selected from cortisones, dexamethasone, hydrocortisones, methylprednisolones, prednisolones, and prednisone. In a particular example, the glucocorticoid is dexamethasone.

[0476] In some embodiments, the agent is a corticosteroid and is administered in an amount that is therapeutically effective to treat, alleviate, or reduce one or more symptoms of toxicity in response to immunotherapy and/or cell therapy, such as CRS or neurotoxicity. In some embodiments, indicators of improvement or treatment success include determining whether an appropriate toxicity score (e.g., CRS or neurotoxicity score) is not present, such as a score of less than 3, or a change in score or severity on the score as discussed here, such as a change from a score of 4 to a score of 3, or a change from a score of 4 to a score of 2, 1, or 0.

[0477] In some aspects, the corticosteroid is administered at a therapeutically effective dose. A therapeutically effective concentration can be empirically determined by testing in known in vitro or in vivo systems (eg, animal models). For example, the amount of a selected corticosteroid that is administered to alleviate the symptoms or adverse effects of toxicity in response to immunotherapy and/or cell therapy, such as CRS or neurotoxicity, can be determined by standard clinical methods. In addition, animal models can be used to help identify optimal dosage ranges. The precise dosage, which may be empirically determined, may depend on the particular therapeutic agent, dosage regimen and regimen, route of administration, and severity of disease.

[0478] The corticosteroid may be administered in any amount that is effective in alleviating one or more symptoms associated with toxicity, such as CRS or neurotoxicity. A corticosteroid, e.g., a glucocorticoid, may be administered, for example, in an amount of from or about 0.1 to 100 mg, per dose, from 0.1 to 80 mg, from 0.1 to 60 mg, from 0.1 to 40 mg, 0.1 to 30 mg, 0.1 to 20 mg, 0.1 to 15 mg, 0.1 to 10 mg, 0.1 to 5 mg, 0.2 to 40 mg, 0.2 to 30 mg, 0.2 to 20 mg, 0.2 to 15 mg, 0.2 to 10 mg, 0.2 to 5 mg, 0.4 to 40 mg, 0 .4 to 30 mg, 0.4 to 20 mg, 0.4 to 15 mg, 0.4 to 10 mg, 0.4 to 5 mg, 0.4 to 4 mg, 1 to 20 mg, 1 to 15 mg or 1 to 10 mg per 70 kg adult. Typically, a corticosteroid, such as a glucocorticoid, is administered in an amount of from or about 0.4 to 20 mg, such as or about 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg , 13mg, 14mg, 15mg, 16mg, 17mg, 18mg, 19mg or 20mg per dose, average adult.

[0479] In some embodiments, the corticosteroid may be administered, for example, at a dosage of, or approximately, 0.001 mg/kg (patient), 0.002 mg/kg, 0.003 mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg, 0.008 mg/kg, 0.009 mg/kg, 0.01 mg/kg, 0.015 mg/kg, 0.02 mg/kg, 0.025 mg/kg, 0.03 mg/kg, 0.035 mg/kg, 0.04 mg/kg, 0.045 mg/kg, 0.05 mg/kg, 0.055 mg/kg, 0.06 mg/kg, 0.065 mg/kg, 0.07 mg/kg, 0.075 mg /kg, 0.08 mg/kg, 0.085 mg/kg, 0.09 mg/kg, 0.095 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0, 25 mg/kg, 0.30 mg/kg, 0.35 mg/kg, 0.40 mg/kg, 0.45 mg/kg, 0.50 mg/kg, 0.55 mg/kg, 0.60 mg/kg, 0.65 mg/kg, 0.70 mg/kg, 0.75 mg/kg, 0.80 mg/kg, 0.85 mg/kg, 0.90 mg/kg, 0.95 mg /kg, 1 mg/kg, 1.05 mg/kg, 1.1 mg/kg, 1.15 mg/kg, 1.20 mg/kg, 1.25 mg/kg, 1.3 mg/kg, 1.35 mg/kg or 1.4 mg/kg, for an average adult, typically weighing between about 70 kg and 75 kg.

[0480] A corticosteroid or glucocorticoid, such as dexamethasone, may be administered orally (tablets, liquid, or liquid concentrate), PO, intravenously (IV), intramuscularly, or by any other route known or described herein (eg, with respect to pharmaceutical formulations). In some aspects, the corticosteroid is administered as a bolus, and in other aspects it may be administered over a period of time.

[0481] In some aspects, the glucocorticoid may be administered over a period of more than one day, such as more than two days, more than 3 days, or more than 4 or more days. In some embodiments, the corticosteroid may be administered once a day, twice a day, or three or more times a day. For example, a corticosteroid, such as dexamethasone, may, in some examples, be administered at 10 mg (or equivalent) IV twice daily for three days.

[0482] In some embodiments, a dosage of a corticosteroid, such as a glucocorticoid, is administered at successively lower dosages during treatment. Consequently, in some of these regimens, the dose of corticosteroid is tapered. For example, the corticosteroid may be administered at an initial dose (or equivalent dose, e.g., in terms of dexamethasone) of 4 mg, and at each subsequent administration the dose may be reduced so that the dose is 3 mg for the next administration, 2 mg for the next administration and 1 mg for the next injection

[0483] Usually, the dose of corticosteroid administered depends on the particular corticosteroid, since there is a difference in activity between different corticosteroids. It is generally understood that preparations vary in potency and that dosages may therefore be varied to obtain equivalent effects. Table 6 shows equivalence in terms of potency for various glucocorticoids and routes of administration. Equivalent activity at clinical dosage is known. Information regarding equivalent dosing of steroids (in a non-chronotherapeutic manner) can be found in the British National Formulary (BNF) 37, March 1999.

Table 6: Administration of glucocorticoids Glucocorticoid (path) Activity equivalent Hydrocortisone (IV or PO) 20 Prednisone 5 Prednisolone (IV or PO) 5 Methylprednisolone sodium succinate (IV) 4 Dexamethasone (IV or PO) 0.5-0.75

[0484] Thus, in some embodiments, the steroid is administered in an equivalent dosage amount of from or about 1.0 mg to 20 mg of dexamethasone per day, for example, from 1.0 mg to 15 mg of dexamethasone per day, from 1.0 mg to dexamethasone at 10 mg per day, from 2.0 mg to 8 mg of dexamethasone per day, or from 2.0 mg to 6.0 mg of dexamethasone per day, inclusive. In some cases, the steroid is administered at an equivalent dose of 4 mg or about dexamethasone or about 8 mg per day.

[0485] In some embodiments, the implementation of the steroid is administered if the temperature increase persists after treatment with tocilizumab. For example, in some embodiments, dexamethasone is administered orally or intravenously at a dosage of 5-10 mg up to every 6-12 hours with prolonged fever. In some embodiments, tocilizumab is administered simultaneously with or after the addition of oxygen.

B. Microglial cell inhibitor

[0486] In some embodiments, the implementation of the inhibitor in combination therapy is an inhibitor of microglial cell activity. In some embodiments, administration of an inhibitor modulates microglial activity. In some embodiments, the implementation of the inhibitor is an antagonist that inhibits the activity of the signaling pathway in microglia. In some embodiments, the microglial inhibitor affects microglial homeostasis, survival, and/or proliferation. In some embodiments, the inhibitor targets the CSF1R signaling pathway. In some embodiments, the inhibitor is a CSF1R inhibitor. In some embodiments, the implementation of the inhibitor is a small molecule. In some cases, the inhibitor is an antibody.

[0487] In some aspects, administration of an inhibitor results in one or more of effects selected from alteration of microglial homeostasis and viability, reduction or blockade of microglial cell proliferation, reduction or elimination of microglial cells, reduction of microglial activation, reduction of microglial nitric oxide production, reduction of synthase activity nitric oxide in microglia or protection of motor neurons caused by microglial activation. In some embodiments, the agent modifies the serum or blood biomarker level of CSF1R inhibition, or a decrease in cross-linked N-telopeptide (NTX) urinary type 1 collagen, from a time point prior to initiation of inhibitor administration. In some embodiments, the administration of the agent temporarily inhibits the activity of microglial activity and/or the inhibition of microglial activity is not permanent. In some embodiments, administration of the agent temporarily inhibits CSF1R activity and/or the inhibition of CSF1R activity is not permanent.

[0488] In some embodiments, the microglial cell depleting agent is a small molecule, peptide, protein, antibody, or antigen-binding fragment thereof, antibody mimetic, aptamer, or nucleic acid molecule. In some embodiments, the method includes administering an inhibitor of microglial activity. In some embodiments, the agent is an antagonist that inhibits signaling pathway activity in microglia. In some embodiments, an agent that reduces the activity of microglial cells affects microglial homeostasis, survival and/or proliferation.

[0489] In some embodiments, the agent that reduces microglial cell activation is selected from an anti-inflammatory agent, a NADPH oxidase (NOX2) inhibitor, a calcium channel blocker, a sodium channel blocker, inhibits GM-CSF, inhibits CSF1R, specifically binds to CSF-1, specifically binds to IL-34, inhibits nuclear factor kappa-B (NF-κB) activation, activates the CB2 receptor and/or is a CB2 agonist, phosphodiesterase inhibitor, inhibits miRNA-155 ((miR-155), upregulates miRNA-124 (( miR-124), inhibits nitric oxide production in microglia, inhibits nitric oxide synthase, or activates the transcription factor NRF2 (also called similar to nuclear factor (derived from erythroid-2)-, or NFE2L2).

[0490] In some embodiments, the microglial cell depleting agent targets CSF1 (also called macrophage colony stimulating factor MCSF). In some embodiments, an agent that reduces the activity of microglial cells affects MCSF-stimulated phosphorylation of the M-CSF receptor (Pryer et al. Proc Am Assoc Cancer Res, AACR Abstract nr DDT02-2 (2009)). In some cases, the agent that reduces the activity of microglial cells is MCS110 (international patent application publication number WO2014001802; Clinical Trial Study Record Nos.: A1 NCT00757757; NCT02807844; NCT02435680; NCT01643850).

[0491] In some embodiments, the implementation of the agent that reduces the activity of microglial cells is a small molecule that targets the CSF1 pathway. In some embodiments, the agent is a small molecule that binds to CSF1R. In some embodiments, the agent is a small molecule that inhibits CSF1R kinase activity by competing with ATP that binds to CSF1R kinase. In some embodiments, the agent is a small molecule that inhibits the activation of the CFS1R receptor. In some cases, the binding of CSF-1 ligand to CSF1R is inhibited. In some embodiments, the microglial cell depleting agent is any of the inhibitors described in US Patent Application Publication No. US20160032248.

[0492] In some embodiments, the agent is a small molecule inhibitor selected from PLX-3397, PLX7486, JNJ-40346527, JNJ28312141, ARRY-382, PLX73086 (AC-708), DCC-3014, AZD6495, GW2580, Ki20227 , BLZ945, PLX647, PLX5622. In some embodiments, the agent is any of the inhibitors described in Conway et al.,Proc Natl Acad Sci USA, 102(44):16078-83 (2005); Dagher et al.Journal of Neuroinflammation, 12:139 (2015); Ohno et al.Mol Cancer Ther. 5(11):2634-43 (2006); von Tresckow et al.Clin Cancer Res.,21(8) (2015); Manthey et al.Mol Cancer Ther. (8(11):3151-61 (2009); Pyonteck et al.,Nat Med. 19(10): 1264-1272 (2013); Haegel et al.,Cancer ResAACR Abstract nr 288 (2015); Smith et al., Cancer Res AACR Abstract nr 4889 (2016); Clinical Trial Study Record No.: NCT01525602; NCT02734433; NCT02777710; NCT01804530; NCT01597739; NCT01572519; NCT01054014; NCT01316822; NCT02880371; NCT02673736; International Patent Application Publication Numbers WO2008063888A2, WO2006009755A2, US Patent Application Publication Numbers US20110044998, US 2014/0065141 and US 2015/0119267.

[0493] In some embodiments, the microglial cell depleting agent is 4-((2-(((1R,2R)-2-hydroxycyclohexyl)amino)benzo[d]thiazol-6-yl)oxy)-N -methylpicolinamide (BLZ945) or a pharmaceutically acceptable salt or derivatives thereof. In some embodiments, the agent is the following compound:

in which R ¹ denotes alkylpyrazole or alkylcarboxamide, and R ² denotes hydroxycycloalkyl

or a pharmaceutically acceptable salt thereof.

[0494] In some embodiments, the microglial cell depleting agent is 5-((5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl)-N-((6-( trifluoromethyl)pyridin-3-yl)methyl)pyridin-2-amine, N-[5-[(5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl]-2-pyridinyl] -6-(trifluoromethyl)-3-pyridinemethanamine) (PLX 3397) or a pharmaceutically acceptable salt or derivatives thereof. In some embodiments, the agent is 5-(1H-pyrrolo[2,3-b]pyridin-3-ylmethyl)-N-[[4-(trifluoromethyl)phenyl]methyl]-2-pyridinamine dihydrochloride (PLX647) or its pharmaceutically acceptable salt or derivatives. In some embodiments, the implementation of the agent that reduces the activity of microglial cells is the following compound:

or a pharmaceutically acceptable salt thereof. In some embodiments, the implementation of the agent that reduces the activity of microglial cells is the following compound:

or a pharmaceutically acceptable salt thereof. In some embodiments, the agent is any of the inhibitors described in US Pat. No. US7893075.

[0495] In some embodiments, the microglial cell depleting agent is 4-cyano-N-[2-(1-cyclohexen-1-yl)-4-[1-[(dimethylamino)acetyl]-4-piperidinyl ]phenyl]-1H-imidazole-2-carboxamide hydrochloride (JNJ28312141) or a pharmaceutically acceptable salt or derivatives thereof. In some embodiments, the agent is the following compound:

or a pharmaceutically acceptable salt thereof. In some embodiments, the agent is any of the inhibitors described in US Pat. No. US7645755.

[0496] In some embodiments, the microglial cell depleting agent is 1H-imidazole-2-carboxamide, N-(2-(4,4-dimethylcyclohex-1-enyl)-6-(2,2,6, 6-tetramethyltetrahydropyran-4-yl)pyridin-3-yl)amide 5-cyano-N-(2-(4,4-dimethyl-1-cyclohexen-1-yl)-6-(tetrahydro-2,2,6 ,6-Tetramethyl-2H-pyran-4-yl)-3-pyridinyl)-4-cyano-1H-imidazole-2-carboxylic acid, 4-Cyano-N-(2-(4,4-dimethylcyclohex-1- en-1-yl)-6-(2,2,6,6-tetramethyl-tetrahydro-2H-pyran-4-yl)pyridin-3-yl)-1H-imidazole-2-carboxamide (JNJ-40346527) or their pharmaceutically acceptable salt or derivatives. In some embodiments, the agent is the following compound:

or a pharmaceutically acceptable salt thereof.

[0497] In another embodiment, the microglial cell depleting agent is 5-(3-Methoxy-4-(4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine (GW2580) or a pharmaceutically acceptable salt thereof, or derivatives. In some embodiments, the agent is the following compound:

or a pharmaceutically acceptable salt thereof (International Patent Application Publication No. WO2009099553).

[0498] In some embodiments, the agent that reduces the activity of microglial cells is 4-(2,4-difluoroaniline)-7-ethoxy-6-(4-methylpiperazin-1-yl)quinoline-3-carboxamide (AZD6495) or its pharmaceutically acceptable salt or derivatives. In some embodiments, the agent is the following compound:

or a pharmaceutically acceptable salt thereof.

[0499] In some embodiments, the microglial cell depleting agent is N-{4-[(6,7-dimethoxy-4-quinolyl)oxy]-2-methoxyphenyl}-N0-[1-(1,3 -thiazol-2-yl)ethyl]urea (Ki20227) or a pharmaceutically acceptable salt or derivatives thereof. In some embodiments, the agent is the following compound:

or a pharmaceutically acceptable salt thereof.

[0500] In some embodiments, the agent that reduces microglial cell activation is an antibody that targets the CSF1 pathway. In some embodiments, the agent is an antibody that binds to CSF1R. In some embodiments, the anti-CSF1R antibody blocks the dimerization of CSF1R. In some embodiments, the anti-CSF1R antibody blocks the inner surface of CSF1R dimerization formed by the D4 and D5 regions (Ries et al. Cancer Cell 25(6):846-59 (2014)). In some cases, the agent is selected from emactuzumab (RG7155; RO5509554), cabiralizumab (FPA 008), LY-3022855 (IMC-CS4), AMG-820, TG-3003, MCS110, H27K15, 12-2D6, 2-4A5 (Rovida and Sbarba, J Clin Cell Immunol,6 :6 (2015); 01444404; NCT02713529; NCT00757757; NCT02807844; NCT02435680; NCT01643850).

[0501] In some embodiments, the microglial cell activation reducing agent is a tetracycline antibiotic. For example, the agent affects the concentration of IL-1b, IL-6, TNF-α, or iNOS in microglial cells (Yrjänheikki et al. PNAS 95(26): 15769-15774 (1998); Clinical Trial Study Record No: NCT01120899). In some embodiments, the agent is an opioid antagonist (Younger et al. Pain Med . 10(4):663-672 (2009). In some embodiments, the agent reduces glutamatergic nerve impulse transmission (U.S. Patent Number 5,527,814). In some embodiments, the agent modulates NFkB signaling pathway (Valera et al J. Neuroinflammation 12:93 (2015); Clinical Trial Study Record No: NCT00231140). In some embodiments, the agent targets cannabinoid receptors (Ramírez et al. J. Neurosci 25(8):1904 -13(2005)).In some embodiments, the agent is selected from minocycline, naloxone, riluzole, lenalidomide, and a cannabinoid (optionally WIN55 or 212-2).

[0502] It is believed that the production of nitric oxide in microglia in some cases leads to neurotoxicity or increases it. In some embodiments, the agent modulates or inhibits nitric oxide production in microglia. In some embodiments, the agent inhibits nitric oxide synthase (NOS). In some embodiments, the NOS inhibitor is ronopterin (VAS 203), also known as 4-amino-tetrahydrobiopterin (4-ABH4). In some embodiments, the NOS inhibitor is cindunistat, A-84643, ONO-1714, L-NOARG, NCX-456, VAS 2381, GW-273629, NXN-462, CKD-712, KD-7040, or guanidinoethyl disulfide. In some embodiments, the agent is any of the inhibitors described in Höing et al., Cell Stem Cell. 2012 Nov 2;11(5):620-32.

[0503] In some embodiments, the agent blocks T cell traffic, such as to the central nervous system. In some embodiments, blocking T cell traffic can reduce or eliminate the passage of immune cells through blood vessel walls into the central nervous system, including crossing the blood-brain barrier. In some cases, activated antigen-specific T cells produce pro-inflammatory cytokines, including IFN-γ and TNF, upon reactivation in the CNS, leading to the activation of resident cells such as microglia and astrocytes. See Kivisäkk et al., Neurology. 2009 Jun 2; 72(22): 1922-1930. Thus, in some embodiments, isolating activated T cells from microglial cells, for example by blocking the traffic and/or inhibiting the ability of such cells to cross the blood-brain barrier, may reduce or eliminate microglial activation. In some embodiments, the agent inhibits adhesion molecules on immune cells, including T cells. In some embodiments, the agent inhibits an integrin. In some embodiments, the implementation of the integrin is alpha-4 integrins. In some embodiments, the agent is natalizumab (Tysabri®). In some embodiments, the agent modulates a cell surface receptor. In some embodiments, the agent modulates a sphingosine-1-phosphate (S1P) receptor, such as S1PR1 or S1PR ⁵ . In some embodiments, the agent causes the internalization of a cellular receptor, such as the sphingosine-1-phosphate (S1P) receptor, such as S1PR1 or S1PR ⁵ . In some embodiments, the agent is fingolimod (Gilenya®) or ozanimod (RPC-1063).

[0504] The transcription factor NRF2 is believed to regulate the antioxidant response, for example, by turning on genes containing a cis-acting element in their promoter region. An example of such an element includes an antioxidant response element (ARE). In some embodiments, the agent activates NRF2. In some embodiments, activation of NRF2 in microglial cells reduces the reactivity of microglial cells in response to IFN and LPS. In some embodiments, activation of NRF2 inhibition slows or reduces demyelination, axonal loss, neuronal death, and/or oligodendrocyte death. In some embodiments, the agent upregulates a cellular cytoprotective pathway regulated by NRF2. In some embodiments, the NRF2 activating agent is dimethyl fumarate (Tecfidera®). In some embodiments, the agent is any of the inhibitors described in US Pat. No. 8,399,514. In some embodiments, the agent is any of the inhibitors described in Höing et al., Cell Stem Cell. 2012 Nov 2;11(5):620-32.

[0505] In some embodiments, the agent that reduces microglial cell activation is (4S,4aS,5aR,12aS)-4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo -1,4,4a,5,5a,6,11,12a-octahydrotetracen-2-carboxamide (Minocycline) or a pharmaceutically acceptable salt or derivatives thereof. In some embodiments, the agent is any of the compounds described in US Patent Application Publication Number US20100190755. In some embodiments, the agent is the following compound:

or a pharmaceutically acceptable salt thereof.

[0506] In some embodiments, the agent that reduces microglial cell activation is 3-(7-amino-3-oxo-1H-isoindol-2-yl)piperidine-2,6-dione (lenalidomide) or a pharmaceutically acceptable salt thereof or derivatives. In some embodiments, the agent is the following compound:

or a pharmaceutically acceptable salt thereof.

[0507] In some embodiments, the microglial cell activation reducing agent is (4R,4aS,7aR,12bS)-4a,9-dihydroxy-3-prop-2-enyl-2,4,5,6,7a, 13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7-one (naloxone) or a pharmaceutically acceptable salt or derivatives thereof. In some embodiments, the agent is any of the compounds described in US Pat. No. US8247425. In some embodiments, the agent is the following compound:

or a pharmaceutically acceptable salt thereof.

[0508] In some embodiments, the agent that reduces microglial cell activation is 2-amino-6-(trifluoromethoxy)benzothiazole, 6-(trifluoromethoxy)benzo[d]thiazol-2-amine, or 6-(trifluoromethoxy)-1, 3-benzothiazol-2-amine (riluzole), or a pharmaceutically acceptable salt or derivative thereof, as described in US Pat. No. US5527814. In some embodiments, the agent is the following compound:

or a pharmaceutically acceptable salt thereof.

[0509] In some embodiments, the implementation of the agent that reduces the activation of microglial cells is a signaling pathway modulator in microglia. In some cases, the agent reduces the microglial signaling pathway. In some embodiments, the agent is a GM-CSF (CSF2) inhibitor. In other embodiments, the agent that reduces microglial cell activation is an ion channel blocker. In certain specific embodiments, the agent is a calcium channel blocker. For example, in some specific examples, the agent is a dihydropyridine calcium channel blocker. In some embodiments, the agent is a miRNA inhibitor. For example, an agent targets miR-155. In some embodiments, the agent that reduces microglial cell activation is selected from MOR103, Nimodipine, IVIg, and LNA-anti-miR-155 (Butoxsky et al.Ann Neurol., 77(1):75-99 (2015) and Sanz et al.,Br J Pharmacol. 167(8): 1702-1711 (2012); Winter et al.,Ann Clin and Transl Neurol.2328-9503 (2016); Clinical Trial Study Record Nos.: NCT01517282, NCT00750867).

[0510] In some embodiments, the agent that reduces microglial cell activation is 3-(2-methoxyethyl)5-propan-2-yl-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine -3,5-dicarboxylate (nimodipine) or a pharmaceutically acceptable salt or derivatives thereof. In some embodiments, the agent is any of the inhibitors described in US Pat. No. US3,799,934. In some embodiments, the agent is the following compound:

or a pharmaceutically acceptable salt thereof.

[0511] In some cases, the agent that reduces the activation of microglial cells is administered in a form that affects only the central nervous system and/or does not affect tumor-associated macrophages. In some embodiments, the agent promotes an inactive state of microglia but does not eliminate or reduce the amount of microglia. In some embodiments, the method includes inhibiting microglial activity specifically in the brain, such as that described in Ponomarev et al., Nature Medicine , (1):64-70 (2011).

[0512] Examples of substances that reduce the activation of microglial cells, and examples of dosing regimens for the introduction of such agents, are presented in Table 7 below.

Table 7. Examples of microglial inhibitors and dosing regimens Inhibitor Example Molecule type Target(s) molecules Example of dosing regimen(s) Pexidartinib (PLX3397) small molecule CSF1R; c-kit; FLT3 200 mg tablets, twice a day for 28 days; Administer daily as a split dose regimen, five dose levels possible in terms of dose escalation: 400 mg 5 days with 2 days off (fluctuating schedule), 400 mg, 600 mg, 800 mg, or 1000 mg; 1000 mg/day for 2 weeks, then 800 mg/day for 22 weeks Emactuzumab (RG1755; RO5509554) monoclonal antibody CSF1R 100-3000 mg once every 2 weeks Caberilizumab (FPA-008) antibody CSF1R IV infusion over 30 minutes every 2 weeks LY-3022855
(IMC-CS4) monoclonal antibody CSF1R IV delivery 1.25 mg/kg every 2 weeks for 6 weeks JNJ-40346527 small molecule CSF1R 100 mg twice a day for 12 weeks; 100-1000 mg capsule daily MCS110 antibody MCSF (CSF1) Up to 4 doses of MCS110 at 10 mg/kg IV once every 4 weeks starting on Day 1 MOR103 antibody GM-CSF 6 doses 0.5-2.0 mg/kg for 70 days IVIg immunoglobulin unknown IV infusion 0.4 g/kg every month for 6 months Minocilin small molecule broad spectrum antibiotic: IL-1b; IL-6, TNF-a; iNOS Oral dose of minocycline 100 mg twice daily for 24 months Naloxone small molecule Opioid receptors Naltrexone hydrochloride 4.5 mg capsules once/day for 8 weeks Lenalidomide / thalidomide small molecule NFkB signaling pathway 100-400 mg per day Riluzole small molecule Release of glutamate by microglia 50 mg twice a day Cannabinoids/Cannabidiol
(for example, WIN55,212-2) small molecule Cannabinoid receptors Orally 10 mg/kg/day for 6 weeks (average 700 mg/day) Dimethyl fumarate (Tecfidera®). small molecule Nrf2 signaling pathway Initial dose of 120 mg taken orally twice a day for 7 days. The dose is increased to 240 mg taken orally twice a day, thereafter natalizumab (Tysabri®) antibody alpha-4 integrin 300 mg IV infused over one hour every four weeks fingolimod (Gilenya®) small molecule S1P receptors, including S1PR1 0.5 mg orally daily ozanimod (RPC-1063) small molecule S1PR1 and S1PR ⁵ 0.25 mg, 0.5 mg, or 1 mg once a day

C. Other Agents (eg, cytokine targeting agents)

[0513] In some embodiments, the agent, e.g., a toxicity-targeting agent that treats or alleviates the symptoms of immunotherapy and/or cell therapy toxicity, such as CRS or neurotoxicity, is one that targets a cytokine, e.g., is an antagonist or inhibitor cytokines such as transforming growth factor beta (TGF-beta), interleukin 6 (IL-6), interleukin 10 (IL-10), IL-2, MIP1β (CCL4), TNF-alpha, IL-1, interferon (IFN -gamma) or monocytic chemoattractant protein-1 (MCP-1). In some embodiments, the agent that treats or alleviates the symptoms of immunotherapy and/or cell therapy toxicity, such as CRS or neurotoxicity, is one that targets (e.g., inhibits or antagonizes) a cytokine receptor, such as the IL-6 receptor (IL -6R), IL-2 receptor (IL-2R/CD25), MCP-1 (CCL2) receptor (CCR2 or CCR4), TGF-beta receptor (TGF-beta I, II, or III), IFN-gamma receptor ( IFNGR), MIP1β receptor (eg CCR5), TNF-alpha receptor (eg TNFR1), IL-1 receptor (IL1-Rα/IL-1Rβ) or IL-10 receptor (IL-10R).

[0514] The amount of a selected agent that treats or alleviates the symptoms of immunotherapy and/or cell therapy toxicity, such as CRS or neurotoxicity, which is administered to alleviate the symptoms or adverse effects of toxicity in response to immunotherapy and/or cell therapy, such as CRS or neurotoxicity , can be determined by standard clinical methods. Examples of adverse events include, but are not limited to, an increase in alanine aminotransferase, an increase in aspartate aminotransferase, chills, febrile neutropenia, headache, hypotension, left ventricular dysfunction, encephalopathy, hydrocephalus, convulsions, and/or tremor.

[0515] In some embodiments, the agent is administered in a dosage amount of from or about 30 mg to 5000 mg, e.g., 50 mg to 1000 mg, 50 mg to 500 mg, 50 mg to 200 mg, 50 mg to 100 mg. mg, 100 mg to 1000 mg, 100 mg to 500 mg, 100 mg to 200 mg, 200 mg to 1000 mg, 200 mg to 500 mg, or 500 mg to 1000 mg.

[0516] In some embodiments, the agent is administered from or from about 0.5 mg/kg to 100 mg/kg, for example, from or from about 1 mg/kg to 50 mg/kg, from 1 mg/kg to 25 mg/kg. kg, 1 mg/kg to 10 mg/kg, 1 mg/kg to 5 mg/kg, 5 mg/kg to 100 mg/kg, 5 mg/kg to 50 mg/kg, 5 mg/kg kg to 25 mg/kg, 5 mg/kg to 10 mg/kg, 10 mg/kg to 100 mg/kg, 10 mg/kg to 50 mg/kg, 10 mg/kg to 25 mg/kg , 25 mg/kg to 100 mg/kg, 25 mg/kg to 50 mg/kg to 50 mg/kg to 100 mg/kg. In some embodiments, the agent is administered in a dosage amount of from or about 1 mg/kg to 10 mg/kg, 2 mg/kg to 8 mg/kg, 2 mg/kg to 6 mg/kg, 2 mg/kg up to 4 mg/kg or from 6 mg/kg to 8 mg/kg, inclusive. In some aspects, the agent is administered in a dosage amount of at least or at least about or about 1 mg/kg, 2 mg/kg, 4 mg/kg, 6 mg/kg, 8 mg/kg, 10 mg/kg or more. In some embodiments, the agent is administered at a dose of 4 mg/kg or 8 mg/kg.

[0517] In some embodiments, the agent is administered by injection, e.g., intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjunctival injection, subconjunctival injection, subtenonic injection , retrobulbar injection, peribulbar injection, or posterior parascleral delivery. In some embodiments, it is administered by parenteral, intrapulmonary, and intranasal routes, and, if topical treatment is desired, intralesional administration. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal or subcutaneous administration.

[0518] In some embodiments, the amount of agent is administered about or about twice a day, once a day, every other day, three times a week, once a week, once every two weeks, or once a month.

[0519] In some embodiments, the agent is administered as part of a composition or formulation, such as a pharmaceutical composition or formulation, as described below. Thus, in some cases, the composition comprising the agent is administered as described below. In other aspects, the agent is administered individually and may be administered by any known acceptable route of administration or by the route described herein, such as those described in relation to compositions and pharmaceutical formulations.

[0520] In some embodiments, the agent that treats or alleviates symptoms of immunotherapy and/or cell therapy toxicity, such as CRS or neurotoxicity, is an antibody or antigen-binding fragment. In some embodiments, the agent is tocilizumab, siltuximab, sarilumab, olokizumab (CDP6038), elsilimoab, ALD518/BMS-945429, sirukumab (CNTO 136), CPSI-2634, ARGX-109, FE301, or FM101.

[0521] In some embodiments, the agent is an antagonist or inhibitor of IL-6 or IL-6 receptor (IL-6R). In some aspects, the agent is an antibody that neutralizes IL-6 activity, such as an antibody or antigen-binding fragment that binds to IL-6 or IL-6R. For example, in some embodiments, the agent is or includes tocilizumab (atlizumab) or sarilumab, an anti-IL-6R antibody. In some embodiments, the agent is an anti-IL-6R antibody described in US Patent number: 8,562,991. In some cases, the IL-6 targeting agent is an anti-IL-6 antibody such as siltuximab, elsilimomab, ALD518/BMS-945429, sirukumab (CNTO 136), CPSI-2634, ARGX-109, FE301, FM101, or olokizumab (CDP6038). In some aspects, the agent can neutralize IL-6 activity by inhibiting ligand-receptor interactions. The feasibility of this general type of approach has been demonstrated with a naturally occurring interleukin-1 receptor antagonist. See Harmurn, C. H. et al., Nature (1990) 343:336-340. In some aspects, the IL-6/IL-6R antagonist or inhibitor is an IL-6 mutein, such as that described in US Patent No. 5,591,827. In some embodiments, an agent that is an IL-6/IL-6R antagonist or inhibitor is a small molecule, protein, or peptide, or nucleic acid.

[0522] In some embodiments, the agent is tocilizumab. In some embodiments, tocilizumab is administered as an early intervention in accordance with the methods of the invention and/or using the articles or compositions of the invention, at a dosage of from or about 1 mg/kg to 12 mg/kg, for example, a component or about a component 4 mg/kg, 8 mg/kg or 10 mg/kg. In some embodiments, tocilizumab is administered by intravenous infusion. In some embodiments, the implementation of tocilizumab is administered in the event of persistent temperature rise to more than 39°C lasting 10 hours, which is resistant to acetaminophen. In some embodiments, a second administration of tocilizumab is given if symptoms recur 48 hours after the initial dose.

[0523] In some embodiments, the agent is a TGF-β or TGF-β receptor (eg, TGF-β receptor I, II, or III) agonist or stimulator. In some aspects, the agent is an antibody that increases TGF-β activity, such as an antibody or antigen-binding fragment that binds to TGF-β or one of its receptors. In some embodiments, an agent that is an agonist or stimulator of TGF-β and/or its receptor is a small molecule, protein or peptide or nucleic acid.

[0524] In some embodiments, the agent is an antagonist or inhibitor of MCP-1 (CCL2) or an MCP-1 receptor (eg, MCP-1 receptor CCR2 or CCR4). In some aspects, the agent is an antibody that neutralizes MCP-1 activity, such as an antibody or antigen-binding fragment that binds to MCP-1 or one of its receptors (CCR2 or CCR4). In some embodiments, the MCP-1 antagonist or inhibitor is as described in Gong et al. J Exp Med. 1997 Jul 7; 186(1): 131-137 or Shahrara et al. J Immunol 2008; 180:3447-3456. In some embodiments, an agent that is an antagonist or inhibitor of MCP-1 and/or its receptor (CCR2 or CCR4) is a small molecule, protein or peptide or nucleic acid.

[0525] In some embodiments, the agent is an antagonist or inhibitor of IFN-γ or IFN-γ receptor (IFNGR). In some aspects, the agent is an antibody that neutralizes IFN-γ activity, such as an antibody or antigen-binding fragment that binds to IFN-γ or its receptor (IFNGR). In some aspects, the IFN-gamma neutralizing antibody is any of those described in Dobber et al. Cell Immunol. 1995 Feb;160(2):185-92 or Ozmen et al. J Immunol. 1993 Apr 1;150(7):2698-705. In some embodiments, an agent that is an antagonist or inhibitor of IFN-γ/IFNGR is a small molecule, protein or peptide or nucleic acid.

[0526] In some embodiments, the agent is an antagonist or inhibitor of IL-10 or IL-10 receptor (IL-10R). In some aspects, the agent is an antibody that neutralizes IL-10 activity, such as an antibody or antigen-binding fragment that binds to IL-10 or IL-10R. In some aspects, the IL-10 neutralizing antibody is any of those described in Dobber et al. Cell Immunol. 1995 Feb;160(2):185-92 or Hunter et al. J Immunol. 2005 Jun 1;174(11):7368-75. In some embodiments, an agent that is an IL-10/IL-10R antagonist or inhibitor is a small molecule, protein, or peptide or nucleic acid.

[0527] In some embodiments, the agent is an antagonist or inhibitor of IL-1 or IL-1 receptor (IL-1R). In some aspects, the agent is an IL-1 receptor antagonist, which is a modified form of IL-1R, such as anakinra (see, for example, Fleischmann et al., (2006) Annals of the rheumatic diseases. 65(8):1006-12 ). In some aspects, the agent is an antibody that neutralizes IL-1 activity, such as an antibody or antigen-binding fragment that binds to IL-1 or IL-1R, such as canakinumab (see also EP 2277543). In some embodiments, an agent that is an IL-1/IL-1R antagonist or inhibitor is a small molecule, protein, or peptide or nucleic acid.

[0528] In some embodiments, the agent is a tumor necrosis factor (TNF) or tumor necrosis factor receptor (TNFR) antagonist or inhibitor. In some aspects, the agent is an antibody that blocks TNF activity, such as an antibody or antigen-binding fragment that binds to TNF, such as TNFα or its receptor (TNFR, eg, TNFRp55 or TNFRp75). In some aspects, the agent is selected from infliximab, adalimumab, certolizumab pegol, golimumab, and etanercept. In some embodiments, the agent that is a TNF/TNFR antagonist or inhibitor is a small molecule, protein, or peptide or nucleic acid. In some embodiments, the agent is a small molecule that affects TNF, such as lenalidomide (see, for example, Muller et al. (1999) Bioorganic & Medicinal Chemistry Letters. 9(11):1625).

[0529] In some embodiments, the agent is an antagonist or inhibitor of signal transduction through Janus kinase (JAK) and a two-element signal transduction cascade: a signal transducer and a transcriptional activator (STAT). JAK/STAT proteins are common components of cytokine signal transduction and the cytokine receptor. In some embodiments, an agent that is a JAK/STAT antagonist or inhibitor such as ruxolitinib (see e.g. Mesa et al. (2012) Nature Reviews Drug Discovery. 11(2):103-104), tofacitinib (also known as Xeljanz, Jakvinus tasocitinib and CP 690550), baricitinib (also known as LY-3009104, INCB 28050), filgotinib (G-146034, GLPG-0634), gandotinib (LY-2784544), lestaurtinib (CEP 701), momelotinib ( GS-0387, CYT-387), pacritinib (SB1518), and upadacitinib (ABT 494). In some embodiments, the agent is a small molecule, protein or peptide or nucleic acid.

[0530] In some embodiments, the agent is a kinase inhibitor. In some embodiments, the agent is a Bruton's tyrosine kinase (BTK) inhibitor. In some embodiments, the inhibitor is or includes ibrutinib or acalabrutinib (see, e.g., Barrett et al., ASH 58th Annual Meeting San Diego, CA December 3-6, 2016, Abstract 654; Ruella et al., ASH 58th Annual Meeting San Diego, CA December 3-6, 2016, Abstract 2159). In some embodiments, the agent is an inhibitor as described in U.S. Patent Nos. 7,514,444; 8,008,309; 8,476,284; 8,497,277; 8,697,711; 8,703,780; 8,735,403; 8,754,090; 8,754,091; 8,957,079; 8,999,999; 9,125,889; 9,181,257; or 9,296,753.

[0531] In some embodiments, a device, such as blood or plasma filtering absorbent resin technology, can be used to reduce cytokine levels. In some embodiments, the device used to reduce cytokine levels is a physical cytokine absorber, such as an extracorporeal cytokine absorber. In some embodiments, a physical cytokine absorber may be used to eliminate cytokines from the bloodstream ex vivo in an in vivo manner. In some embodiments, the substance is a porous polymer. In some embodiments, the agent is CytoSorb (see, for example, Basu et al. Indian J Crit Care Med. (2014) 18(12): 822-824).

III. RECOMBINANT ANTIGENIC RECEPTORS EXPRESSED BY CELLS

[0532] In some embodiments, cells for use in or administered in connection with the methods of the invention contain or are engineered to contain a genetically engineered receptor, for example, a genetically engineered antigen receptor, such as a chimeric antigen receptor (CAR) or T- cell receptor (TCR). The invention also relates to a population of such cells, compositions containing such cells and/or enriched with such cells, such as in which cells of a certain type, such as T cells or CD8 ⁺ or CD4 ⁺ cells, are enriched or selected. Among the compositions may be mentioned pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. The invention also relates to therapeutic methods for administering cells and compositions to individuals, eg patients, in accordance with the methods of the invention and/or using articles or compositions of the invention.

[0533] In some embodiments, cells include one or more genetically engineered nucleic acids and thus express the recombinant gene or genetically engineered products of such nucleic acids. In some embodiments, gene transfer is performed by first stimulating the cells, for example by combining them with a stimulus that elicits a response such as proliferation, survival and/or activation, for example as measured by the expression of a cytokine or an activation marker, followed by transduction of the activated cells and growth into culture to numbers sufficient for clinical applications.

[0534] Cells typically express recombinant receptors, such as antigen receptors, including functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs) and other antigen-binding receptors, such as transgenic T cell receptors (TCRs). Other chimeric receptors may also be mentioned among the receptors.

A. Chimeric antigen receptors (CARs)

In some embodiments of the methods and uses of the invention, chimeric receptors, such as chimeric antigen receptors, comprise one or more domains comprising a ligand-binding domain (e.g., an antibody or antibody fragment) that provides specificity for a desired antigen (e.g., a tumor antigen), with intracellular signaling domains. In some embodiments, the intracellular signaling domain is a stimulatory or activating portion of an intracellular domain, such as a T cell stimulating or activating domain, providing a primary activation signal or primary signal. In some embodiments, the implementation of the intracellular signaling domain contains or additionally contains a co-stimulatory signaling domain to facilitate effector functions. In some embodiments, chimeric receptors, when genetically engineered into immune cells, can modulate T cell activity and, in some cases, can modulate T cell differentiation or homeostasis, thus leading to genetically engineered cells with improved lifespan. , survival and/or persistence in vivo, for example, for use in methods of adoptive cell therapy.

[0535] Examples of antigen receptors, including CARs, and methods for designing and introducing such receptors into cells include those described, for example, in International Patent Applications WO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/07115 4, WO2013 /123061, US Patent Publications US2002131960, US2013287748, US20130149337 , 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 EP2537416 and /or described by Sadelain et al., Cancer Discov. April 2013; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, antigen receptors include CARs as described in US Patent: 7,446,190 and described in International Patent Application Publication: WO/2014055668 A1. Examples of CARs include CAR as disclosed in any of the above publications such as WO2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, US Patent: 7,446,190, US Patent: 8,389,282, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, US Patent: 7,446,190 and US Patent: 8,389,282.

[0536] Chimeric receptors, such as CARs, typically include an extracellular antigen-binding domain, such as part of an antibody molecule, typically a heavy chain variable region (V _H ) and/or a light chain variable region (V _L ) of an antibody, e.g., an antibody scFv fragment.

[0537] In some embodiments, the antigen targeted by the receptor is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, an antigen is selectively expressed or overexpressed on cells of a disease or condition, such as a tumor or pathogenic cells, over normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or is expressed on genetically engineered cells.

[0538] In some embodiments, the antigen targeted by the receptor is or includes an αvβ6 integrin (avb6 integrin), B-cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as as CAIX or G250), testicular cancer antigen, cancer/testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, Chemokine Motif Ligand CC 1 (CCL -1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, proteoglycan 4 with chondroitin sulfates (CSPG4), epidermal growth factor protein (EGFR ), mutations in epidermal growth factor receptor type III (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin A2 receptor (EPHa2), estrogen receptor, Fc-like receptor 5 ( FCRL5; also known as the homologue of the Fc 5 receptor or FCRH5), embryonic acetylcholine receptor (embryonic AchR), folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 ( gp100), glypican-3 (GPC3), G protein-coupled receptor 5D (GPCR5D), Her2/neu (erb-B2 receptor tyrosine kinase), Her3 (erb-B3), HeR ⁴ (erb-B4), erbB dimers , human high molecular weight melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22Rα), alpha IL-13 receptor 2 (IL-13Rα2), kinase insert domain-containing receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, family 8 member A containing leucine-rich repeats (LRRC8A), Ley antigen, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, mouse cytomegalovirus (CMV), mucin 1 (MUC1) , MUC16, natural killer cell group 2 D member ligands (NKG2D), melan (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, preferentially expressed melanoma antigen (PRAME), progesterone receptor, prostate-specific antigen, stem cell antigen prostate cells (PSCA), prostate-specific membrane antigen (PSMA), orphan receptor tyrosine kinase-like 1 (ROR1), survivin, trophoblastic glycoprotein (TPBG, also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-dependent protein 1 ( TRP1, also known as TYRP1 or gp75), Tyrosinase-Dependent Protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2) , Wilms tumor 1 (WT-1), a pathogen-specific or pathogen-expressed antigen or an antigen associated with a universal label, and/or biotinylated molecules and/or molecules expressed by HIV, HCV, HBV or other pathogens. Receptor-targeted antigens, in some embodiments, include antigens associated with B-cell cancer, such as any of the many known B-cell markers. In some embodiments, the antigen targeted by the receptor is CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b, or CD30.

[0539] In some embodiments, the scFv and/or V _H domains are derived from FMC63. FMC63 generally refers to a mouse IgG1 monoclonal antibody produced against Nalm-1 and -16 cells expressing CD19 of human origin (Ling, NR, et al . (1987). Leucocyte typing III . 302). The FMC63 antibody includes CDRH1 and H2, represented in SEQ ID NOS: 38, 39, respectively, and CDRH3, presented in SEQ ID NOS: 40 or 54, and CDRL1, presented in SEQ ID NOS: 35 and CDR L2 36 or 55 and CDR L3 of sequence 37 or 56. The FMC63 antibody comprises a heavy chain variable region (V _H ) comprising the amino acid sequence of SEQ ID NO: 41 and a light chain variable region (V _L ) comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, implementation svFv includes a variable light chain containing a CDRL1 sequence of SEQ ID NO:35, a CDRL2 sequence of SEQ ID NO:36 and a CDRL3 sequence of SEQ ID NO:37 and/or a variable heavy chain containing a CDRH1 sequence of SEQ ID NO:38, a CDRH2 sequence of SEQ ID NO:39 and the sequence of CDRH3 SEQ ID NO:40. In some embodiments, the scFv comprises the FMC63 heavy chain variable region set forth in SEQ ID NO:41 and the FMC63 light chain variable region set forth in SEQ ID NO:42. In some embodiments, the variable heavy and variable light chains are linked by a linker. In some embodiments, the implementation of the linker is presented in SEQ ID NO:24. In some embodiments, the scFv includes, in that order, V _H , a linker, and V _L . In some embodiments, the scFv includes, in that order, V _L , a linker, and V _H . In some embodiments, the svFc is encoded by the nucleotide sequence shown in SEQ ID NO:25 or by a sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:25. In some embodiments, the scFv comprises the amino acid sequence shown in SEQ ID NO:43 or a sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:43.

[0540] In some embodiments, the scFv is derived from SJ25C1. SJ25C1 is a mouse IgG1 monoclonal antibody produced against Nalm-1 and -16 cells expressing CD19 of human origin (Ling, NR, et al . (1987). Leucocyte typing III . 302). The SJ25C1 antibody comprises CDRH1, H2 and H3 as shown in SEQ ID NOS: 47-49, respectively, and CDRL1, sequences L2 and L3 as shown in SEQ ID NOS: 44-46, respectively. The SJ25C1 antibody comprises a heavy chain variable region (V _H ) comprising the amino acid sequence of SEQ ID NO: 50 and a light chain variable region (V _L ) comprising the amino acid sequence of SEQ ID NO: 51. In some embodiments, the svFv comprises a variable light chain, containing a CDRL1 sequence of SEQ ID NO:44, a CDRL2 sequence of SEQ ID NO:45 and a CDRL3 sequence of SEQ ID NO:46 and/or a variable heavy chain containing a CDRH1 sequence of SEQ ID NO:47, a CDRH2 sequence of SEQ ID NO:48 and a CDRH3 sequence SEQ ID NO:49. In some embodiments, the scFv comprises the SJ25C1 heavy chain variable region set forth in SEQ ID NO:50 and the SJ25C1 light chain variable region set forth in SEQ ID NO:51. In some embodiments, the variable heavy and variable light chains are linked by a linker. In some embodiments, the implementation of the linker is presented in SEQ ID NO:52. In some embodiments, the scFv includes, in that order, V _H , a linker, and V _L . In some embodiments, the scFv includes, in that order, V _L , a linker, and V _H . In some embodiments, the scFv comprises the amino acid sequence shown in SEQ ID NO:53 or a sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:53.

[0541] In some embodiments, the chimeric antigen receptor includes an extracellular portion containing an antibody fragment or antibody. In some aspects, the chimeric antigen receptor includes an extracellular portion containing an antibody or fragment and an intracellular signaling domain. In some embodiments, the antibody or fragment comprises an scFv.

[0542] In some embodiments, the recombinant receptor antibody portion, e.g., CAR, further comprises at least a portion of an immunoglobulin constant region, such as a hinge region, e.g., an IgG4 hinge region, and/or an Fc and/or CH1/CL region. In some embodiments, the constant region or portion is derived from a human IgG, such as IgG4 or IgG1. In some aspects, a portion of the constant region serves as a spacer region between the antigen recognition component, eg, scFv, and the transmembrane domain. The spacer may be of a length that provides increased cell reactivity after antigen binding compared to no spacer. Examples of spacers include, but are not limited to, those described in Hudecek et al . (2013) Clin. Cancer Res ., 19:3153, International Patent Publication WO2014031687, U.S. Patent No. 8,822,647 or published application no. US2014/0271635.

[0543] In some embodiments, the constant region or portion is derived from a human IgG, such as IgG4 or IgG1. In some embodiments, the spacer has the sequence ESKYGPPCPPCP (represented in SEQ ID NO: 1) and is encoded by the sequence shown in SEQ ID NO: 2. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 3. In some embodiments, the implementation the spacer has the sequence shown in SEQ ID NO: 4. In some embodiments, the constant region or portion is derived from IgD. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 5. In some embodiments, the spacer has an amino acid sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any SEQ ID NOS: 1, 3, 4, or 5. In some embodiments, the spacer has a sequence represented by in SEQ ID NOS: 26-34. In some embodiments, the spacer has an amino acid sequence indicating at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or more sequence identity with any SEQ ID NOS: 26-34.

[0544] In some embodiments, the implementation of the antigen receptor includes an intracellular domain associated directly or indirectly with the extracellular domain. In some embodiments, the implementation of the chimeric antigen receptor includes a transmembrane domain that binds an extracellular domain and an intracellular signaling domain. In some embodiments, the implementation of the intracellular signaling domain includes ITAM. For example, in some aspects, an antigen recognition domain (e.g., an extracellular domain) is typically associated with one or more intracellular signaling components, such as signaling components that are mimics of activation through an antigen receptor complex, such as the TCR complex, in the case of a CAR and/or a signal through another cell surface receptor. In some embodiments, the chimeric receptor includes a transmembrane domain linked or fused between an extracellular domain (eg, scFv) and an intracellular signaling domain. Thus, in some embodiments, the antigen-binding component (eg, antibody) is associated with one or more transmembrane and intracellular signaling domains.

[0545] In one embodiment, a transmembrane domain is used, naturally associated with one of the domains in the receptor, for example, CAR. In some instances, a transmembrane domain is selected or modified by amino acid substitution to avoid binding such domains to transmembrane domains of the same or different surface membrane proteins to reduce interactions with other members of the receptor complex.

[0546] The transmembrane domain, in some embodiments, is derived from a natural or synthetic source. When the source is natural, the domain is in some aspects derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e., include at least the transmembrane region(s)), T cell receptor alpha, beta, or zeta chains, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the transmembrane domain, in some embodiments, is a synthetic product. In some aspects, the synthetic transmembrane domain includes predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine is found at each end of the synthetic transmembrane domain. In some embodiments, communication is via linkers, spacers, and/or transmembrane domain(s). In some aspects, the transmembrane domain contains the transmembrane portion of CD28.

[0547] In some embodiments, the extracellular domain and the transmembrane domain may be linked directly or indirectly. In some embodiments, the extracellular domain and the transmembrane domain are linked by a spacer, such as any described herein. In some embodiments, the receptor comprises an extracellular portion of the molecule from which the transmembrane domain is derived, such as the extracellular portion of CD28.

[0548] Intracellular signaling domains include those that are mimetics or approximators of a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, e.g., a linker 2 to 10 amino acids in length, such as a linker containing glycines and serines, e.g., a glycine-serine doublet, is present and forms a link between the transmembrane domain and the CAR cytoplasmic signaling domain. .

[0549] T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signal sequences: those that initiate antigen-dependent primary activation via TCRs (primary cytoplasmic signal sequences), and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signal sequences). In some aspects, the CAR includes one or both of these signaling components.

[0550] A receptor, such as a CAR, typically includes at least one intracellular signaling component or components. In some aspects, the CAR includes a primary cytoplasmic signal sequence that regulates the primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs, which are known as immunoreceptor tyrosine activating motifs, or ITAMs. Examples of ITAMs containing primary cytoplasmic signal sequences include those derived from CD3 zeta chains, FcR gamma, CD3 gamma, CD3 delta, and CD3 epsilon. In some embodiments, the cytoplasmic signaling molecule(s) in the CAR comprises a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3 zeta.

[0551] In some embodiments, the implementation of the receptor includes an intracellular component of the TCR complex, such as the CD3 TCR chain, which mediates T-cell activation and cytotoxicity, for example, the CD3 zeta chain. Thus, in some aspects, the antigen-binding portion is associated with one or more cell signaling modules. In some embodiments, cell signaling modules include a CD3 transmembrane domain, CD3 intracellular signaling regions, and/or other CD3 transmembrane regions. In some embodiments, the implementation of the receptor, for example, CAR, further includes part of one or more additional molecules, such as the receptor Fc γ, CD8, CD4, CD25 or CD16. For example, in some aspects, the CAR or other chimeric receptor comprises a chimeric molecule between CD3-zeta (CD3-ζ) or Fcγ receptor and CD8, CD4, CD25, or CD16.

[0552] In some embodiments, upon ligation of a CAR or other chimeric receptor, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or immune cell responses, e.g., T cells modified to express CAR. For example, in some contexts, CAR induces T cell function, such as cytolytic activity or T helper activity, such as the secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example if it transduces an effector function signal. In some embodiments, the intracellular signaling domain or domains include cytoplasmic T cell receptor (TCR) sequences, and in some aspects also those co-receptors that naturally co-act with such receptors to initiate signal transduction upon antigen receptor activation.

[0553] In the context of a natural TCR, complete activation typically requires not only signal transduction through the TCR, but also a co-stimulatory signal. Thus, in some embodiments, a component for generating a secondary or costimulatory signal is also included in the CAR to promote full activation. In other embodiments, the CAR does not include a component for generating a costimulatory 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.

[0554] In some embodiments, the implementation of the chimeric antigen receptor contains the intracellular domain of the T-cell co-stimulatory molecule. In some embodiments, the CAR includes the signaling domain and/or the transmembrane portion of a co-stimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some aspects, the same CAR includes both activating and costimulatory components. In some embodiments, the chimeric antigen receptor comprises an intracellular domain derived from a costimulatory T cell molecule, or a functional variant thereof, such as between a transmembrane domain and an intracellular signaling domain. In some aspects, the costimulatory T cell molecule is CD28 or 41BB.

[0555] In some embodiments, the activating domain is included in one CAR while the co-stimulatory component is provided by another CAR recognizing a different antigen. In some embodiments, the implementation of the CAR includes an activating or stimulating CAR, costimulatory CAR, which are both expressed on the same cell (see WO2014/055668). In some aspects, the cells include one or more stimulatory or activating CARs and/or costimulatory CARs. In some embodiments, the cells further comprise inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such as a CAR recognizing an antigen other than the associated and/or specific antigen. for a disease or condition whereby an activating signal delivered through a disease-targeting CAR is reduced or inhibited by binding the inhibitory CAR to its ligand, for example, to reduce off-target effects.

[0556] In some embodiments, the intracellular signaling domain includes a CD28 transmembrane and signaling domain associated with an intracellular CD3 domain (eg, CD3-zeta). In some embodiments, the intracellular signaling domain includes CD28 and CD137 chimeric co-stimulatory domains (4-1BB, TNFRSF9) associated with the CD3 zeta intracellular domain.

[0557] In some embodiments, a CAR encompasses one or more, eg, two or more, co-stimulatory domains and an activating domain, eg, a primary activating domain, in the cytoplasmic portion. Examples of CARs include the intracellular components of CD3-zeta, CD28 and 4-1BB.

[0558] In some embodiments, the antigen receptor further includes a marker and/or cells expressing CAR or other antigen receptor further includes a surrogate marker, such as a cell surface marker, that can be used to confirm transduction or engineering of the cell to express the receptor. In some aspects, the marker includes all or part (eg, a truncated form) of CD34, NGFR, or an epidermal growth factor receptor, such as a truncated version of such a cell surface receptor (eg, tEGFR). In some embodiments, a nucleic acid encoding a marker is operably linked to a polynucleotide encoding a linker sequence, such as a cleavable linker sequence, for example, T2A. For example, the marker and optionally the linker sequence can be any as disclosed in Published Patent Application No. WO2014031687. For example, the marker may be a truncated EGFR (tEGFR), ie, optionally linked to a linker sequence, such as a cleavable T2A linker sequence.

[0559] An exemplary polypeptide for truncated EGFR (e.g., tEGFR) includes the amino acid sequence shown in SEQ ID NO: 7 or 16, or an amino acid sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 7 or 16. An example T2A linker sequence includes the amino acid sequence, shown in SEQ ID NO: 6 or 17, or an amino acid sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 6 or 17.

[0560] In some embodiments, the marker is a molecule, such as a cell surface protein, not naturally occurring on T cells or not naturally occurring on the surface of T cells, or a portion thereof. In some embodiments, the molecule is a foreign molecule, eg, a foreign protein, ie, not recognized as "self" by the immune system of the host into which the cells will be adoptively transferred.

[0561] In some embodiments, the implementation of the marker does not serve any therapeutic function and/or has no effect other than use as a marker for genetic engineering, for example, to select cells that have been successfully modified by genetic engineering. In other embodiments, the marker may be a therapeutic molecule or a molecule otherwise exhibiting some desired effect, such as a ligand for the cell to be interacted with in vivo, such as a co-stimulatory or immune checkpoint molecule to enhance and/or attenuate a response. cells after adoptive transfer and interaction with the ligand.

[0562] In some instances, CARs are referred to as first, second, and/or third generation CARs. In some aspects, a first generation CAR is one that exclusively provides a CD3 chain-inducible signal after antigen binding; in some aspects, a second generation CAR is one that provides such a signal and a costimulatory signal, such as comprising an intracellular signaling domain from a costimulatory receptor, such as CD28 or CD137; in some aspects, the third generation CAR is one that includes multiple costimulatory domains of various costimulatory receptors.

[0563] For example, in some embodiments, a CAR comprises an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains a CD28 transmembrane portion or a functional variant thereof, and an intracellular signaling domain comprising a CD28 signal portion or a functional variant thereof and a signal portion CD3 zeta or its functional variant. In some embodiments, the CAR comprises an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains a CD28 transmembrane portion or a functional variant thereof, and an intracellular signaling domain comprising a 4-1BB signaling portion or a functional variant thereof, and a CD3 zeta signaling portion. or its functional variant. In some such embodiments, the receptor further includes a spacer comprising a portion of an Ig molecule, such as a human Ig molecule, such as an Ig hinge, eg, an IgG4 hinge, such as a hinge-only spacer.

[0564] In some embodiments, the transmembrane domain of the recombinant receptor, e.g., CAR, is or includes the transmembrane domain of human CD28 (e.g., Accession No. P01747.1) or a variant thereof, such as a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or more sequence identity with SEQ ID NO: 8; in some embodiments, the transmembrane domain containing the recombinant receptor portion comprises the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with it.

[0565] In some embodiments, the intracellular signaling component(s) of the recombinant receptor, e.g., CAR, comprises an intracellular costimulatory signaling domain of human CD28, or a functional variant or portion thereof, such as a domain with an LL to GG substitution at positions 186-187 of the native CD28 protein . For example, an intracellular signaling domain may include an amino acid sequence shown in SEQ ID NO: 10 or 11, or an amino acid sequence showing 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: 10 or 11. In some embodiments, the intracellular domain includes the 4-1BB intracellular costimulatory signaling domain ( for example, (Access No. Q07011,1) or a functional variant or part thereof, such as the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence showing 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: 12.

[0566] In some embodiments, the intracellular signaling domain of a recombinant receptor, e.g., CAR, includes a human CD3 zeta stimulatory signaling domain or a functional variant thereof, such as the human CD3ζ isoform 3 cytoplasmic domain 112 AA (accession number: P20963,2) or the signaling domain CD3 zeta as described in US Patent: 7,446,190 or US Patent No. 8,911,993. For example, in some embodiments, the intracellular signaling domain comprises an amino acid sequence as set forth in SEQ ID NO: 13, 14, or 15, or an amino acid sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 13, 14 or 15.

[0567] In some aspects, the spacer comprises an IgG hinge only, such as an IgG4 or IgG1 hinge only, such as the hinge-only spacer shown in SEQ ID NO: 1. In other embodiments, the spacer is or contains an Ig hinge, e.g. , an IgG4-derived hinge optionally associated with C _H 2 and/or C _H 3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge associated with C _H 2 and C _H 3 domains, such as those shown in SEQ ID NO: 4. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge associated only with a C _H 3 domain, such as shown in SEQ ID NO: 3. In some embodiments, the spacer is or includes a glycine rich sequence -serine, or other flexible linker such as known flexible linkers.

[0568] For example, in some embodiments, a CAR includes an antibody, such as an antibody fragment, including scFvs, a spacer, such as a spacer containing a portion of an immunoglobulin molecule, such as a hinge region and/or one or more constant regions of a heavy chain molecule, such as as an Ig-hinge spacer, a transmembrane domain containing all or part of a CD28-derived transmembrane domain, a CD28-derived intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises an antibody or fragment such as an scFv, a spacer such as any of the spacers containing an Ig hinge, a CD28 derived transmembrane domain, a 4-1BB derived intracellular signaling domain, and a CD3 derived zeta signaling domain.

[0569] In some embodiments, nucleic acid molecules encoding such CAR constructs further comprise a sequence encoding a T2A ribosomal skip element and/or a tEGFR sequence, e.g., a downstream portion of a CAR encoding sequence. In some embodiments, the sequence encodes a T2A ribosomal skip element shown in SEQ ID NO: 6 or 17, or an amino acid sequence showing 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, antigen receptor-expressing T cells (e.g., CAR) can also be generated to express truncated EGFR (EGFRt) as a non-immunogenic epitope of choice (eg, by introducing a construct encoding CAR and EGFRt separated by the T2A ribosomal switch to express two proteins from the same construct), which can then be used as a marker for the detection of such cells (see, for example, US Patent No. 8,802,374). In some embodiments, the sequence encodes a tEGFR sequence shown in SEQ ID NO: 7 or 16, or an amino acid sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 7 or 16. In some cases, a peptide such as T2A can cause a ribosome to skip (skip in ribosome) synthesis of a peptide bond at the C-terminus of the 2A element, leading to separation between the end of the 2A sequence and the next downstream peptide (see, for example, de Felipe. Genetic Vaccines and Ther. 2:13 (2004) and deFelipe et al. Traffic 5 :616-626 (2004)). Many 2A elements are known. Examples of 2A sequences that can be used in the methods and nucleic acids disclosed herein include, without limitation, the 2A sequences of foot and mouth disease virus (F2A, e.g., SEQ ID NO: 21), equine rhinitis A virus (E2A, e.g., SEQ ID NO: 20), Thosea asigna virus (T2A, eg, SEQ ID NO: 6 or 17) and porcine teshovirus-1 (P2A, eg, SEQ ID NO: 18 or 19), as described in US Patent Publication No. 20070116690.

[0570] Recombinant receptors, such as CARs, expressed by cells administered to a patient typically recognize or bind specifically to a molecule expressed in, associated with, and/or specific to the disease or condition being treated or its cells. Upon specific binding to a molecule, such as an antigen, the receptor typically delivers an immunostimulatory signal, such as an ITAM-transduced signal, into the cell, thereby promoting an immune response directed at the disease or condition. For example, in some embodiments, the cells express a CAR that specifically binds to an antigen expressed by the cell or tissue of the disease or condition, or is associated with the disease or condition.

B. T cell receptors (TCRs)

[0571] In some embodiments, the engineered cells, such as T cells, used in connection with the methods, uses, articles, or compositions of the invention are cells that express a T cell receptor (TCR) or an epitope-recognizing antigen-binding portion thereof. peptide or T-cell epitope of the target polypeptide, such as a tumor antigen, viral or autoimmune protein.

[0572] In some embodiments, a "T cell receptor" or "TCR" is a molecule containing variable α and β chains (also known as TCRα and TCRβ, respectively) or variable γ and δ chains (also known as TCRα and TCRβ , respectively), or their antigen-binding portions, and which is capable of specific binding to a peptide that binds to an MHC molecule. In some embodiments, the TCR is in the αβ form. Typically, TCRs that exist in αβ and γδ forms are generally structurally similar, but T cells expressing them may have different anatomical locations or functions. TCR can be found on the surface of cells or in soluble form. TCR is normally found on the surface of T cells (or T lymphocytes), where it is usually responsible for recognizing antigens associated with major histocompatibility complex (MHC) molecules.

[0573] Unless otherwise indicated, the term "TCR" is to be understood as referring to complete TCRs as well as their antigen-binding portions or antigen-binding fragments. In some embodiments, the TCR is intact or full length TCR, including TCR in αβ form or γδ form. In some embodiments, the TCR is an antigen-binding portion that is smaller than the full-length TCR, but binds to a specific peptide bound to the MHC molecule, eg, binds to an MHC-peptide complex. In some instances, an antigen-binding portion or fragment of a TCR may contain only a subset of the structural regions of a full-length or intact TCR, but be able to bind to an epitope of a peptide, such as an MHC-peptide complex, to which the entire TCR binds. In some instances, the antigen-binding portion contains TCR variable regions, such as a TCR variable α chain and a TCR variable β chain, sufficient to form a binding site for binding to a specific MHC-peptide complex. Typically, TCR variable chains contain complementarity-determining regions involved in peptide, MHC and/or MHC-peptide recognition.

[0574] In some embodiments, the TCR variable regions comprise hypervariable loops, or complementarity determining regions (CDRs), which are typically primary components contributing to antigen recognition and binding abilities and specificity. In some embodiments, the CDRs of a TCR, or a combination thereof, form all or substantially all of the antigen-binding site of a given TCR molecule. The different CDRs in the variable region of the TCR chain are usually separated by frame-reading regions (FRs), usually showing less variability among TCR molecules compared to CDRs (see, e.g., Jores et al., Proc. Nat'l Acad. Sci. USA 87: 9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, CDR3 is the primary CDR responsible for antigen binding or specificity, or is the most important of the three CDRs on a given TCR variable region for antigen recognition and/or interaction with the processed peptide portion of the peptide-MHC complex. In some contexts, the alpha chain CDR1 may interact with the N-terminal portion of certain antigenic peptides. In some contexts, the CDR1 beta chain may interact with the C-terminal portion of the peptide. In some contexts, CDR2 contributes most strongly, or is the primary CDR responsible for interacting with or recognizing the MHC portion of the MHC-peptide complex. In some embodiments, the β-chain variable region may contain an additional hypervariable region (CDR ⁴ or HVR ⁴ ) that is normally involved in superantigen binding rather than antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).

[0575] In some embodiments, the implementation of the TCR may also contain a constant domain, a transmembrane domain and/or a short cytoplasmic tail (see, for example, Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications , p. 4:33, 1997). In some aspects, each TCR chain may have one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, the TCR is associated with invariant proteins of the CD3 complex involved in mediating signal transduction.

[0576] In some embodiments, the TCR chain contains one or more constant domains. For example, the extracellular portion of a given TCR chain (eg, α-chain or β-chain) may contain two immunoglobulin-like domains, such as a variable domain (eg, Vα or Vb; typically amino acids 1-116 based on Kabat numbering, Kabat et al. ., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed) and a constant domain (e.g., α-chain constant domain, or Cα, typically position 117 -259 strands based on Kabat numbering or a β-chain or Cβ constant domain, typically positions 117-295 of the Kabat-based strand) adjacent to the cell membrane. For example, in some cases, the extracellular portion of the TCR formed by these two chains contains two membrane-proximal constant domains and two membrane-distal variable domains, with each of the variable domains containing a CDR. The TCR constant domain may contain short junction sequences in which a cysteine residue forms a disulfide bond, thus linking two TCR strands. In some embodiments, the TCR may have an additional cysteine residue in each of the α and β chains such that the TCR contains two disulfide bonds in the constant domains.

[0577] In some embodiments, the TCR chains comprise a transmembrane domain. In some embodiments, the transmembrane domain has a positive charge. In some cases, the TCR chain contains a cytoplasmic tail. In some cases, the structure allows the TCR to bind to other molecules such as CD3 and its subunits. For example, a TCR containing constant domains with a transmembrane region can fix a protein in the cell membrane and associate with invariant signaling subunits or the CD3 complex. The intracellular tails of the CD3 signaling subunits (eg, the CD3γ, CD3δ, CD3ε, and CD3ζ chains) contain one or more immunoreceptor tyrosine activation motifs, or ITAMs, involved in the signaling ability of the TCR complex.

[0578] In some embodiments, the TCR may be a heterodimer of two chains α and β (or optionally γ and δ), or it may be a TCR of a single chain construct. In some embodiments, the TCR is a heterodimer containing two separate chains (α and β chains or γ and δ chains) that are linked, for example, by a disulfide bond or disulfide bonds.

[0579] In some embodiments, the TCR may be generated from known TCR sequence(s), such as Vα,β chain sequences, for which a substantially full length coding sequence is readily available. Methods for obtaining full-length TCR sequences, including V chain sequences, from cell sources are known. In some embodiments, TCR-encoding nucleic acids can be obtained from a variety of sources, such as polymerase chain reaction (PCR) amplification of TCR-encoding nucleic acids in or isolated from a given cell or cells, or synthesis of publicly available TCR DNA sequences.

[0580] In some embodiments, the TCR is derived from a biological source, such as cells, such as T cells (eg, cytotoxic T cells), T cell hybridomas, or other publicly available source. In some embodiments, T cells can be obtained from in vivo isolated cells. In some embodiments, the TCR is thymus-selected TCR. In some embodiments, the TCR is a limited TCR neoepitope. In some embodiments, the T cells may be a cultured T cell hybridoma or a clone. In some embodiments, the TCR, or an antigen-binding portion thereof, or an antigen-binding fragment thereof, may be artificially generated from a known TCR sequence.

[0581] In some embodiments, a TCR is generated from a TCR identified or selected by screening a candidate TCR library against a target polypeptide antigen or target T cell epitope. TCR libraries can be generated by amplifying the Vα and Vβ repertoire from T cells isolated from a patient, including cells present in the PBMC, spleen, or other lymphoid organ. In some cases, T cells can be amplified from their tumor infiltrating lymphocytes (TILs). In some embodiments, TCR libraries can be generated from CD4 ⁺ or CD8 ⁺ cells. In some embodiments, TCRs can be amplified from a normal or healthy patient's T cell source, ie, normal TCR libraries. In some embodiments, TCRs can be amplified from a source of T cells from a sick patient, i.e., patients' TCR libraries. In some embodiments, degenerate primers are used to amplify the Vα and Vβ gene repertoire, such as by RT-PCR, in samples, such as human T cells. In some embodiments, scTv libraries can be assembled from intact Vα and Vβ libraries in which the amplified products are cloned or assembled to be separated by a linker. Depending on the source of the patient and the cells, the libraries may be HLA allele-specific. Also in some embodiments, TCR libraries can be generated by mutagenesis or diversification of a parent or backbone TCR molecule. In some aspects, the TCRs are targeted, such as by mutagenesis, for example, α or β chains. In some aspects, certain residues in the CDR TCR are changed. In some embodiments, selected TCRs can be modified by affinity maturation. In some embodiments, antigen-specific T cells can be selected, for example, by screening to evaluate CTL activity against a peptide. In some aspects, TCRs, such as those present on antigen-specific T cells, may be selected, for example, by binding activity, such as a particular affinity or avidity for the antigen.

[0582] In some embodiments, the implementation of the TCR or its antigennegative are modified or subjected to genetic engineering. In some embodiments, targeting methods are used to generate TCRs with altered properties, such as higher affinity for a specific MHC-peptide complex. In some embodiments, directed development is achieved by display methods, including but not limited to yeast display (Holler et al. (2003) Nat Immunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci U S A, 97 , 5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54), or T cell display (Chervin et al. (2008) J Immunol Methods, 339, 175-84). In some embodiments, display approaches include developing, or modifying, a known, parent, or reference TCR. For example, in some cases, wild-type TCRs can be used as a template for the production of mutagenized TCRs in which the CDRs are mutated at one or more residues and mutants with the desired altered property, such as higher affinity for the desired target antigen, are selected.

[0583] In some embodiments, the peptides of the target polypeptide for use in making or generating the TCR of interest are known or readily identifiable. In some embodiments, peptides suitable for use in generating TCRs or antigen-binding portions can be determined based on the presence of an HLA-restricted motif in a target polypeptide, such as the target polypeptide described below. In some embodiments, peptides are identified using available computer predictive models. In some embodiments, for predicting class I MHC binding sites, such models include, but are not limited to, ProPred1 (Singh and Raghava (2001) Bioinformatics 17(12):1236-1237), and SYFPEITHI (see Schuler et al. (2007 ) Immunoinformatics Methods in Molecular Biology, 409(1): 75-93 2007) In some embodiments, the MHC-restricted epitope is HLA-A0201, which is expressed in approximately 39-46% of all Caucasians and therefore represents a suitable MHC antigen choice for use in the production of a TCR or other MHC peptide binding molecule.

[0584] HLA-A0201 binding motifs and cleavage sites for proteasomes and immune proteasomes are known by computer predictive models. For class I MHC binding site prediction, such models include, but are not limited to, ProPred1 (described in more detail in Singh and Raghava, ProPred: prediction of HLA-DR binding sites. BIOINFORMATICS 17(12):1236-1237 2001), and SYFPEITHI (see Schuler et al. SYFPEITHI, Database for Searching and T-Cell Epitope Prediction. in Immunoinformatics Methods in Molecular Biology, vol 409(1): 75-93 2007).

[0585] In some embodiments, the TCR, or an antigen-binding portion thereof, may be a recombinantly produced natural protein or a mutant form thereof in which one or more properties, such as binding characteristics, have been altered. In some embodiments, the TCR may be derived from one of a variety of animal species, such as a human, mouse, rat, or other mammal. The TCR may be cell-bound or in soluble form. In some embodiments, for purposes of the methods of the invention, the TCR is in a cell-associated form expressed on the cell surface.

[0586] In some embodiments, the TCR is a full length TCR. In some embodiments, the TCR is an antigen-binding portion. In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the TCR is a single chain TCR (sc-TCR). In some embodiments, the dTCR or scTCR have structures as described in WO 03/020763, WO 04/033685, WO2011/044186.

[0587] In some embodiments, the TCR contains a sequence corresponding to a transmembrane sequence. In some embodiments, the implementation of the TCR does contain a sequence corresponding to the cytoplasmic sequences. In some embodiments, the TCR is capable of complexing the TCR with CD3. In some embodiments, any of the TCRs, including dTCRs or scTCRs, can be associated with active TCR signaling regions on the T cell surface. In some embodiments, the TCR is expressed on the surface of cells.

[0588] In some embodiments, the dTCR comprises a first polypeptide in which a sequence corresponding to the TCR α chain variable region sequence is fused to the N-terminus of a sequence corresponding to the TCR α chain constant extracellular region sequence, and a second polypeptide in which the sequence corresponding to the sequence variable region of the β chain of the TCR, fused to the N-terminus of the sequence corresponding to the sequence of the constant extracellular region of the β chain of the TCR, and the first and second polypeptides are linked by a disulfide bond. In some embodiments, the bond may correspond to a native interchain disulfide bond present in native dimeric αβ TCRs. In some embodiments, interchain disulfide bonds are not present in native TCR. For example, in some embodiments, one or more cysteines may be included in the constant region of the extracellular dTCR sequences of the polypeptide pair. In some cases, both native and non-native disulfide bonds may be desirable. In some embodiments, the implementation of the TCR contains a transmembrane sequence for fixation to the membrane.

[0589] In some embodiments, the dTCR comprises a TCR α chain comprising a variable α region, a constant α region, and a first dimerization motif attached to the C-terminus of the constant α region, and a TCR β chain comprising a variable β region, a constant β region, and a first a dimerization motif attached to the C-terminus of a constant β region, wherein the first and second dimerization motifs readily interact to create a covalent bond between the amino acid in the first dimerization motif and the amino acid in the second dimerization motif linking the TCR α chain and the TCR β chain together.

[0590] In some embodiments, the TCR is scTCR. Typically, scTCR can be generated using known methods, see, for example, Soo Hoo, W. F. et al. PNAS (USA) 89, 4759 (1992); Wülfing, C. and Plückthun, A., J. Mol. Biol. 242, 655 (1994); Kurucz, I. et al. PNAS (USA) 90 3830 (1993); PCT International Publications WO 96/13593, WO 96/18105, WO99/60120, WO99/18129, WO 03/020763, WO2011/044186; and Schlueter, C. J. et al. J. Mol. Biol. 256, 859 (1996). In some embodiments, the scTCR contains an introduced non-native interchain disulfide bond to facilitate association of the TCR chains (see, for example, PCT International Application Publication WO 03/020763). In some embodiments, the scTCR is a non-disulfide linked truncated TCR in which heterologous leucine zippers fused to its C-terminus facilitate chain association (see, for example, PCT International Publication No. WO99/60120). In some embodiments, the scTCR comprises a TCRα variable region covalently linked to the TCRβ variable region via a peptide linker (see, for example, PCT International Application Publication No. WO99/18129).

[0591] In some embodiments, the scTCR comprises a first segment constituted by an amino acid sequence corresponding to the variable region α chain of the TCR, a second segment constituted by an amino acid sequence corresponding to the sequence of the variable region β chain of the TCR fused to the N-terminus of the amino acid sequence corresponding to the sequence of the extracellular constant the β domain of the TCR chain, and a linker sequence linking the C-terminus of the first segment to the N-terminus of the second segment.

[0592] In some embodiments, the scTCR comprises a first segment constituted by an α chain variable region sequence fused to the N-terminus of an α chain extracellular constant domain sequence, and a second segment constituted by a β chain variable region sequence fused to the N-terminus of an extracellular constant α chain sequence. a β chain domain, and a transmembrane sequence, and optionally a linker sequence linking the C-terminus of the first segment to the N-terminus of the second segment.

[0593] In some embodiments, the scTCR comprises a first segment composed of a TCR β chain variable region sequence fused to the N-terminus of a β chain extracellular constant domain sequence and a second segment composed of an α chain variable region sequence fused to the N-terminus of an extracellular a concatenation domain of the α chain, and a transmembrane sequence, and optionally a linker sequence linking the C-terminus of the first segment to the N-terminus of the second segment.

[0594] In some embodiments, the scTCR linker binding the first and second TCR segments can be any linker capable of forming a single polypeptide chain while maintaining TCR binding specificity. In some embodiments, the linker sequence may, for example, have the formula -P-AA-P- where P is proline and AA is an amino acid sequence where the amino acids are glycine and serine. In some embodiments, the implementation of the first and second segments are connected so that the sequence of the variable regions are oriented for such binding. Therefore, in some cases, the linker is long enough to span the distance between the C-terminus of the first segment and the N-terminus of the second segment, or vice versa, but not too long to block or reduce scTCR binding to the target ligand. In some embodiments, the implementation of the linker may contain from or from about 10 to 45 amino acids, for example, from 10 to 30 amino acids or from 26 to 41 amino acid residues, for example, 29, 30, 31 or 32 amino acids. In some embodiments, the implementation of the linker has the formula -PGGG-(SGGGG) ₅ -P-, in which P is proline, G is glycine, and S is serine (SEQ ID NO:28). In some embodiments, the linker has the sequence GSADDAKKDAAKKDGKS (SEQ ID NO:29)

[0595] In some embodiments, the implementation of the scTCR contains a covalent disulfide bond linking the remainder of the immunoglobulin region of the α chain constant domain to the remainder of the immunoglobulin region of the β chain constant domain. In some embodiments, no interchain disulfide bond is present in native TCR. For example, in some embodiments, one or more cysteines may be included in the extracellular constant region sequences of the first and second segments of the scTCR polypeptide. In some cases, both native and non-native disulfide bonds may be desirable.

[0596] In some embodiments, the implementation of dTCR or scTCR containing introduced interchain disulfide bonds, native disulfide bonds are not present. In some embodiments, one or more native cysteines forming native interchain disulfide bonds are replaced by another residue, such as serine or alanine. In some embodiments, the introduced disulfide bond can be formed by replacing non-cysteine residues on the first and second segments with cysteine. Examples of non-native TCR disulfide bonds are described in PCT International Application Publication No. WO2006/000830.

[0597] In some embodiments, the TCR, or antigen-binding fragment thereof, exhibits an affinity with an equilibrium binding constant for the target antigen of from or about 10 ^-5 to 10 ^-12 M and all individual values and ranges within that range. In some embodiments, the target antigen is an MHC-peptide complex or ligand.

[0598] In some embodiments, a nucleic acid or nucleic acids encoding a TCR, such as α and β chains, can be amplified by PCR, cloning, or other suitable means and cloned into a suitable expression vector or vectors. The expression vector may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and growth or for expression or both, such as plasmids and viruses.

[0599] In some embodiments, the vector may be a vector of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden) or the pEX series (Clontech, Palo Alto, CA). In some cases, bacteriophage-based vectors such as λG10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 may also be used. In some embodiments, plant expression vectors can be used and include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). In some embodiments, animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). In some embodiments, a viral vector is used, such as a retroviral vector.

[0600] In some embodiments, the implementation of recombinant expression vectors can be obtained using standard methods of recombinant DNA. In some embodiments, vectors may contain regulatory sequences, such as transcription and translation initiators and termination codons, that are specific to the type of host (e.g., bacteria, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and considering whether the vector is a DNA or RNA vector. In some embodiments, the vector may contain a non-native promoter operably linked to a nucleotide sequence encoding a TCR or antigen binding portion (or other MHC peptide binding molecule). In some embodiments, the promoter may be a non-viral promoter or a viral promoter such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and the promoter found in the long terminal repeat of murine stem cell virus. Other known promoters are also considered.

[0601] In some embodiments, to generate a vector encoding a TCR, the α and β chains are PCR amplified from total cDNA isolated from a T cell clone expressing the TCR of interest and cloned into an expression vector. In some embodiments, the α and β chains are cloned into the same vector. In some embodiments, the α and β chains are cloned into different vectors. In some embodiments, the generated α and β chains are incorporated into a retroviral, eg, lentiviral, vector.

Genetically engineered cells and methods for producing cells

[0602] In some embodiments, the methods of the invention include administering to a patient having a disease or condition, cells expressing a recombinant antigen receptor. Various methods of introducing genetically engineered components, for example, recombinant receptors, such as CAR or TCR, are known and can be used with the methods and compositions of the invention. Examples of methods include those for the transfer of nucleic acids encoding receptors, including transfer by viral, eg, retroviral or lentiviral, transduction, transposons, and electroporation.

[0603] Among the cells expressing the receptors and administered by the methods of the invention, genetically engineered cells can be mentioned. Genetic engineering typically involves introducing a nucleic acid encoding a recombinant or genetically engineered component into a composition containing cells, such as by retroviral transduction, transfection, or transformation.

C. Chimeric autoantibody receptors (CAARs)

[0604] In some embodiments, among the recombinant receptors expressed by engineered cells used in connection with the methods, uses, articles and compositions of the invention, chimeric autoantibody receptor (CAAR) can be mentioned. In some embodiments, the CAAR is specific for an autoantibody. In some embodiments, a cell expressing CAAR, such as a T cell engineered to express CAAR, can be used to specifically bind to and kill autoantibody-expressing cells but not normal antibody-expressing cells. In some embodiments, CAAR-expressing cells can be used to treat an autoimmune disease associated with the expression of autoantigens, such as autoimmune diseases. In some embodiments, CAAR-expressing cells can target B cells that eventually produce autoantibodies and display autoantibodies on their cell surface, label those B cells as disease-specific targets for therapeutic intervention. In some embodiments, CAAR-expressing cells can be used to effectively target and kill pathogenic B cells in autoimmune diseases by targeting disease-causing B cells with an antigen-specific chimeric autoantibody receptor. In some embodiments, the recombinant receptor is a CAAR, such as any described in US Patent Application Publication US 2017/0051035.

[0605] In some embodiments, the CAAR includes an autoantibody-binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling region includes an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or includes a primary signaling domain, a signaling domain that is capable of inducing a primary activation signal in a T cell, a T cell receptor (TCR) component signaling domain, and/or a signaling domain comprising an immunoreceptor tyrosine activating motif (ITAM). In some embodiments, the intracellular signaling domain includes a secondary or costimulatory signaling domain (secondary intracellular signaling regions).

[0606] In some embodiments, the autoantibody-binding domain includes a self-antigen or a fragment thereof. The choice of autoantigen may depend on the type of autoantibody being targeted. For example, an autoantigen may be selected because it recognizes an autoantibody on a target cell, such as a B cell, associated with a particular disease state, eg, an autoimmune disease, such as an autoantibody-mediated autoimmune disease. In some embodiments, the autoimmune disease includes pemphigus vulgaris (PV). Examples of self antigens include desmoglein 1 (Dsg1) and Dsg3.

D. Multitargeting

[0607] In some embodiments, cells used in connection with the methods, uses, articles, and compositions of the invention include cell-based multi-targeting strategies, such as expressing two or more engineered receptors on a cell, each of which recognizes the same the same or different antigens, and as a rule each includes different intracellular signaling components. Such multitargeting strategies are described, for example, in International Patent Application Publication: WO 2014055668 A1 (which describes the combination of an activating and a co-stimulatory CAR, e.g. targeting two different antigens individually present on non-target e.g. normal cells, but present together only on cells of the disease or condition to be treated) and Fedorov et al., Sci. Transl. Medicine, 5(215) (2013) (describes cells expressing both an activating and an inhibitory CAR, such as those in which an activating CAR binds to a single antigen expressed both on normal or non-disease-related cells and on disease or disease cells). the condition to be treated and the inhibitory CAR binds to another antigen expressed only on normal cells or cells that are not desired for treatment).

[0608] For example, in some embodiments, the cells include a receptor that expresses a first engineered antigen receptor (e.g., CAR or TCR) that is capable of inducing an activating or stimulatory signal in the cell, typically upon specific binding to an antigen recognized by the first receptor, for example, the first antigen. In some embodiments, the cell further comprises a second engineered antigen receptor (e.g., CAR or TCR), e.g., a chimeric costimulatory receptor, that is capable of inducing a costimulatory signal in an immune cell, typically after specific binding to a second antigen recognized by the second receptor. In some embodiments, the first antigen and the second antigen are the same. In some embodiments, the first antigen and the second antigen are different.

[0609] In some embodiments, the first and/or second engineered antigen receptor (eg, CAR or TCR) is capable of inducing an activating signal in a cell. In some embodiments, the implementation of the receptor includes an intracellular signaling component containing ITAM or ITAM-like motifs. In some embodiments, activation induced by the first receptor includes signal transduction or alteration of protein expression in a cell resulting in the initiation of an immune response, such as ITAM phosphorylation and/or initiation of an ITAM-mediated signal transduction cascade, formation of an immunological synapse, and/or grouping of molecules around associated receptor (e.g., CD4 or CD8, etc.), activation of one or more transcription factors such as NF-κB and/or AP-1, and/or induction of gene expression of factors such as cytokines, proliferation and/or survival .

[0610] In some embodiments, the first and/or second receptor includes intracellular signaling domains or regions of costimulatory receptors such as CD28, (4-1BB) CD137, OX40, and/or ICOS. In some embodiments, the first and second receptors comprise a co-stimulatory receptor intracellular signaling domain that are different. In one embodiment, the first receptor contains the CD28 costimulatory signaling domain and the second receptor contains the 4-1BB costimulatory signaling domain, or vice versa.

[0611] In some embodiments, the first and/or second receptor includes both an intracellular signaling domain containing ITAM or ITAM-like motifs and an intracellular signaling domain of a costimulatory receptor.

[0612] In some embodiments, the first receptor contains an intracellular signaling domain containing ITAM or ITAM-like motifs, and the second receptor contains an intracellular signaling domain of a co-stimulatory receptor. A costimulatory signal, in combination with an activation signal induced in the same cell, is a signal that results in an immune response, such as a strong and prolonged immune response such as increased gene expression, secretion of cytokines and other factors, and T-cell mediated effector functions, such as cell destruction.

[0613] In some embodiments, neither ligation of the first receptor alone nor ligation of the second receptor alone elicits a strong immune response. In some aspects, if only one receptor is ligated, the cell becomes tolerant or resistant to the antigen, or inhibited, and/or not induced to proliferate or secrete factors or exercise effector functions. In some such embodiments, however, when a plurality of receptors are ligated, for example, after interaction with a cell expressing the first and second antigens, the desired response is achieved, such as complete immune activation or stimulation, for example, as indicated by secretion of one or more cytokines, proliferation , persistence and/or performance of an immune effector function such as cytotoxic killing of target cells.

[0614] In some embodiments, these two receptors induce, respectively, an activating and inhibitory signal to a cell, such that binding of one of the receptors to its antigen activates the cell or induces a response, but binding of the second inhibitory receptor to its antigen induces a signal that suppresses or dampening this answer. Examples are combinations of activating CARs and inhibitory CARs or iCARs. Such a strategy can be used, for example, in which the activating CAR binds to an antigen expressed in the disease or condition, but which is also expressed in normal cells, and the inhibitory receptor binds to a single antigen expressed in normal cells, but not on cells of the disease or condition.

[0615] In some embodiments, a multi-targeting strategy is used where an antigen associated with a particular disease or condition is expressed on a non-diseased cell and/or expressed on the genetically engineered cell itself, transiently (e.g., after stimulation in association with genetic engineering) or constantly. In such cases, by requiring the ligation of two separate and individually specific antigen receptors, specificity, selectivity and/or efficacy can be improved.

[0616] In some embodiments, a plurality of antigens, such as first and second antigens, are expressed on a cell, tissue, or target disease or condition, such as a cancer cell. In some aspects, the cell, tissue, disease, or condition is multiple myeloma or a multiple myeloma cell. In some embodiments, one or more of the plurality of antigens is typically also expressed on a cell for which targeting by cell therapy is not desired, such as a normal or non-diseased cell or tissue and/or the genetically engineered cells themselves. In such embodiments, due to the need for ligation of multiple receptors to achieve a cellular response, specificity and/or efficacy is achieved.

E. Vectors and methods for genetic engineering

[0617] In some embodiments, engineered cells, such as T cells, used in connection with the methods, uses, articles, or compositions of the invention are cells that have been genetically modified to express a recombinant receptor, e.g., CAR or TCR, described here. In some embodiments, cells are modified by introducing, delivering, or transferring nucleic acid sequences encoding a recombinant receptor and/or other molecules.

[0618] In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious viral particles, such as, for example, vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred to T cells using recombinant lentiviral vectors or retroviral vectors such as retroviral gamma vectors (see, for example, Koste et al. (2014) Gene Therapy 2014 Apr 3. doi: 10, 1038/gt, 2014, 25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol 2011 November 29(11): 550-557).

[0619] In some embodiments, the retroviral vector has a long terminal repeat (LTR) sequence, e.g., a retroviral vector derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), virus mouse stem cells (MSCV), spleen necrosis virus (SFFV) or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include avian or mammalian cells derived from any source. Retroviruses are generally amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol and/or env sequences of the retrovirus. Numerous exemplary retroviral systems have been described (e.g., US Pat. 1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3: 102-109).

[0620] Methods for lentiviral transduction are known. Exemplary methods are described in, for example, Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506:97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.

[0621] In some embodiments, recombinant nucleic acids are 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 Therapy 7( 16): 1431-1437). In some embodiments, recombinant nucleic acids are transferred into T cells by transposition (see, for example, Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2 , e74 and Huang et al (2009) Methods Mol Biol 506: 115-126). Other methods for introducing and expressing genetic material in immune cells include calcium phosphate transfection (eg, as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York, New York), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-assisted microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and coprecipitation of DNA with strontium phosphate (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

[0622] Other approaches and vectors for transferring nucleic acids encoding recombinant products are described, for example, in International Patent Application Publication: WO2014055668 and US Pat. 7,446,190.

[0623] In some embodiments, cells, eg, T cells, can be transfected either during or after growth, eg, with a T cell receptor (TCR) or a chimeric antigen receptor (CAR). This transfection to introduce the gene of the desired receptor can be carried out with, for example, any suitable retroviral vector. The population of genetically modified cells can then be released from the initial stimulus (anti-CD3/anti-CD28 stimulus, for example) and then stimulated with a second type of stimulus, eg, via a de novo introduced receptor). This second type of stimulus may include an antigenic stimulus in the form of a peptide/MHC molecule, a cognate (cross-linking) ligand of a genetically introduced receptor (eg, a natural CAR ligand), or any ligand (such as an antibody) that directly binds within the novel receptor (eg, by recognition of constant regions in the receptor). See, for example, Cheadle et al, “Chimeric antigen receptors for T-cell based therapy” Methods Mol Biol. 2012; 907:645-66 or Barrett et al., Chimeric Antigen Receptor Therapy for Cancer Annual Review of Medicine Vol. 65:333-347 (2014).

[0624] In some cases, a vector may be used that does not require cells, such as T cells, to be activated. In some such cases, cells may be selected and/or transduced prior to activation. Thus, cells can be modified before or after cell culture, and in some cases at the same time as or during at least part of the culture.

[0625] Among additional nucleic acids, for example, genes to be introduced are those that improve the effectiveness of therapy, for example, by promoting the viability and/or function of the transferred cells; genes for providing a genetic marker for cell selection and/or evaluation, eg for in vivo survival or localization evaluation; genes to improve safety, for example, making cells capable of negative selection in vivo, as described by Lupton SD et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also PCT/US91/08442 and PCT/US94/05601, 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, for example, Riddell et al., U.S. Patent No. 6,040,177, in columns 14-17.

F. Cells and cell production for genetic engineering

[0626] In some embodiments, cells, such as T cells, used in connection with the methods, uses, articles, or compositions of the invention are cells that have been genetically modified to express a recombinant receptor, such as CAR or TCR, as described herein. In some embodiments, the genetically engineered cells are used in the context of cell therapy, such as adoptive cell therapy. In some embodiments, the genetically engineered cells are immune cells. In some embodiments, the engineered cells are T cells, such as CD4+ or CD8+ T cells.

[0627] In some embodiments, nucleic acids, such as nucleic acids encoding a recombinant receptor, are heterologous, i.e., not normally present in a cell or sample derived from a cell, such as derived from another organism or a cell that, for example, usually not found in a genetically engineered cell and/or the organism from which such a cell is derived. In some embodiments, the nucleic acids are non-natural, such as nucleic acids not found in nature, including combinations of chimeric nucleic acids encoding different regions from many different cell types.

[0628] The cells are typically eukaryotic cells, such as mammalian cells, and typically are human cells. In some embodiments, the cells are derived from blood, bone marrow, lymph or lymphoid organs, cells of the immune system, such as cells of innate or adaptive immunity, for example, myeloid or lymphoid cells, including lymphocytes, usually T cells and/or NK cells. Other examples of cells include stem cells such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells are typically primary cells, such as those isolated directly from the patient and/or isolated from the patient and frozen. In some embodiments, the cells include one or more subpopulations of T cells or other cell types, such as the total population of T cells, CD4 ⁺ cells, CD8 ⁺ cells, and subpopulations thereof, such as defined by function, activation state, maturity, potential for differentiation , growth, recycling, localization and/or persistence, antigen specificity, antigen receptor type, presence in a particular organ or compartment, cytokine secretion marker or profile, and/or degree of differentiation. In relation to the patient receiving treatment, the cells may be allogeneic and/or autologous. Among the methods are the standard methods. In some aspects, such as for standard technologies, the cells are pluripotent and/or multipotent, such as stem cells such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from a patient, dissecting, processing, culturing and/or genetically modifying them, and reintroducing them to the same patient before or after cryopreservation.

[0629] Among the subtypes and subpopulations of T cells and/or CD4 ⁺ and/or CD8 ⁺ T cells can be called intact T (T _N ) cells, effector T cells (T _EFF ), memory T cells and their subtypes, such as stem cell memory T (T _SCM ), central memory T (T _CM ), effector memory T (T _EM ), or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, T helpers, cytotoxic T cells, mucosal invariant T (MAIT) cells, natural and adaptive regulatory T (Treg) cells, T helpers such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, cells TH9, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.

[0630] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.

[0631] In some embodiments, cells include one or more genetically engineered nucleic acids and thus express recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., not normally present in a cell or sample derived from a cell, such as derived from another organism or cell, which, for example, is not normally found in a genetically engineered cell and/or organism from which the cell is derived. In some embodiments, the nucleic acids are non-natural, such as non-naturally occurring nucleic acids, including those containing chimeric combinations of nucleic acids encoding different domains from many different cell types.

[0632] In some embodiments, the production of genetically engineered cells includes one or more stages of cultivation and/or production. Cells for administration of a nucleic acid encoding a transgenic receptor, such as a CAR, may be isolated from a sample, such as a biological sample, eg obtained from or derived from a patient. In some embodiments, the patient from whom the cell is isolated is having a disease or condition, or in need of cell therapy, or to whom cell therapy will be administered. A patient, in some embodiments, is a human in need of a particular therapeutic intervention, such as adoptive cell therapy for which cells are isolated, processed, and/or engineered.

[0633] Accordingly, the cells, in some embodiments, are primary cells, eg, human primary cells. Samples include tissue, fluid, and other samples taken directly from the patient, as well as samples obtained from one or more processing steps such as separation, centrifugation, genetic engineering (eg, transduction with a viral vector), washing, and/or incubation. The biological sample may be a sample obtained directly from a biological source, or a sample after processing. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0634] In some aspects, the sample from which cells are derived or isolated is blood or a blood-derived sample, or is or is derived from a leukapheresis or apheresis product. Sample specimens include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, intestinal lymphoid tissue, mucosal lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestines, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil or other organ and/or cells derived therefrom. Samples include, in the context of cell therapy, such as adoptive cell therapy, samples from autologous and allogeneic sources.

[0635] In some embodiments, cells are derived from cell lines, eg, T cell lines. The cells, in some embodiments, are derived from a xenogeneic source, such as a mouse, rat, non-human primate, and pig.

[0636] In some embodiments, cell isolation includes one or more steps of obtaining and/or separating non-affinity based cells. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich with desired components, decompose, or remove cells sensitive to certain reagents. In some examples, cells are separated based on one or more properties such as density, adhesive properties, size, sensitivity, and/or resistance to certain components.

[0637] In some examples, cells are obtained from the patient's circulating blood, for example, by apheresis or leukapheresis. Samples in some aspects contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes and/or platelets, and in some aspects contain cells other than erythrocytes and platelets.

[0638] In some embodiments, the blood cells collected from the patient are washed, for example, to remove the plasma fraction, and the cells are placed in a suitable buffer or medium for subsequent processing steps. In some embodiments, cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution is free of calcium and/or magnesium and/or many or all of the divalent cations. In some aspects, a washing step is achieved, a semi-automatic "pump" centrifuge (eg Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, the washing step is performed by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca ⁺⁺ /Mg ⁺⁺ free PBS. In some embodiments, components of the blood cell sample are removed and the cells directly resuspended in culture media.

[0639] In some embodiments, the methods include density-based cell separation methods, such as obtaining leukocytes from peripheral blood by digesting erythrocytes and centrifuging through a Percoll or Ficoll gradient.

[0640] In some embodiments, isolation methods include separating different cell types based on the expression or presence of one or more specific molecules in the cell, such as surface markers, such as surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known separation method based on such markers may be used. In some embodiments, the separation is an affinity or immunoaffinity separation. For example, isolation in some aspects involves separating cells and cell populations based on cell expression or the level of expression of one or more markers, typically cell surface markers, e.g., by incubation with an antibody or binding partner that specifically binds to such markers, usually followed by steps washing and separating cells bound to the antibody or binding partner from cells not bound to the antibody or binding partner.

[0641] Such separation steps can be based on positive selection, in which cells bound to the reagents are retained for later use, and/or negative selection, in which cells not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for later use. In some aspects, a negative selection can be particularly useful if no antibody is available that specifically identifies a cell type in a heterogeneous population such that separation is best done based on markers expressed by cells other than the desired population.

[0642] Separation should not lead to 100% enrichment or removal of a specific population of cells or cells expressing a specific marker. For example, positive selection or enrichment for a particular cell type, such as those expressing a marker, refers to an increase in the number or percentage of such cells, but should not lead to a complete absence of cells not expressing that marker. Similarly, negative selection, removal or depletion of cells of a certain type, such as those expressing a marker, refers to a reduction in the number or percentage of such cells, but should not result in the complete removal of all such cells.

[0643] In some examples, multiple cycles of separation steps are performed, where positively or negatively selected fractions from one stage are subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, for example, by incubating the cells with multiple antibodies or binding partners, each of which is specific for the marker targeted for negative selection. Similarly, multiple cell types can be positively selected at the same time by incubating cells with multiple antibodies or binding partners expressed on different cell types.

[0644] For example, in some aspects, certain subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, for example, CD28 ⁺ , CD62L ⁺ , CCR7 + , CD27 ⁺ , CD127 ^{+ ,} CD4 ⁺ , CD8 ⁺ , CD45RA ⁺ , and/or CD45RO ⁺ T cells are isolated by positive or negative selection methods.

[0645] For example, CD3 ⁺ , CD28 ⁺ T cells can be positively selected using anti-CD3/anti-CD28 conjugated magnetic particles (eg, DYNABEADS® M-450 CD3/CD28 T Cell Expander).

[0646] In some embodiments, isolation is performed by enriching a certain cell population with a positive choice or depleting a certain cell population with a negative choice. In some embodiments, a positive or negative selection is made by incubating cells with one or more antibodies or other binder that specifically binds to one or more surface markers expressed (marker ⁺ ) or expressed at a relatively higher level (marker ^high ) at a positive or negatively selected cells, respectively.

[0647] In some embodiments, T cells are separated from the PBMC sample by a negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other leukocytes such as CD14. In some aspects, the CD4 ⁺ or CD8 ⁺ selection step is used to separate CD4 ⁺ helper and CD8 ⁺ cytotoxic T cells. Such a population of CD4 ⁺ and CD8 ⁺ can be further sorted into a subset of positive or negative selection for markers expressed or expressed to a relatively high degree on one or more intact, memory and/or effector T cell subpopulations.

[0648] In some embodiments, CD8 ⁺ cells are further enriched or depleted for intact, central memory, effector memory, and/or central memory stem cells, e.g., by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for T central memory cells (T _CM ) is performed to increase efficacy, for example, to improve long-term survival, growth, and/or engraftment after administration, which in some aspects is particularly potent 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 of _TCM -enriched CD8 ⁺ T cells and CD4 ⁺ T cells further enhances efficacy.

[0649] In embodiments, memory T cells are present in both CD62L ⁺ and CD62L ^- subpopulations of CD8 ⁺ peripheral blood lymphocytes. PBMC can be enriched or depleted in relation to CD62L ^- CD8 ⁺ and/or CD62L ⁺ CD8 ⁺ fractions, for example, using anti-CD8 and anti-CD62L antibodies.

[0650] In some embodiments, the enrichment in central memory T cells (T _CM ) is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3 and/or CD127; in some aspects it is based on a negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a population of CD8 ⁺ enriched in T _CM cells is performed by depleting cells expressing CD4, CD14, CD45RA and positively selecting or enrichment of cells expressing CD62L. In one aspect, enrichment for central memory T ( _TCM ) cells is performed based on a negative selection based on CD4 expression, negative selection based on CD14 and CD45RA expression, and positive selection based on CD62L. Such elections are in some aspects carried out simultaneously, and in other aspects are carried out sequentially, in any order. In some aspects, the same expression-based CD4 selection step used in generating a CD8 ⁺ population or subset of cells is also used to generate a CD4 ⁺ population or subset of cells such that both positive and negative fractions from the CD4-based separation are preserved and are used in subsequent steps of the methods, optionally after one or more additional positive or negative selection steps.

[0651] In a specific example, a PBMC sample or other leukocyte sample is subjected to CD4 ⁺ cell selection, where both negative and positive fractions are retained. The negative fraction is then subjected to negative selection based on CD14 and CD45RA or CD19 expression, and positive selection based on central memory T cell marker characteristics such as CD62L or CCR7, where positive and negative selections are made in any order.

[0652] CD4 ⁺ T helper cells are sorted into intact, central memory, and effector cells by identifying a population of cells having cell surface antigens. CD4 ⁺ lymphocytes can be obtained by standard methods. In some embodiments, the intact CD4 ⁺ T cells are CD45RO ^- , CD45RA ⁺ , CD62L ⁺ , CD4 ⁺ T cells. In some embodiments, the CD4 ⁺ central memory cells are CD62L ⁺ and CD45RO ⁺ . In some embodiments, the effector CD4 ⁺ cells are CD62L ^- and CD45RO ^- .

[0653] In one example, to enrich CD4 ⁺ cells with a negative selection, the monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner binds to a solid support or matrix, such as a magnetic particle or paramagnetic particle, to allow cell separation for positive and/or negative selection. For example, in some embodiments, cells and a population of cells are separated or isolated using immunomagnetic (or affinity magnetic) separation methods (discussed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: SA Brooks and U. Schumacher © Humana Press Inc., Totova, NJ).

[0654] In some aspects, the sample or cell composition to be separated is incubated with a small magnetizable or magnetically sensitive material, such as magnetically sensitive particles or microparticles, such as paramagnetic particles (eg, such as Dynalbeads or MACS particles). The magnetically sensitive material, such as a particle, is usually attached directly or indirectly to a binding partner, such as an antibody, that specifically binds to a molecule, such as a surface marker, present on the cell, cells, or population of cells that one wishes to separate, such as negative or positive selection of which wish to implement.

[0655] In some embodiments, the magnetic particle or bead includes a magnetically sensitive material that binds to a specific binding partner, such as an antibody or other binding partner. There are many known magnetically sensitive materials used in magnetic separation processes. Suitable magnetic particles include those described in Molday US 4,452,773 and EP 452342 B, which are incorporated herein by reference. Colloidal particles such as those described in Owen, US 4,795,698 and Liberti et al., US 5,200,084 are other examples.

[0656] Incubation is typically carried out under conditions whereby antibodies or binding partners, or molecules such as secondary antibodies or other reagents that specifically bind to such antibodies or binding partners that are attached to the magnetic particle or bead, specifically bind to surface molecules. cells if they are present on the cells in the sample.

[0657] In some aspects, the sample is placed in a magnetic field, and cells having magnetically sensitive or magnetizable particles attached to them will be attracted to the magnet and separated from unlabeled cells. For positive selection, retain the cells attracted to the magnet; for negative selection, unattracted cells (unlabeled cells) are retained. In some aspects, the combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subjected to further separation steps.

[0658] In some embodiments, the magnetically sensitive particles are coated with primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In some embodiments, the magnetic particles are attached to cells through a coating of primary antibodies specific for one or more markers. In some embodiments, cells, rather than particles, are labeled with a primary antibody or binding partner, and then coated with a cell type-specific secondary antibody or other binding partner (eg, streptavidin) magnetic particles are added. In some embodiments, streptavidin-coated magnetic particles are used in combination with biotinylated primary or secondary antibodies.

[0659] In some embodiments, the magnetically sensitive particles are left attached to cells that are to be subsequently incubated, cultured and/or genetically modified; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, magnetizable or magnetically sensitive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of competing unlabeled antibodies and magnetizable particles or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0660] In some embodiments, affinity-based selection is via magnetically activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). Magnetically Activated Cell Sorting Systems (MACS) are capable of high purity selection of cells to which magnetizing particles are attached. In some embodiments, MACS is performed in a manner in which non-target and target species are sequentially eluted after application of an external magnetic field. That is, cells attached to magnetized particles remain in place while non-attached species are eluted. Then, after this first elution step is complete, the species that were held in the magnetic field and not eluted are isolated in some way so that they can be eluted and recovered. In some embodiments, non-target cells are labeled and depleted from a heterogeneous population of cells.

[0661] In some embodiments, isolation or separation is performed by a system, device, or apparatus that performs one or more of the steps of isolation, cell production, separation, processing, incubation, culture, and/or formulation. In some aspects, the system is used to perform each of these steps in a closed or sterile environment, such as to minimize error, customization, and/or contamination. In one example, the system is the system described in International Patent Application Publication WO2009/072003 or US 20110003380 A1.

[0662] In some embodiments, the system or apparatus performs one or more, for example, all of the isolation, processing, engineering, and formulation steps in an integrated or separate system, and/or in an automated or programmable manner. In some aspects, the system or apparatus includes a computer and/or a computer program in communication with the system or apparatus allowing a user to program, control, evaluate and/or customize various aspects of the processing, extraction, engineering, and formulation steps.

[0663] In some aspects, separation and/or other steps are performed using the CliniMACS system (Miltenyi Biotec), for example, for automated separation of cells at the clinical scale level in a closed and sterile system. The components may include an integrated microcomputer, a magnetic separation unit, a peristaltic pump and various boost valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a selection column holder. The peristaltic pump controls the flow rate throughout the tubing system and, together with boost valves, ensures controlled buffer flow through the system and continuous cell suspension.

[0664] The CliniMACS system, in some aspects, uses magnetizable particles coupled to an antibody, supplied in a sterile, non-pyrogenic solution. In some embodiments, after labeling cells with magnetic particles, the cells are washed to remove excess particles. The cell collection bag is then attached to a tubing in turn connected to a buffer containing bag and a cell collection bag. The tubing system consists of a pre-assembled sterile tubing set including a pre-column and separation column and is for single use only. Upon initiation of the separation program, the system automatically applies the cell sample to the separation column. Labeled cells are retained in the column while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell population for use with the methods described herein is not labeled and stored in a column. In some embodiments, the cell population for use with the methods described herein is labeled and stored in a column. In some embodiments, the cell population for use with the methods described herein is eluted from the column after removal of the magnetic field and collected in a cell collection bag.

[0665] In some embodiments, separation and/or other steps are performed using a CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy System is equipped in some aspects with a cell processing module that provides automated cell washing and cell fractionation by centrifugation. The CliniMACS Prodigy system can also include an on-board camera and image recognition software that determines the optimal cell fractionation result by distinguishing macroscopic layers of the original cell product. For example, peripheral blood is automatically divided into erythrocytes, leukocytes and plasma layers. The CliniMACS Prodigy system may also include an integrated cell culture chamber that provides cell culture protocols such as, for example, cell differentiation and growth, antigen loading, and long-term cell culture. Entry ports can allow for sterile removal and replenishment of media, and cells can be inspected using an integrated microscope. See, for example, Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood, 1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.

[0666] In some embodiments, the cell population described herein is harvested and enriched (or depleted) via flow cytometry, in which cells stained with multiple cell surface markers are in a liquid stream. In some embodiments, the cell population described herein is harvested and enriched (or depleted) via preparative scale (FACS) sorting. In some embodiments, the cell population described herein is harvested and enriched (or depleted) using microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, for example, 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 multiple markers, allowing the isolation of well-defined subpopulations of T cells in high purity.

[0667] In some embodiments, the antibodies or binding partners are labeled with one or more detectable markers to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, cell separation based on the binding of antibodies or other binding partners specific for one or more cell surface markers is carried out in a liquid stream, such as fluorescence-activated cell sorting (FACS), including preparative-scale (FACS) and/or microelectromechanical systems chips. (MEMS), for example, in combination with a flow cytometric detection system. Such methods provide positive and negative selection based on multiple markers simultaneously.

[0668] In some embodiments, methods of preparation include the steps of freezing, eg, freezing preservation, of cells before or after isolation, incubation, and/or engineering. In some embodiments, the freezing and subsequent thawing step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a cooling solution, for example, by performing a washing step to remove plasma and platelets. Any of a variety of known cooling solutions and parameters may be used in some aspects. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA) or other suitable cell freezing media. They are then diluted 1:1 with the media so that the final concentration of DMSO and HSA is 10% and 4%, respectively. Cells are typically then frozen to -80° C. at a rate of 1° C. per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[0669] In some embodiments, cells are incubated and/or cultured prior to or in connection with genetic engineering. The incubation steps may include culture, culturing, stimulation, activation and/or growth. Incubation and/or engineering may be performed in a culture vessel such as a module, chamber, well, column, tube, tubing, valve, vial, culture dish, bag, or other culture or cell culture container. In some embodiments, the compositions or cells are incubated in the presence of stimulation conditions or a stimulating agent. Such conditions include those designed to induce proliferation, growth, activation and/or survival of cells in a population, stimulate antigen exposure, and/or prime cells for genetic engineering, for example, to introduce a recombinant antigen receptor.

[0670] Conditions may include one or more specific environments, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions, and/or stimulatory factors such as cytokines, chemokines, antigens, partners. bindings, fusion proteins, recombinant soluble receptors, and any other substances designed to activate cells.

[0671] In some embodiments, the implementation of the stimulating conditions or agents include one or more agents, for example, ligands, that are capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the agent turns on or initiates an intracellular TCR/CD3 signaling cascade in a T cell. Such agents may include antibodies such as those specific for TCR, eg anti-CD3. In some embodiments, the stimulatory conditions include one or more agents, eg, a ligand, that is capable of stimulating a co-stimulatory receptor, eg, anti-CD28. In some embodiments, such agents and/or ligands may be associated with a solid support, such as a particle and/or one or more cytokines. Optionally, the expansion method may further comprise the step of adding an anti-CD3 and/or anti-CD28 antibody to the culture medium (eg, at a concentration of at least about 0.5 ng/mL). In some embodiments, stimulants include IL-2, IL-15 and/or IL-7. In some aspects, the concentration of IL-2 is at least about 10 units/ml.

[0672] In some aspects, the incubation is carried out in accordance with methods such as those described in US Patent No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood, 1:72-82, and/or Wang et al. (2012) J Immunother. 35(9):689-701.

[0673] In some embodiments, T cells are grown by adding to the initiating culture composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs) (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC nurse cells for each T cell in the initial growth population); and incubating the culture (eg, for a period of time sufficient to increase the number of T cells). In some aspects, non-dividing nurse cells may include gamma irradiated PBMC nurse cells. In some embodiments, the implementation of PBMC irradiated with gamma rays in the range of approximately 3000-3600 rad to prevent cell division. In some aspects, the feeder cells are added to the culture medium prior to the addition of the T cell population.

[0674] In some embodiments, stimulating conditions include a temperature suitable for human T cell growth, e.g., at least about 25 degrees Celsius, typically at least about 30 degrees, and typically at or about 37 degrees Celsius. Optionally, the incubation may further include the addition of non-dividing EBV-transformed lymphoblastic cells (LCL) as nurse cells. LCLs can be irradiated with gamma rays in the range of approximately 6000-10000 rads. Feeder LCL cells are in some aspects used in any suitable amount, such as a ratio of feeder LCL cells to primordial T lymphocytes of at least about 10:1.

[0675] In embodiments, antigen-specific T cells, such as antigen-specific CD4 ⁺ and/or CD8 ⁺ T cells, are obtained by stimulating intact or antigen-specific T cells with an antigen. For example, lines or clones of antigen-specific T cells can be generated for cytomegalovirus antigens by isolating T cells from infected patients and stimulating the cells in vitro with the same antigen.

IV. COMPOSITIONS AND COMPOSITIONS

[0676] In some embodiments, a dose of cells, including cells genetically modified with a recombinant antigen receptor, such as CAR or TCR, is provided as a composition or formulation, such as a pharmaceutical composition or formulation. Such compositions can be used in accordance with the methods or uses of the invention and/or using the articles or compositions of the invention, for example, in the prevention or treatment of diseases, conditions and disorders, or in methods of detection, diagnosis and prognosis.

[0677] The term "pharmaceutical composition" refers to a preparation that is in a form that provides the effectiveness of the biological activity of the active ingredient contained therein, and does not contain additional components that are unacceptably toxic to the patient to whom the composition is administered.

[0678] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than the active ingredient, that is non-toxic to the patient. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0679] In some aspects, the choice of carrier is determined in part by a specific cell or agent and/or route of administration. Accordingly, there are many suitable formulations. For example, the pharmaceutical composition may contain preservatives. Suitable preservatives may include, for example, methyl paraben, propyl paraben, sodium benzoate and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative, or mixtures thereof, is typically present in an amount of from about 0.0001% to about 2% of the total weight of the composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (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 phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and meta-cresol) ; low molecular weight (less than about 10 residues) polypeptides; 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 (eg Zn-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG).

[0680] Buffer substances in some aspects are included in the compositions. Suitable buffering agents 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 buffer substances is used. The buffering agent, or mixtures thereof, is typically present in an amount of from about 0.001% to about 4% by weight of the total composition. Methods for preparing pharmaceutical compositions for administration are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins; 21st ed. (May 1, 2005).

[0681] The composition or composition may also contain more than one active ingredient useful for a particular indication, disease or condition prevented or cured by cells or agents, where the respective actions do not adversely affect each other. Such active ingredients are preferably present in combination in amounts that are effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharmaceutically active substances or drugs such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab , vinblastine, vincristine, etc. In some embodiments, the agents or cells are administered in the form of a salt, such as a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, and organic acids such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic and arylsulfonic acid, for example p-toluenesulfonic acid.

[0682] The pharmaceutical composition in some embodiments contains substances or cells in amounts effective to treat or prevent a disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy is, in some embodiments, verified by periodic evaluation of treated patients. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until the desired suppression of the symptoms of the disease occurs. However, other dosage regimens may be useful and may be determined. The desired dosage may be delivered by a single bolus of the composition, multiple boluses of the composition, or continuous administration of the composition by infusion.

[0683] The agents or cells may be administered by any suitable means, e.g., bolus infusion, injection, e.g., intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intracameral injection , subconjunctival injection, subconjunctival injection, subtenon injection, retrobulbar injection, peribulbar injection, or posterior parascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary and intranasal, and if desired for topical treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal or subcutaneous administration. In some embodiments, the administered dose is administered as a single bolus of cells or agent. In some embodiments, they are administered by multiple bolus injections of cells or agent, for example, over no more than 3 days, or by continuous administration of cells or agent by infusion.

[0684] For the prevention or treatment of a disease, the appropriate dosage may depend on the type of disease being treated, the type of agent or agents, the type of cell or recombinant receptor, the severity and course of the disease, whether the agent or cells are being administered prophylactically or therapeutically, previous therapy, the patient's medical history and response to the agent or cells, and the discretion of the attending physician. Compositions in some embodiments, the implementation is preferably administered to the patient once or in a series of treatments.

[0685] Cells or agents can be administered using standard administration methods, formulations, and/or devices. The invention relates to formulations and devices such as syringes and vials for storing and administering compositions. With respect to cells, administration may be autologous or heterologous. For example, immunoreactive cells or progenitors can be obtained from one patient and administered to the same patient or another compatible patient. Peripherally derived immunoreactive cells or progeny thereof (eg, obtained in vivo, ex vivo, or in vitro) can be administered by localized injection, including catheter, systemic injection, localized injection, intravenous injection, or parenteral administration. When a therapeutic composition (eg, a pharmaceutical composition containing a genetically modified immunoreactive cell or an agent that treats or alleviates the symptoms of neurotoxicity) is administered, it is usually formulated in an injectable unit dosage form (solution, suspension, emulsion).

[0686] 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", within the scope of the invention, includes intravenous, intramuscular, subcutaneous, rectal, vaginal and intraperitoneal administration. In some embodiments, the agent or cell population is administered to a patient using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0687] Compositions in some embodiments are in the form of sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which in some aspects may be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous formulations, and solid formulations. In addition, liquid formulations are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated in a suitable viscosity range to provide longer periods of contact with specific tissues. Liquid or viscous compositions may include carriers, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, a polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0688] Sterile injectable solutions can be prepared by incorporating the agent or cells in a solvent, for example, in admixture with a suitable carrier, diluent, or excipient such as sterilized water, physiological saline, glucose, dextrose, and the like.

[0689] Formulations that are used for in vivo administration are usually sterile. Sterility can be easily achieved, for example, by filtration through sterile filtration membranes.

V. COMBINATION THERAPY

[0690] In some embodiments of methods, articles, uses, or compositions, cell therapy, e.g., a dose of T cells (e.g., CAR ⁺ T cells), is administered to patients in combination with an additional therapeutic agent or therapy, usually other than cell therapy or other cell therapy, such as other than CAR ⁺ T cell therapy. In some embodiments, cell therapy, e.g., a dose of engineered T cells, such as CAR ⁺ T cells, in the methods or uses of the invention and/or with articles or compositions, is administered as part of a combination treatment or combination therapy, e.g., simultaneously with, sequentially with or intermittently with, in any order, one or more additional therapeutic interventions. In some embodiments, one or more additional therapeutic interventions includes any agent or treatment for the treatment or prevention of a disease or condition, such as B-cell cancer, e.g., NHL, and/or any agent or treatment for increasing efficacy, persistence, and/ or activity of genetically engineered cell therapy.

[0691] In some embodiments, an additional therapeutic agent or therapy is administered to patients who are, or are likely to be, or are predicted to be, immunocompromised organisms and/or who do not respond, are likely not responding, and/or are predicted to be will not respond or will not respond within a certain time and/or to some extent to treatment with cell therapy, for example, a dose of T cells (eg, CAR ⁺ T cells). In some embodiments, an additional therapeutic agent is administered to patients who do not or are unlikely or predicted to have a complete response or an overall response, for example, within 1 month, within two months, or within three months of initiating cell therapy administration. In some embodiments, an additional therapeutic agent is administered to patients who show or are likely to show or are predicted to have disease progression (PD), for example, within 1 month, two months, or three months after the introduction of cell therapy. In some embodiments, a patient is likely or predicted to be non-responder or some response based on a plurality of similar patients receiving or having previously received the same cell therapy.

[0692] In some embodiments, it is noted that a patient who may or is more likely to show a poor response to cell therapy, e.g., a dose of T cells (e.g., CAR ⁺ T cells), includes an NHL patient who is or has been identified as having stable or progressive disease (SD/PD) after treatment with prior therapy, optionally prior therapy with a chemotherapeutic agent that is or has been identified as having Eastern Cooperative Oncology Group Performance Status (ECOG) status 2, which is or has been identified as having transformed follicular lymphoma (tFL), or which is or has been identified as having DLBCL transformed from MZL and CLL. In some embodiments, the methods of the invention include selecting a patient who is or is likely to show a poor response to cellular therapy when the cellular therapy is administered alone, and administering the cellular therapy in combination with an additional agent or therapy such as any as described. In some embodiments, the patient for treatment in the combination therapy methods of the invention is a patient selected as having a B-cell cancer, such as NHL, and who has stable or progressive disease (SD/PD) following treatment with prior therapy, optionally prior chemotherapy therapy. an Eastern Cooperative Oncology Group Performance Status (ECOG) status 2 agent that has transformed follicular lymphoma (tFL), or DLBCL transformed from MZL and CLL. In some embodiments, an additional agent or therapy may be administered prior to, concomitant with, or simultaneously with and/or after the initiation of administration of a cell therapy, eg, a dose of T cells (eg, CAR ⁺ T cells).

[0693] In some embodiments, the pharmacokinetics (PK) of cell therapy in the blood of patients after administration of cell therapy is found to be similar or not significantly different between responders (e.g., showing CR or OR) versus non-responders (e.g., showing PD ) for cell therapy. In some embodiments, such observations indicate that the cell therapy has or is expanding in the patient, but may not show optimal efficacy.

[0694] In some contexts, the optimal efficacy of cell therapy may depend on the ability of the administered cells to recognize and bind to a target, such as a target antigen, to transport, localize, and successfully enter appropriate sites in the patient, tumors, and their environments. In some contexts, optimal efficacy may depend on the ability of the administered cells to become activated, grow, exhibit various effector functions, including cytotoxic killing, and secretion of various factors such as cytokines, for persistence, including long-term, differentiation, transition, or participation in reprogramming into certain phenotypic states. (such as long-term memory, less differentiated and effector state), avoid or reduce immunosuppressive conditions in the local microenvironment of the disease, to provide effective and strong secondary immune responses after clearance and re-exposure to the target ligand or antigen, and avoid or reduce debilitation, anergy, peripheral tolerance, terminal differentiation and/or differentiation into a suppressive state.

[0695] In some aspects, the effectiveness of immunotherapy, such as T-cell therapy, may be limited by immunosuppressive activity or factors present in the local microenvironment of the disease or disorder, such as TME. In some aspects, the TME contains or produces factors or conditions that can inhibit the activity, function, proliferation, survival, and/or persistence of T cells administered for T cell therapy.

[0696] In some embodiments, administration of an additional agent or therapy prior to, concomitant with, or simultaneously with and/or after initiation of administration of a cell therapy, e.g., a dose of T cells (e.g., CAR ⁺ T cells), may result in improved potency, efficacy and/or persistence of cell therapy and/or improved response of the treated patient. In some embodiments, an additional agent for combination treatment or combination therapy enhances, enhances and/or promotes the efficacy and/or safety of the therapeutic effect of cell therapy, for example, genetically engineered T cell therapy, such as CAR ⁺ T cells. In some embodiments, the implementation of the additional agent enhances or enhances the efficiency, survival or persistence of the introduced cells, for example, cells expressing a recombinant receptor, for example, CAR.

[0697] In some embodiments, the additional therapy agent is an antibody or a cytotoxic or therapeutic agent, such as a chemotherapeutic agent. In some embodiments, the one or more additional agents for treatment or therapy are an immunomodulatory agent, an immune checkpoint inhibitor, an adenosine signaling pathway, or an adenosine receptor antagonist or agonist, and kinase inhibitors. In some embodiments, the combination treatment or combination therapy includes post-treatment such as surgery, transplantation, and/or radiation therapy.

[0698] In some embodiments, the additional agent is selected from a protein phosphatase inhibitor, a kinase inhibitor, a cytokine, an immunomodulator, or an agent that reduces the level or activity of regulatory T (Treg) cells. In some embodiments, the implementation of the additional agent enhances safety based on the reduction or improvement of the adverse effects of the administered cell therapy. In some embodiments, an additional agent may treat the same disease, condition, or comorbidity. In some embodiments, an additional agent may alleviate, reduce, or eliminate one or more of the toxicities, adverse effects, or side effects associated with the administration of cells, eg, CAR-expressing cells.

[0699] In some embodiments, the complementary therapy, treatment, or agent includes chemotherapy, radiation therapy, surgery, transplantation, adoptive cell therapy, antibodies, cytotoxic agents, chemotherapeutic agents, cytokines, growth-suppressing agents, antihormonal agents, kinase inhibitors, antiangiogenic agents, cardioprotectors, immunostimulants, immunosuppressants, immune checkpoint inhibitors, antibiotics, angiogenesis inhibitors, metabolic modulators, or other therapeutic agents, or any combination thereof. In some embodiments, the additional agent is a protein, peptide, nucleic acid, small molecule substance, cell, toxin, lipid, carbohydrate, or combinations thereof, or any other type of therapeutic agent, such as radiation. In some embodiments, the complementary therapy, agent, or treatment includes surgery, chemotherapy, radiation therapy, transplantation, administration of cells expressing a recombinant receptor, e.g., CAR, kinase inhibitor, immune checkpoint inhibitor, mTOR pathway inhibitor, immunosuppressants, immunomodulators, antibodies, immunodestructive agents, antibodies and/or antigen-binding fragments thereof, antibody conjugates, other antibody therapies, cytotoxins, steroids, cytokines, peptide vaccines, hormone therapy, antimetabolites, metabolic modulators, drugs that inhibit either calcium-dependent calcineurin phosphatase or p70S6 kinase FK506, or p70S6 kinase inhibitors, alkylating agents, anthracyclines, vinca alkaloids, proteasome inhibitors, GITR agonists, protein tyrosine phosphatase inhibitors, protein kinase inhibitors, oncolytic virus, and/or other types of immunotherapy. In some embodiments, the additional agent or treatment is bone marrow transplantation, ablative T cell therapy using chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, and/or antibody therapy.

[0700] In some embodiments, the additional agent is a kinase inhibitor, such as a Bruton's tyrosine kinase (Btk) inhibitor, such as ibrutinib. In some embodiments, the additional agent is an adenosine pathway or adenosine receptor antagonist or agonist. In some embodiments, the additional agent is an immunomodulator, such as thalidomide or a thalidomide derivative (eg, lenalidomide). In some embodiments, the complementary therapy, agent, or treatment is a cytotoxic agent, or an agent for chemotherapy, biological therapy (eg, an antibody, eg, a monoclonal antibody or cell therapy), or an inhibitor (eg, a kinase inhibitor).

[0701] In some embodiments, the additional agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include an anthracycline (eg, doxorubicin, such as liposomal doxorubicin); vinca alkaloid (eg vinblastine, vincristine, vindesine, vinorelbine); an alkylating agent (eg, cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide); anti-immune cell antibody (eg, alemtuzumab, gemtuzumab, rituximab, tositumomab); an antimetabolite (including, for example, folic acid antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors such as fludarabine); a TNFR glucocorticoid-induced TNFR-related protein (GITR) agonist; a proteasome inhibitor (eg, aclacinomycin A, gliotoxin, or bortezomib); an immunomodulator such as thalidomide or a thalidomide derivative (eg lenalidomide).

[0702] In some embodiments, the additional agent is an immunomodulatory agent. In some embodiments, the combination therapy includes an immunomodulatory agent that can stimulate, enhance, and/or otherwise improve an antitumor immune response, e.g., an antitumor immune response from administered genetically engineered cells, for example, by inhibiting immunosuppressive signal transduction or improving immunostimulatory signal transduction. In some embodiments, the implementation of the immunomodulatory agent is a peptide, protein, or is a small molecule. In some embodiments, the protein may be a fusion protein or a recombinant protein. In some embodiments, the immunomodulatory agent binds to an immunological target, such as a cell surface receptor expressed on immune cells, such as T cells, B cells, or antigen presenting cells. For example, in some embodiments, the immunomodulatory agent is an antibody or antigen-binding antibody fragment, fusion protein, small molecule, or polypeptide. In some embodiments, binding molecules, recombinant receptors, cells, and/or compositions are administered in combination with an additional agent that is an antibody or antigen-binding fragment thereof, such as a monoclonal antibody.

[0703] In some embodiments, the immunomodulatory agent blocks, inhibits, or antagonizes a component of the immune checkpoint pathway. The immune system has multiple inhibitory pathways involved in maintaining self-tolerance and in modulating immune responses. Tumors may use some immune checkpoint pathways as the main immune mechanism, especially against T cells that are specific for tumor antigens (Pardoll (2012) Nature Reviews Cancer 12:252-264), for example, genetically engineered cells such as CAR-expressing cells. Because many of these immune checkpoints are initiated by ligand-receptor interactions, they can be easily blocked by antibodies to the ligands and/or their receptors. Unlike most anticancer agents, checkpoint inhibitors do not necessarily target tumor cells directly, but rather target lymphocyte receptors or their ligands to improve the endogenous antitumor activity of the immune system.

[0704] In some embodiments, the additional agent is an immunomodulatory agent that is an antagonistic molecule or is an immune checkpoint inhibitor capable of inhibiting or blocking the function of a molecule or signaling pathway, including an immune checkpoint molecule. In some embodiments, the immune checkpoint or pathway molecule is PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM3, VISTA, adenosine 2A receptor (A2AR), or adenosine, or a pathway comprising any from those listed. In some embodiments, antagonistic molecules that block the immune checkpoint pathway, such as small molecules, nucleic acid inhibitors (eg, RNAi), or antibody molecules, are emerging as promising agents for immunotherapy for cancer and other diseases.

[0705] In some embodiments, an immune checkpoint inhibitor is a molecule that wholly or partially reduces, inhibits, interferes with, or modulates one or more checkpoint proteins. Checkpoint proteins regulate T cell activation or function. These proteins are responsible for the costimulatory or inhibitory interactions of T cell responses. Immune checkpoint proteins regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses.

[0706] Immune checkpoint inhibitors include any agent that blocks or inhibits inhibitory pathways of the immune system in a statistically significant manner. Such inhibitors may include small molecule inhibitors or may include antibodies or antigen-binding fragments thereof that bind to and block or inhibit immune checkpoint receptors, ligands and/or receptor-ligand interaction. In some embodiments, modulation, enhancement, and/or stimulation of certain receptors can overcome components of the immune checkpoint pathway. Exemplary immune checkpoint molecules that can be targeted to block, inhibit, modulate, enhance and/or stimulate include, but are not limited to, PD-1 (CD279), PD-L1 (CD274, B7-H1), PDL2 ( CD273, B7-DC), CTLA-4, LAG-3 (CD223), TIM-3, 4-1BB (CD137), 4-1BBL (CD137L), GITR (TNFRSF18, AITR), CD40, OX40 (CD134, TNFRSF4 ), CXCR2, tumor-associated antigens (TAA), B7-H3, B7-H4, BTLA, HVEM, GAL9, B7H3, B7H4, VISTA, KIR, 2B4 (belongs to the CD2 family of molecules and is expressed on all NK, γδ and CD8 memory ⁺ (αβ) T cells), CD160 (also referred to as BY55), CGEN-15049, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), TIGIT, LAIR1, CD160, 2B4, CD80, CD86 , B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and transforming growth factor receptor (TGFR; e.g., beta TGFR) . Immune checkpoint inhibitors include antibodies or antigen-binding fragments thereof or other binding proteins that bind to and block or inhibit and/or enhance or stimulate the activity of one or more of these molecules.

[0707] Examples of immune checkpoint inhibitors include tremelimumab (CTLA-4 blocking antibody, also known as ticilimumab, CP-675,206), anti-OX40, PD-L1 monoclonal antibody (Anti-B7-H1; MEDI4736), MK-3475 ( PD-1 blocker), nivolumab (anti-PD-1 antibody), CT-011 (anti-PD-1 antibody), BY55 monoclonal antibody, AMP224 (anti-PD-L1 antibody), BMS-936559 (anti-PD -L1), MPLDL3280A (anti-PD-L1 antibody), MSB0010718C (anti-PD-L1 antibody) and ipilimumab (anti-CTLA-4 antibody also known as Yervoy®, MDX-010 and MDX-101). Examples of immunomodulatory antibodies include, but are not limited to, daclizumab (Zenapax), bevacizumab (Avastin®), basiliximab, ipilimumab, nivolumab, pembrolizumab, MPDL3280A, pidilizumab (CT-011), MK-3475, BMS-936559, MPDL3280A (atezolizumab) Tremelimumab, IMP321, BMS-986016, LAG525, URELUMAB, PF-05082566, TRX518, MK-4166, DCETUZUMAB (SGN-40), Lukatumumab (HCD122), Sea-CD40, CP-870, CP-893, Medi6, Mediy, Mediy 469, Medi6383 , MOXR0916, AMP 224, MSB0010718C (Avelumab), MEDI4736, PDR001, rHIgM12B7, ulokuplumab, BKT140, varlilumab (CDX-1127), ARGX-110, MGA271, lirilumab (BMS-986015, IPH2101), IPH2201, ARGX-115, emactuzumab , CC-90002 and MNRP1685A or their antibody-binding fragment. Other examples of immunomodulators include, for example, afutuzumab (available from Roche®); pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®); thalidomide (Thalomid®), actimide (CC4047); and IRX-2 (a mixture of human cytokines including interleukin 1, interleukin 2 and interferon-gamma, CAS 951209-71-5, available from IRX Therapeutics).

[0708] In some embodiments, the additional agent administered according to the methods of the invention and/or using the articles or compositions of the invention is an agent that binds to and/or inhibits Programmed Cell Death 1 (PD-1). PD-1 is an immune checkpoint protein expressed in B cells, NK cells and T cells (Shinohara et al., 1995, Genomics 23:704-6; Blank et al., 2007, Cancer Immunol Immunother 56:739- 45; Finger et al., 1997, Gene 197:177-87; Pardoll (2012) Nature Reviews Cancer 12:252-264). The main role of PD-1 is to limit the activity of T cells in peripheral tissues during inflammation in response to infection, as well as to limit autoimmunity. Expression of PD-1 is induced in activated T cells, and binding of PD-1 to one of its endogenous ligands inhibits T cell activation by inhibiting stimulatory kinases. PD-1 also inhibits the TCR stop signal. PD-1 is highly expressed on Treg cells and can increase their proliferation in the presence of a ligand (Pardoll (2012) Nature Reviews Cancer 12:252-264). Anti-PD-1 antibody has been used to treat melanoma, non-small cell lung cancer, bladder cancer, prostate cancer, colorectal cancer, head and neck cancer, triple negative breast cancer, leukemia, lymphoma, and renal cell cancer (Topalian et al ., 2012, N Engl J Med 366:2443-54; Lipson et al., 2013, Clin Cancer Res 19:462-8; Berger et al., 2008, Clin Cancer Res 14:3044-51; Gildener-Leapman et al., 2013, Oral Oncol 49:1089-96; Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85). Examples of anti-PD-1 antibodies include nivolumab (Opdivo, BMS), pembrolizumab (Keytruda, Merck), pidilizumab (CT-011, Cure Tech), lambrolizumab (MK-3475, Merck) and AMP 224 (Merck), nivolumab (also called Opdivo, BMS-936558 or MDX1106; Bristol-Myers Squibb), a fully human IgG4 monoclonal antibody that specifically blocks PD-1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are described in US 8,008,449 and WO2006/121168. Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD-1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are described in WO2009/101611. Pembrolizumab (formerly known as lambrolizumab, and also called Keytruda, MK03475; Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1. Pembrolizumab and other humanized anti-PD-1 antibodies are described in US 8,354,509 and WO2009/114335. Other anti-PD-1 antibodies include AMP 514 (Amplimmune), among others, for example, anti-PD-1 antibodies described in US 8,609,089, US 2010028330, US 20120114649 and/or US 20150210769. AMP 224 (B7-DCIg; Amplimmune ; for example, described in WO2010/027827 and WO2011/066342) is a PD-L2 Fc fused soluble receptor that blocks the interaction between PD-1 and B7-H1.

[0709] In some embodiments, the additional agent administered in accordance with the methods of the invention and/or using the articles or compositions of the invention is an agent that binds to or inhibits PD-L1 (also known as CD274 and B7-H1) and /or PD-L2 (also known as CD273 and B7-DC). PD-L1 and PD-L2 are ligands for PD-1 found on activated T cells, B cells, myeloid cells, macrophages, and some types of tumor cells. Anticancer therapies focus on anti-PD-L1 antibodies. The complex of PD-1 and PD-L1 inhibits CD8 ⁺ T cell proliferation and reduces the immune response (Topalian et al., 2012, N Engl J Med 366:2443-54; Brahmer et al., 2012, N Eng J Med 366: 2455-65). Anti-PD-L1 antibodies have been used to treat non-small cell lung cancer, melanoma, colorectal cancer, renal cell carcinoma, pancreatic cancer, gastric cancer, ovarian cancer, breast cancer, and hematological malignancies (Brahmer et al., 2012 , N Eng J Med 366:2455-65; Ott et al., 2013, Clin Cancer Res 19:5300-9; Radvanyi et al., 2013, Clin Cancer Res 19:5541; Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85; Berger et al., 2008, Clin Cancer Res 14:13044-51). Examples of anti-PD-L1 antibodies include MDX-1105 (Medarex), MEDI4736 (Medimmune) MPDL3280A (Genentech), BMS-935559 (Bristol-Myers Squibb) and MSB0010718C. MEDI4736 (Medimmune) is a human monoclonal antibody that binds to PD-L1 and inhibits ligand interaction with PD-1. MDPL3280A (Genentech/Roche) is a monoclonal human Fc optimized IgG1 antibody that binds to PD-L1. MDPL3280A and other human PD-L1 monoclonal antibodies are described in US Patent Publication No. 20120039906 and no. 7,943,743. Other anti-PD-L1 binders include YW243.55. S70 (see WO2010/077634), and MDX-1105 (also referred to as BMS-936559, and for example the anti-PD-L1 binders described in WO2007/005874).

[0710] In some embodiments, the additional agent administered in accordance with the methods of the invention and/or using the articles or compositions of the invention is an agent that is an inhibitor of cytotoxic T-lymphocyte-associated antigen (CTLA-4), also known as CD152, or binds to CTLA-4. CTLA-4 is a co-inhibitory molecule that regulates T cell activation. CTLA-4 is a member of the immunoglobulin superfamily, exclusively expressed on T cells. CTLA-4 inhibits T cell activation and is reported to inhibit T helper activity and enhance regulatory T cell immunosuppressive activity. Although the exact mechanism of action of CTLA-4 remains unclear, it is thought to inhibit T cell activation by displacing CD28 in binding to CD80 and CD86, as well as actively delivering inhibitory signals to the T cell (Pardoll (2012) Nature Reviews Cancer 12:252-264). Anti-CTLA-4 antibodies have been used in clinical trials to treat melanoma, prostate cancer, small cell lung cancer, non-small cell lung cancer (Robert & Ghiringhelli, 2009, Oncologist 14:848-61; Ott et al., 2013, Clin Cancer Res 19 :5300; Weber, 2007, Oncologist 12:864-72; Wada et al., 2013, J Transl Med 11:89). An essential feature of anti-CTLA-4 is the kinetics of the antitumor effect with a delay period of up to 6 months after the initial treatment, which was required for a physiological response. In some cases, tumors may actually increase in size after initiation of treatment before shrinkage is seen (Pardoll (2012) Nature Reviews Cancer 12:252-264). Examples of anti-CTLA-4 antibodies include ipilimumab (Bristol-Myers Squibb) and tremelimumab (Pfizer). Ipilimumab has recently received FDA approval for the treatment of metastatic melanoma (Wada et al., 2013, J Transl Med 11:89).

[0711] In some embodiments, the additional agent administered in accordance with the methods of the invention and/or using the articles or compositions of the invention is an agent that binds to and/or inhibits the lymphocyte activation gene 3 (LAG-3), also known like CD223. LAG-3 is another immune checkpoint protein. LAG-3 has been associated with inhibition of lymphocyte activity and in some cases induction of lymphocyte anergy. LAG-3 is expressed on various cells in the immune system, including B cells, NK cells, and dendritic cells. LAG-3 is a natural ligand for the class II MHC receptor, essentially expressed on melanoma infiltrating T cells, including those provided with potent immune suppressive activity. Examples of anti-LAG-3 antibodies include BMS-986016 (Bristol-Myers Squib), which is a monoclonal antibody that targets LAG-3. IMP701 (Immutep) is a LAG-3 antagonist antibody, and IMP731 (Immutep and GlaxoSmithKline) is a LAG-3 depleting antibody. Other LAG-3 inhibitors include IMP321 (Immutep), which is a recombinant LAG-3 soluble portion and Ig fusion protein that binds to MHC class II molecules and activates antigen presenting cells (APCs). Other antibodies are described, for example, in WO2010/019570 and US 2015/0259420.

[0712] In some embodiments, the additional agent administered in accordance with the methods of the invention and/or using the articles or compositions of the invention is an agent that binds to and/or inhibits the T-cell immunoglobulin domain and mucin domain-3 (TIM -3). TIM-3 was originally identified on activated Th1 cells and has been shown to be a negative regulator of the immune response. Blockade of TIM-3 mediates T cell-promoted antitumor immunity and has antitumor activity in various mouse tumor models. Combinations of TIM-3 blockade with other immunotherapeutic agents, such as TSR-042, anti-CD137 antibodies, and others, may additively or synergistically enhance antitumor effects. TIM-3 expression has been associated with many different tumor types, including melanoma, NSCLC, and renal cancer, and intratumoral TIM-3 expression has additionally been shown to correlate with poor prognosis in various tumor types, including NSCLC, cervical cancer, and gastric cancer. Blockade of TIM-3 is also of interest in terms of promoting increased immunity to many chronic viral diseases. TIM-3 has also been shown to interact with many ligands, including galectin-9, phosphatidylserine, and HMGB1, although it is not currently clear which of these, if any, are relevant in regulating anticarcinogenic effects. In some embodiments, antibodies, antibody fragments, small molecules, or peptidic inhibitors that target TIM-3 can bind to the IgV domain of TIM-3, inhibiting interactions with its ligands. Examples of antibodies and peptides that inhibit TIM-3 are described in US 2015/0218274, WO2013/006490 and US 2010/0247521. Other anti-TIM-3 antibodies include humanized versions of RMT3-23 (Ngiow et al., 2011, Cancer Res, 71:3540-3551), and clone 8B,2C12 (Monney et al., 2002, Nature, 415:536- 541). Bispecific antibodies that inhibit TIM-3 and PD-1 are described in US 2013/0156774.

[0713] In some embodiments, the additional agent administered in accordance with the methods of the invention and/or using the articles or compositions of the invention is an agent that is a CEACAM inhibitor (e.g., CEACAM-1, CEACAM-3, and/or a CEACAM inhibitor). -5). In some embodiments, the CEACAM inhibitor is an anti-CEACAM antibody molecule. Examples of anti-CEACAM-1 antibodies are described in WO 2010/125571, WO 2013/082366, WO 2014/059251 and WO 2014/022332, for example, monoclonal antibody 34B1, 26H7 and 5F4; or its recombinant form as described in, for example, US 2004/0047858, US 7,132,255 and WO 99/052552. In some embodiments, the anti-CEACAM antibody binds to CEACAM-5 as described in, for example, Zheng et al. PLOS One. (2011) 6(6): e21146) or cross-reacts with CEACAM-1 and CEACAM-5 as described in, for example, WO 2013/054331 and US 2014/0271618.

[0714] In some embodiments, the additional agent administered in accordance with the methods of the invention and/or using the articles or compositions of the invention is a 4-1BB binding and/or inhibitory agent, also known as CD137. 4-1BB is a transmembrane glycoprotein belonging to the TNFR superfamily. 4-1BB receptors are present on activated T and B cells and monocytes. An example of an anti-4-1BB antibody is urelumab (BMS-663513), which has potential immunostimulatory and antitumor activities.

[0715] In some embodiments, the additional agent administered in accordance with the methods of the invention and/or using the articles or compositions of the invention is an agent that binds to and/or inhibits the tumor necrosis factor receptor member 4 (TNFRSF4) superfamily, also known as OX40 and CD134. TNFRSF4 is another member of the TNFR superfamily. OX40 is not expressed constitutively on intact T cells at rest and acts as a secondary costimulatory immune checkpoint molecule. Examples of anti-OX40 antibodies are MEDI6469 and MOXR0916 (RG7888, Genentech).

[0716] In some embodiments, the additional agent administered in accordance with the methods of the invention and/or using the articles or compositions of the invention is an agent or molecule that reduces the population of regulatory T cells (Treg). Methods to reduce (eg, deplete) Treg cells are known in the art and include, for example, CD25 depletion, cyclophosphamide administration, and modulation of glucocorticoid-induced TNFR family gene (GITR) function. GITR is a member of the TNFR superfamily, upregulated on activated T cells, which enhances the immune system. Reducing the number of Treg cells in a patient prior to apheresis or prior to the introduction of genetically engineered cells, such as CAR-expressing cells, can reduce the number of unwanted immune cells (eg, Tregs) in the tumor microenvironment and reduce the patient's risk of recurrence. In some embodiments, the additional agent comprises a molecule that targets GITR and/or modulates GITR functions, such as a GITR agonist and/or a GITR antibody that depletes regulatory T cells (Tregs). In some embodiments, the implementation of the additional agent includes cyclophosphamide. In some embodiments, a GITR binding molecule and/or a molecule that modulates GITR function (eg, a GITR agonist and/or Treg depleting GITR antibodies) is administered prior to engineered cells, eg, CAR-expressing cells. For example, in some embodiments, the GITR agonist may be administered prior to cell apheresis. In some embodiments, cyclophosphamide is administered to the patient prior to administration (eg, infusion or re-infusion) of engineered cells, eg, CAR-expressing cells, or prior to cell apheresis. In some embodiments, the cyclophosphamide and the anti-GITR antibody are administered to the patient prior to administration (eg, infusion or re-infusion) of engineered cells, eg, CAR-expressing cells, or prior to cell apheresis.

[0717] In some embodiments, the additional agent administered in accordance with the methods of the invention and/or using the articles or compositions of the invention is an agent that is a GITR agonist. Examples of GITR agonists include, for example, GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies) such as, for example, the GITR fusion protein described in U.S. Patent No. 6,111,090, European Patent No. 090505 B1, US Patent No. 8,586,023, PCT Publications: WO 2010/003118 and 2011/090754, or an anti-GITR antibody as described in, for example, US Pat. 7,025,962, European Patent No. 1947183 B1, US Patent No. 7,812,135, US Patent No. 8,388,967, U.S. Patent No. 8,591,886, European Patent Number EP 1866339, PCT Publication WO 2011/028683, PCT Publication WO 2013/039954, PCT Publication WO2005/007190, PCT Publication WO 2007/133822, PCT Publication WO2005/055 808, PCT Publications WO 99/40196, PCT Publications WO 2001/03720, PCT Publication WO99/20758, PCT Publication WO2006/083289, PCT Publication WO 2005/115451, US Patent No. 7,618,632 and PCT Publication WO 2011/051726. An example of an anti-GITR antibody is TRX518.

[0718] In some embodiments, an additional agent administered in accordance with the methods of the invention and/or using the articles or compositions of the invention enhances tumor infiltration or migration of the injected cells, eg, CAR-expressing cells. For example, in some embodiments, the additional agent stimulates CD40, such as CD40L, eg, recombinant human CD40L. Cluster of differentiation 40 (CD40) is also a member of the TNFR superfamily. CD40 is a costimulatory protein found on antigen presenting cells and mediates a wide range of immune and inflammatory responses. CD40 is also expressed on some cancers where it proliferates. Examples of anti-CD40 antibodies are dacetuzumab (SGN-40), lucatumumab (Novartis, antagonist), SEA-CD40 (Seattle Genetics) and CP-870,893. In some embodiments, the additional tumor infiltration-improving agent comprises the tyrosine kinase inhibitor sunitnib, heparanase, and/or chemokine receptors such as CCR2, CCR4, and CCR7.

[0719] In some embodiments, the additional agent administered in accordance with the methods of the invention and/or using the articles or compositions of the invention is an immunomodulatory agent that is a structural or functional analog or derivative of thalidomide and/or an E3 ubiquitin ligase inhibitor. In some embodiments, an immunomodulatory agent binds to cereblon (CRBN). In some embodiments, the immunomodulatory agent binds to the CRBN E3 ubiquitin ligase complex. In some embodiments, the immunomodulatory agent binds to CRBN and the CRBN E3 ubiquitin ligase complex. In some embodiments, the immunomodulatory agent upregulates the CRBN protein or gene expression. In some aspects, CRBN is a substrate adapter for CRL4 ^CRBN E3 ubiquitin ligase and modulates enzyme specificity. In some embodiments, binding to the CRB or CRBN E3 complex of the ubiquitin ligase inhibits the activity of the E3 ubiquitin ligase. In some embodiments, the immunomodulatory agent causes ubiquitinylation of KZF1 (Ikaros) and IKZF3 (Aiolos) and/or causes degradation of IKZF1 (Ikaros) and IKZF3 (Aiolos). In some embodiments, the immunomodulatory agent causes ubiquitinylation of casein kinase 1A1 (CK1α) CRL4 ^CRBN E3 by ubiquitin ligase. In some embodiments, CK1α ubiquitinylation results in degradation of CK1α.

[0720] In some embodiments, the immunomodulatory agent is an inhibitor of the Ikaros transcription factor (IKZF1). In some embodiments, the immunomodulatory agent enhances Ikaros ubiquitinylation. In some embodiments, the implementation of the immunomodulatory agent enhances the degradation of Ikaros. In some embodiments, the immunomodulatory agent down-regulates the Ikaros protein or gene expression. In some embodiments, administration of an immunomodulatory agent causes a decrease in Ikaros protein.

[0721] In some embodiments, the immunomodulatory agent is an inhibitor of the transcription factor Aiolos (IKZF3). In some embodiments, the immunomodulatory agent enhances Aiolos ubiquitinylation. In some embodiments, the implementation of the immunomodulatory agent enhances the degradation of Aiolos. In some embodiments, the immunomodulatory agent down-regulates the Aiolos protein or gene expression. In some embodiments, administration of an immunomodulatory agent causes a decrease in Aiolos protein.

[0722] In some embodiments, the immunomodulatory agent is an inhibitor of the transcription factors Ikaros (IKZF1) and Aiolos (IKZF3). In some embodiments, the immunomodulatory agent enhances ubiquitinylation of both Ikaros and Aiolos. In some embodiments, the immunomodulatory agent enhances the degradation of both Ikaros and Aiolos. In some embodiments, the immunomodulatory agent enhances ubiquitinylation and degradation of both Ikaros and Aiolos. In some embodiments, administration of an immunomodulatory agent results in a decrease in the levels of both Aiolos and Ikaros proteins.

[0723] In some embodiments, the immunomodulatory agent is a selective cytokine inhibitory drug (SelCID). In some embodiments, the immunomodulatory agent inhibits phosphodiesterase-4 (PDE4) activity. In some embodiments, the immunomodulatory agent inhibits the enzymatic activity of CDC25 phosphatases. In some embodiments, the immunomodulatory agent modifies the intracellular traffic of CDC25 phosphatases.

[0724] In some embodiments, the immunomodulatory agent is thalidomide (2-(2,6-dioxopiperidin-3-yl)-1H-isoindol-1,3(2H)-dione) or an analog or derivative of thalidomide. In some embodiments, the thalidomide derivative includes structural variants of thalidomide having similar biological activity. Examples of thalidomide derivatives include, but are not limited to, lenalidomide (REVLIMMUNOMODULATORY COMPOUND™; Celgene Corporation), pomalidomide (also known as ACTIMMUNOMODULATORY COMPOUND™ or POMALYST™ (Celgene Corporation)), CC-1088, CDC-501 and CDC-801, and compounds disclosed in US Pat. Nos. 5,712,291; 7,320,991; and 8,716,315; US Patent Application 2016/0313300; and Publ. PCT WO 2002/068414 and WO 2008/154252.

[0725] In some embodiments, the immunomodulatory agent is 1-oxo- and 1,3-dioxo-2-(2,6-dioxo-piperidin-3-yl)isoindolines substituted with amino on the benzene ring as described in U.S. Patent 5,635,517, included in the present description by reference.

[0726] In some embodiments, the implementation of the immunomodulatory agent is a compound of the following formula:

in which one of X and Y is -C(O)- and the other X and Y is -C(O)- or -CH ₂ - and R ⁵ is hydrogen or lower alkyl, or a pharmaceutically acceptable salt thereof. In some embodiments, X is -C(O)- and Y is -CH ₂ -. In some embodiments, both X and Y are -C(O)-. In some embodiments, R ⁵ is hydrogen. In other embodiments, R ⁵ is methyl.

[0727] In some embodiments, the immunomodulatory compound is a compound belonging to the class of substituted 2-(2,6-dioxopiperidin-3-yl)phthalimidomodulatory compounds and substituted 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoles , such as those described in US Pat. Nos. 6,281,230; 6,316,471; 6,335,349; and 6,476,052 and International Patent Application No. PCT/US97/13375 (International Publication WO 98/03502), each of which is incorporated herein by reference.

[0728] In some embodiments, the implementation of the immunomodulatory agent is a compound of the following formula:

wherein

one of X and Y is -C(O)- and the other X and Y is -C(O)- or -CH ₂ -;

(1) each of R ¹ , R ² , R ³ and R ⁴ independently represents halogen, alkyl of 1-4 carbon atoms or alkoxy of 1-4 carbon atoms, or

(2) one of R ¹ , R ³ , R ⁴ and R ⁵ is -NHR ^a and the rest of R ¹ , R ² , R ³ and R ⁴ are hydrogen, with R ^a being hydrogen or alkyl of 1-8 atoms carbon;

R ⁵ is hydrogen or alkyl of 1-8 carbon atoms, benzyl or halogen;

with the proviso that R ⁵ is other than hydrogen, if X and Y are —C(O)— and (i) each of R ¹ , R ² , R ³ and R ⁴ is fluoro; or (ii) one of R ¹ , R ² , R ³ and R ⁴ is amino;

or a pharmaceutically acceptable salt thereof.

[0729] In some embodiments, the implementation of the immunomodulatory agent is a compound belonging to the class of isoindole-immunomodulatory compounds disclosed in US patent No. 7,091,353, US Patent Publication No. 2003/0045552 and International Application No. PCT/USOI/50401 (International Publication No. WO02/059106), each of which is incorporated herein by reference. For example, in some embodiments, the immunomodulatory agent is [2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmethyl]-amide ; (2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmethyl)-carbamic acid tert-butyl ester; 4-(aminomethyl)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione; N-(2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmethyl)acetamide; N-{(2-(2,6-dioxo(3-piperidyl)-1,3-dioxoisoindolin-4-yl)methyl}cyclopropylcarboxamide; 2-chloro-N-{(2-(2,6-dioxo (3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}acetamide; N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)- 3-pyridylcarboxamide;3-{1-oxo-4-(benzylamino)isoindolin-2-yl}piperidin-2,6-dione;2-(2,6-dioxo(3-piperidyl))-4-(benzylamino) isoindolin-1,3-dione; N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}propanamide; N-{(2-(2, 6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}-3-pyridylcarboxamide;N-{(2-(2,6-dioxo(3-piperidyl))-1,3- dioxoisoindolin-4-yl)methyl}heptanamide;N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}-2-furylcarboxamide;{N-( 2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)carbamoyl}methyl acetate;N-(2-(2,6-dioxo(3-piperidyl))-1,3 -dioxoisoindolin-4-yl)pentanamide;N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)-2-thienylcarboxamide;N-{[2-(2 ,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(butylamino)carboxamide; N-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(octylamino)carboxamide; or N-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(benzylamino)carboxamide.

[0730] In some embodiments, the immunomodulatory agent is a compound belonging to the class of isoindole immunomodulatory compounds disclosed in U.S. Patent Application Publication No. 2002/0045643, International Publication WO 98/54170, and US Patent No. 6,395,754, each of which is incorporated herein by reference. In some embodiments, the immunomodulatory agent is the tetrasubstituted 2-(2,6-dioxo-piperidin-3-yl)-1-oxoisoindolines described in U.S. Patent No. 5,798,368, incorporated herein by reference. In some embodiments, the immunomodulatory agent is 1-oxo and 1,3-dioxo-2-(2,6-dioxo-piperidin-3-yl)isoindolines as disclosed in U.S. Patent No. 6,403,613, incorporated herein by reference. In some embodiments, the immunomodulatory agent is 1-oxo or 1,3-dioxoisoindoline substituted at position 4 or 5 of the indoline ring, as described in U.S. Patent No. 6,380,239 and US Patent No. 7,244,759, both of which are incorporated herein by reference.

[0731] In some embodiments, the immunomodulatory agent is 2-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-4-carbamoyl-butyric acid or 4-(4-amino-1 -oxo-1,3-dihydro-isoindol-2-yl)-4-carbamoyl-butyric acid. In some embodiments, the immunomodulatory compound is 4-carbamoyl-4-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}- butyric acid, 4-carbamoyl-2-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-butyric acid, 2- {4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-4-phenylcarbamoyl-butyric acid or 2-{4-[( furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-glutaric acid.

[0732] In some embodiments, the immunomodulatory agent is isoindolin-1-one or isoindolin-1,3-dione substituted at the 2-position with 2,6-dioxo-3-hydroxypiperidin-5-yl, as described in U.S. Patent No. 6,458,810, incorporated herein by reference. In some embodiments, the immunomodulatory compound is 3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, or an enantiomer or mixture of enantiomers; or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate or polymorph thereof. In some embodiments, the immunomodulatory compound is 3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dione.

[0733] In some embodiments, the immunomodulatory agent is as described in Oshima, K. et al., Nihon Rinsho., 72(6):1130-5 (2014); Millrine, D. et al., Trends Mol Med., 23(4):348-364 (2017); and Collins, et al., Biochem J., 474(7):1127-1147 (2017).

[0734] In some embodiments, the immunomodulatory agent is lenalidomide, pomalidomide, avadomide, a stereoisomer of lenalidomide, pomalidomide, avadomide, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof. In some embodiments, the immunomodulatory compound is lenalidomide, a stereoisomer of lenalidomide, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof. In some embodiments, the immunomodulatory compound is lenalidomide or ((RS)-3-(4-Amino-1-oxo-1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione).

[0735] In some embodiments, the additional agent includes drugs based on thalidomide or its analogues and/or derivatives, such as lenalidomide, pomalidomide, or apremilast. See, for example, Bertilaccio et al., Blood (2013) 122:4171, Otahal et al., Oncoimmunology (2016) 5(4):e1115940; Fecteau et al., Blood (2014) 124(10):1637-1644 and Kuramitsu et al., Cancer Gene Therapy (2015) 22:487-495). Lenalidomide ((RS)-3-(4-Amino-1-oxo-1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione; also known as revlimide) is a synthetic derivative of thalidomide and has multiple immunomodulatory effects, including mediating immune synapse formation between the T cell and antigen presenting cells (APCs). For example, in some cases, lenalidomide modulates the T cell response and leads to increased production of interleukin (IL)-2 in CD4 ⁺ and CD8 ⁺ T cells, causes a change in the T helper (Th) response from Th2 to Th1, inhibits an increase in the subpopulation of regulatory T cells (Tregs) and enhances the functioning of immunological synapses in follicular lymphoma and chronic lymphocytic leukemia (CLL) (Otahal et al., Oncoimmunology (2016) 5 (4): e1115940). Lenalidomide also has direct tumor cell-killing activity in multiple myeloma (MM) patients and directly and indirectly modulates CLL tumor cell survival by acting on supportive cells, such as nurse-like cells, found in the microenvironment of lymphoid tissues. Lenalidomide can also enhance T cell proliferation and interferon-γ production in response to T cell activation via CD3 ligation or dendritic cell-mediated activation. Lenalidomide can also induce malignant B cells to express higher levels of immunostimulatory molecules such as CD80, CD86, HLA-DR, CD95 and CD40 (Fecteau et al., Blood (2014) 124(10):1637-1644). In some embodiments, lenalidomide is administered at a dosage of from about 1 mg to about 20 mg per day, for example, from about 1 mg to about 10 mg, from about 2.5 mg to about 7.5 mg, from about 5 mg to about 15 mg, for example, about 5 mg, 10 mg, 15 mg or 20 mg per day. In some embodiments, lenalidomide is administered at a dose of from about 10 µg/kg to 5 mg/kg, for example, from about 100 µg/kg to about 2 mg/kg, from about 200 µg/kg to about 1 mg/kg, from about 400 µg/kg to about 600 µg/kg, for example about 500 µg/kg.

[0736] In some embodiments, the additional agent administered in accordance with the methods of the invention and/or using articles or compositions of the invention is a B cell inhibitor. In some embodiments, the additional agent is one or more B cell inhibitors selected from CD10, CD19, CD20, CD22, CD34, CD123, CD79a, CD79b, CD179b, FLT-3, or ROR1 inhibitors, or combinations thereof. In some embodiments, the B cell inhibitor is an antibody (eg, a mono- or bispecific antibody) or an antigen-binding fragment thereof. In some embodiments, the additional agent is a genetically engineered cell expressing recombinant receptors targeting B cell targets, e.g., CD10, CD19, CD20, CD22, CD34, CD123, CD79a, CD79b, CD179b, FLT-3, or ROR1.

[0737] In some embodiments, the additional agent administered in accordance with the methods of the invention and/or using the articles or compositions of the invention is a CD20 inhibitor, e.g., an anti-CD20 antibody (e.g., an anti-CD20 mono- or bispecific antibody) or a fragment of it. Examples of anti-CD20 antibodies include, but are not limited to, rituximab, ofatumomab, ocrelizumab (also known as GA101 or RO5072759), veltuzumab, obinutuzumab, TRU-015 (Trubion Pharmaceuticals), ocaratuzumab (also known as AME-133v or ocaratuzumab), and Pro131921 (Genentech). See, for example, Lim et al. haematologica. (2010) 95(1):135-43. In some embodiments, the anti-CD20 antibody comprises rituximab. Rituximab is a chimeric mouse/human kappa IgG1 monoclonal antibody that binds to CD20 and induces cytolysis of CD20-expressing cells. In some embodiments, the implementation of the additional agent includes rituximab. In some embodiments, the CD20 inhibitor is a small molecule.

[0738] In some embodiments, the additional agent administered according to the methods of the invention and/or using the articles or compositions of the invention is a CD22 inhibitor, e.g., an anti-CD22 antibody (e.g., an anti-CD22 mono- or bispecific antibody) or a fragment of it. Examples of anti-CD22 antibodies include epratuzumab and RFB4. In some embodiments, the CD22 inhibitor is a small molecule. In some embodiments, the antibody is a monospecific antibody, optionally conjugated to a second agent, such as a chemotherapeutic agent. For example, in some embodiments, the antibody is an anti-CD22 monoclonal antibody-MMAE conjugate (eg, DCDT2980S). In some embodiments, the antibody is an anti-CD22 scFv, eg, an RFB4 scFv. In some embodiments, the scFv is fused to all or a fragment of Pseudomonas exotoxin-A (eg, BL22). In some embodiments, the scFv is fused to all or a fragment (eg, a 38 kD fragment) of a Pseudomonas exotoxin-A (eg, moxetumomab pasudotox). In some embodiments, the anti-CD22 antibody is an anti-CD19/CD22 bispecific antibody, optionally conjugated to a toxin. For example, in some embodiments, an anti-CD22 antibody comprises an anti-CD19/CD22 bispecific moiety (e.g., two scFv ligands recognizing human CD19 and CD22) optionally linked to all or part of a diphtheria toxin (DT), e.g., the first 389 amino acids diphtheria toxin (DT), DT 390, for example a ligand-targeted toxin such as DT2219ARL). In some embodiments, the bispecific moiety (eg, anti-CD 19/anti-CD22) is linked to a toxin, such as a deglycosylated ricin chain (eg, Combotox).

[0739] In some embodiments, the additional agent administered in accordance with the methods of the invention and/or using the articles or compositions of the invention is a cytokine or is an agent that causes increased expression of cytokines in the tumor microenvironment. Cytokines have important functions related to growth, differentiation, survival, and homeostasis of T cells. Cytokines that may be administered to a patient receiving combination therapy in the methods or uses of the invention, the recombinant receptors, cells and/or compositions described herein, include one or more of IL-2, IL-4, IL-7, IL-9, IL-15, IL-18 and IL-21. In some embodiments, the cytokine administered is IL-7, IL-15, or IL-21, or a combination thereof. In some embodiments, administration of a cytokine to a patient having a suboptimal response to administration of engineered cells, eg, CAR-expressing cells, improves the efficacy and/or antitumor activity of the administered cells, eg, CAR-expressing cells.

[0740] In some embodiments, the additional agent administered in accordance with the methods of the invention and/or using the articles or compositions of the invention is a cytokine, such as a protein, that acts as intercellular mediators on another cell. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Among the cytokines, growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone are included; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor alpha and beta; müllerian inhibitory substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet growth factor; transforming growth factors (TGF) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon alpha, beta and gamma; colony stimulating factors (CSF) such as macrophage CSF (M-CSF); granulocyte-macrophage CSF (GM-CSF); and granulocyte CSF (G-CSF); interleukins (IL) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10 , IL-11, IL-12; IL-15, tumor necrosis factor such as TNF-alpha or TNF-beta; and other polypeptide factors including LIF and kit ligand (KL). Within the scope of the invention, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of native cytokine sequences. For example, the immunomodulatory agent is a cytokine, and the cytokine is IL-4, TNF-α, GM-CSF, or IL-2.

[0741] In some embodiments, the additional agent administered in accordance with the methods of the invention and/or using the articles or compositions of the invention comprises an interleukin 15 (IL-15) polypeptide, an interleukin 15 receptor alpha (IL-15Rα) polypeptide, or a combination of them, such as hetIL-15 (Admune Therapeutics, LLC). hetIL-15 is a heterodimeric non-covalent complex of IL-15 and IL-15Rα. hetIL-15 is described in, for example, US 8,124,084, US 2012/0177598, US 2009/0082299, US 2012/0141413 and US 2011/0081311. In some embodiments, the implementation of the immunomodulatory agent may contain one or more cytokines. For example, the interleukin may include an injection of leukocyte interleukin (Multikine), which is a combination of naturally occurring cytokines.

[0742] In some embodiments, the additional agent administered according to the methods of the invention and/or using the articles or compositions of the invention is an adenosine level modulator and/or a component of the adenosine pathway. Adenosine can function as an immunomodulatory agent in the body. For example, adenosine and certain adenosine analogs that nonselectively activate adenosine receptor subtypes reduce neutrophil production of inflammatory oxidative products (Cronstein et al., Ann. N.Y. Acad. Sci. 451:291, 1985; Roberts et al., Biochem. J., 227 :669, 1985; Schrier et al., J. Immunol. 137:3284, 1986; Cronstein et al., Clinical Immunol. Immunopath. 42:76, 1987). In some cases, the concentration of extracellular analogs of adenosine or adenosine may increase in certain environments, such as tumor microenvironment (TME). In some instances, signaling of the adenosine analog or adenosine is dependent on hypoxia or factors involved in or regulating hypoxia, such as hypoxia-inducible factor (HIF). In some embodiments, an increase in adenosine signal transduction can increase in intracellular cAMP and cAMP-dependent protein kinase, resulting in inhibition of proinflammatory cytokine production and can lead to the synthesis of immunosuppressive molecules and the development of Tregs (Sitkovsky et al., Cancer Immunol Res (2014) 2( 7):598-605). In some embodiments, an additional agent can reduce or reverse the immunosuppressive effects of adenosine, adenosine analogs, and/or adenosine transduction signals. In some embodiments, an additional agent can reduce or reverse hypoxia-driven immunosuppression of A2 adenosinergic T cells. In some embodiments, the additional agent is selected from adenosine receptor antagonists, extracellular adenosine degrading agents, inhibitors of CD39/CD73 adenosine production by ectoenzymes, and inhibitors of hypoxia-HIF-1α signal transduction. In some embodiments, the additional agent is an adenosine receptor antagonist or agonist.

[0743] In some embodiments, the additional agent administered according to the methods of the invention and/or using the articles or compositions of the invention is an agent that inhibits the activity and/or amount of the adenosine receptor. In some embodiments, inhibition or reduction of extracellular adenosine or an adenosine receptor by an inhibitor of extracellular adenosine (such as an agent that prevents the formation of, destroys, inactivates, and/or reduces extracellular adenosine) and/or an adenosine receptor inhibitor (such as an adenosine receptor antagonist) is contemplated. ) can enhance the immune response, such as macrophage, neutrophil, granulocyte, dendritic cell, T and/or B cell mediated response. In addition, inhibitors of the Gs-mediated cAMP-dependent intracellular pathway and inhibitors of the adenosine receptor-induced Gi protein-mediated intracellular pathways can also increase acute and chronic inflammation.

[0744] In some embodiments, the additional agent administered according to the methods of the invention and/or using the articles or compositions of the invention is an adenosine receptor antagonist or agonist, e.g., an antagonist or agonist of one or more of the A2a, A2b, A1 adenosine receptors. and A3. A1 and A3 inhibit, and A2a and A2b stimulate, respectively, adenylate cyclase activity. Some adenosine receptors, such as A2a, A2b, and A3, can suppress or reduce the immune response during inflammation. Thus, counteracting immunosuppressive adenosine receptors can increase, enhance, or enhance the immune response, eg, the immune response from introduced cells, eg, CAR-expressing T cells. In some embodiments, the additional agent inhibits extracellular adenosine production and adenosine-induced signal transduction through adenosine receptors. For example, enhancement of the immune response, local tissue inflammation, and targeted tissue destruction can be enhanced by inhibiting or attenuating adenosine-producing local tissue hypoxia; by destroying (or inactivating) accumulated extracellular adenosine; by preventing or reducing the expression of adenosine receptors on immune cells; and/or by inhibiting/opposing signal transduction by adenosine ligands via adenosine receptors.

[0745] In some embodiments, the additional agent administered according to the methods of the invention and/or using the articles or compositions of the invention is an adenosine receptor antagonist. In some embodiments, the antagonist is a small molecule or chemical compound of an adenosine receptor, such as an A2a, A2b, or A3 receptor. In some embodiments, the antagonist is a peptide or peptidomimetic that binds to the adenosine receptor but does not trigger a Gi protein-dependent intracellular pathway. Examples of such antagonists are described in US Pat. Nos. 5,565,566; 5.545, 627, 5.981.524; 5,861,405; 6,066,642; 6,326,390; 5,670,501; 6,117,998; 6,232,297; 5,786,360; 5,424,297; 6,313,131, 5,504,090; and 6,322,771.

[0746] In some embodiments, the additional agent is an A2 receptor (A2R) antagonist, such as an A2a antagonist. Examples of an A2R antagonist include, but are not limited to, KW6002 (istradephylline), SCH58261, caffeine, paraxanthine, 3,7-dimethyl-1-propargylxanthine (DMPX), 8-(m-chlorostyryl)caffeine (CSC), MSX-2, MSX-3, MSX-4, CGS-15943, ZM-241385, SCH-442416, preladenant, vipadenant (BII014), V2006, ST-1535, SYN-115, PSB-1115, ZM241365, FSPTP and targetable inhibitory nucleic acid on the expression of A2R, for example, siRNA or shRNA, or any antibodies or antigen-binding fragment thereof that is targeted to A2R. In some embodiments, the additional agent is an A2R antagonist as described in, for example, Ohta et al., Proc Natl Acad Sci U S A (2006) 103:13132-13137; Jin et al., Cancer Res. (2010) 70(6):2245-2255; Leone et al., Computational and Structural Biotechnology Journal (2015) 13:265-272; Beavis et al., Proc Natl Acad Sci U S A (2013) 110:14711-14716; and Pinna, A., Expert Opin Investig Drugs (2009) 18:1619-1631; Sitkovsky et al., Cancer Immunol Res (2014) 2(7):598-605; US 8,080,554; US 8,716,301; US20140056922; WO2008/147482; US 8,883,500; US20140377240; WO02/055083; US 7,141,575; US 7,405,219; US 8,883,500; US 8,450,329 and US 8,987,279).

[0747] In some embodiments, an adenosine receptor antagonist that is an antisense molecule, an inhibitory nucleic acid molecule (e.g., a small inhibitory RNA (siRNA)) or a catalytic nucleic acid molecule (e.g., a ribozyme) that specifically binds to an mRNA encoding an adenosine receptor . In some embodiments, an antisense molecule, an inhibitory nucleic acid molecule, or a catalytic nucleic acid molecule binds to nucleic acids encoding A2a, A2b, or A3. In some embodiments, an antisense molecule, an inhibitory nucleic acid molecule, or a catalytic nucleic acid molecule targets biochemical pathways downstream of the adenosine receptor. For example, an antisense molecule or a catalytic nucleic acid can inhibit an enzyme involved in a Gs protein or Gi protein dependent intracellular pathway. In some embodiments, the implementation of the additional agent includes a dominant negative mutant form of the adenosine receptor, such as A2a, A2b or A3.

[0748] In some embodiments, the additional agent administered in accordance with the methods of the invention and/or using the articles or compositions of the invention is an extracellular adenosine inhibitory agent. Agents that inhibit extracellular adenosine include agents that make extracellular adenosine non-functional (or reduce such function), such as a substance that alters the structure of adenosine to inhibit the ability of adenosine to transduce signals through adenosine receptors. In some embodiments, the additional agent is an extracellular adenosine-generating or adenosine-degrading enzyme, a modified form thereof, or a modulator. For example, in some embodiments, the additional agent is an enzyme (e.g., adenosine deaminase) or other catalytic molecule that selectively binds to and degrades adenosine, thereby suppressing or significantly reducing the ability of endogenously formed adenosine to transduce signals through adenosine receptors and result in inflammation.

[0749] In some embodiments, the additional agent is adenosine deaminase (ADA) or a modified form thereof, such as recombinant ADA and/or polyethylene glycol modified ADA (ADA-PEG), which can inhibit local tissue accumulation of extracellular adenosine. ADA-PEG has been used in the treatment of ADA SCID patients (Hershfield (1995) Hum Mutat. 5:107). In some embodiments, the extracellular adenosine inhibitory agent includes agents that prevent or reduce the formation of extracellular adenosine and/or prevent or reduce the accumulation of extracellular adenosine, thereby eliminating or substantially reducing the immunosuppressive effects of adenosine. In some embodiments, the implementation of the additional agent specifically inhibits enzymes and proteins involved in the regulation of the synthesis and/or secretion of pro-inflammatory molecules, including modulators of nuclear transcription factors. Suppression of adenosine receptor expression or expression of a Gs-dependent protein or Gi protein of an intracellular pathway or a cAMP-dependent intracellular pathway can lead to an increase/strengthening of the immune response.

[0750] In some embodiments, an additional agent may target ectoenzymes that generate or produce extracellular adenosine. In some embodiments, the additional agent targets the CD39 and CD73 ectoenzymes working in tandem to generate extracellular adenosine. CD39 (also called ectonucleoside triphosphate diphosphohydrolase) converts extracellular ATP (or ADP) to 5'AMP. Subsequently, CD73 (also called 5'nucleotidase) converts 5'AMP to adenosine. CD39 activity is reversible under the action of NDP kinase and adenylate kinase, while CD73 activity is irreversible. CD39 and CD73 are expressed on tumor stromal cells, including endothelial cells and Tregs, and also on many cancer cells. For example, expression of CD39 and CD73 on endothelial cells is increased in hypoxic conditions of the tumor microenvironment. Tumor hypoxia may result from inadequate blood supply and disorganized tumor vasculature impairing oxygen delivery (Carroll and Ashcroft (2005), Expert. Rev. Mol. Med. 7(6):1-16). Hypoxia also inhibits adenylate kinase (AK), which converts adenosine to AMP, resulting in very high extracellular concentrations of adenosine. Thus, adenosine is released at high concentrations in response to hypoxia, which is a condition often occurring in the tumor microenvironment (TME), in or around solid tumors. In some embodiments, the additional agent is one or more of an anti-CD39 antibody or antigen-binding fragment thereof, an anti-CD73 antibody or antigen-binding fragment thereof, e.g., MEDI9447 or TY/23, α-β-methylene adenosine diphosphate (ADP) , ARL 67156, POM-3, IPH52 (see e.g. Allard et al. Clin Cancer Res (2013) 19(20):5626-5635; Hausler et al., Am J Transl Res (2014) 6(2): 129-139; Zhang, B., Cancer Res. (2010) 70(16):6407-6411).

[0751] In some embodiments, the additional agent administered in accordance with the methods of the invention and/or using articles or compositions of the invention is a chemotherapeutic agent (sometimes referred to as a cytotoxic agent). In some embodiments, the chemotherapeutic agent is any agent known to those skilled in the art to be effective in the treatment, prevention, or amelioration of hyperproliferative disorders such as cancer. Chemotherapeutic agents include, but are not limited to, small molecules, synthetic drugs, peptides, polypeptides, proteins, nucleic acids (e.g., DNA and RNA polynucleotides, including, but not limited to, antisense nucleotide sequences, triple helixes, and nucleotide sequences encoding biologically active proteins, polypeptides or peptides), antibodies, synthetic or natural inorganic molecules, mimetics and synthetic or natural organic molecules. In some embodiments, the chemotherapeutic drugs include alkylating agents, anthracyclines, cytoskeletal disruptors (taxanes), epothilones, histone deacetylase inhibitors, topoisomerase inhibitors, topoisomerase II inhibitors, kinase inhibitors, nucleotide analogs and precursor analogs, peptide antibiotics, platinum-based agents, and vinca alkaloids and derivatives.

[0752] Chemotherapeutic agents may include, but are not limited to, abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, asparaginase, live BCG, bevacizumab, bexarotene, bleomycin, bortezomib, busulfan, calusterone, camptothecin , capecitabine, carboplatin, carmustine, celecoxib, cetuximab, chlorambucil, cinacalcet, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, darbepoetin alfa, daunorubicin, denileukin diftitox, dexrazoxane, docetaxel, doxorubicin, dromostano lon, Elliot B solution, epirubicin, epoetin alpha, estramustine, etoposide, exemestane, filgrastim, floxuridine, fludarabine, fluorouracil, fulvestrant, gemcitabine, gemtuzumab ozogamicin, gefitinib, goserelin, hydroxyurea, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib, interferon alfa-2a, interferon alfa- 2b, irinotecan, letrozole, leucovorin, levamisole, lomustine, meclorethamine, megestrol, melphalan, mercaptopurine, mesna, methotrexate, methoxsalen, methylprednisolone, mitomycin C, mitotane, mitoxantrone, nandrolone, nofetumomab, oblimersen, oprelvekin, oxaliplatin, paclitaxel, pamidronate, pegade emazu, pegaspargasu, pegfligrastim , pemetrexed, pentostatin, pipobroman, plicamycin, polypheprosan, porfimer, procarbazine, akrikhin, rasburicase, rituximab, sargramostim, streptozocin, talc, tamoxifen, tarceva, temozolid, teniposide, testolactone, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuz umab, tretinoin , uramustine, valrubicin, vinblastine, vincristine, vinorelbine and zoledronate.

[0753] In some embodiments, the additional agent is an inhibitor of the hypoxia-inducible factor 1 alpha (HIF-1α) signaling pathway. Examples of HIF-1α inhibitors include digoxin, acriflavin, sirtuin-7 and ganetespib.

[0754] In some embodiments, the implementation of the additional agent includes a protein tyrosine phosphatase inhibitor, for example, a protein tyrosine phosphatase inhibitor described here. In some embodiments, the protein tyrosine phosphatase inhibitor is an SHP-1 inhibitor, such as an SHP-1 inhibitor described herein, such as, for example, sodium stibogluconate. In some embodiments, the protein tyrosine phosphatase inhibitor is an SHP-2 inhibitor, such as the SHP-2 inhibitor described herein.

[0755] In some embodiments, the additional agent is a kinase inhibitor. Kinase inhibitors, such as a CDK4 kinase inhibitor, a BTK kinase inhibitor, an MNK kinase inhibitor, or a DGK kinase inhibitor, can regulate constitutively active survival pathways present in tumor cells and/or modulate immune cell function. In some embodiments, the kinase inhibitor is a Bruton's tyrosine kinase (BTK) inhibitor, such as ibrutinib. In some embodiments, the kinase inhibitor is a phosphatidylinositol 4,5-biphosphate-3-kinase (PI3K) inhibitor. In some embodiments, the kinase inhibitor is a CDK4 inhibitor, such as a CDK4/6 inhibitor. In some embodiments, the kinase inhibitor is an mTOR inhibitor, such as, for example, rapamycin, a rapamycin analog, OSI-027. An mTOR inhibitor can be, for example, an mTORC1 inhibitor and/or an mTORC2 inhibitor, eg an mTORC1 inhibitor and/or an mTORC2 inhibitor. In some embodiments, the kinase inhibitor is an MNK inhibitor or a dual PI3K/mTOR inhibitor. In some embodiments, other examples of kinase inhibitors include the AKT inhibitor perifosin, the mTOR inhibitor temsirolimus, the Src kinase inhibitors dasatinib and fostamatinib, the JAK2 inhibitors pacritinib and ruxolitinib, the PKCβ inhibitors enzastuaurin and bryostatin, and the AAK inhibitor alisertib.

[0756] In some embodiments, the kinase inhibitor is a BTK inhibitor selected from ibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13. In some embodiments, the BTK inhibitor does not reduce or inhibits interleukin-2 inducible kinase (ITK) kinase activity and is selected from GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13.

[0757] In some embodiments, the kinase inhibitor is a BTK inhibitor, such as ibrutinib (1-[(3R)-3-[4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine -1-yl]piperidin-1-yl]prop-2-en-1-one; also known as PCI-32765). In some embodiments, the kinase inhibitor is a BTK inhibitor, such as ibrutinib (PCI-32765), and ibrutinib is administered at a dose of about 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg (eg, 250 mg, 420 mg, or 560 mg) per day for a period of time, such as daily for a 21-day cycle, or daily for a 28- daily cycle. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of ibrutinib are administered. In some embodiments, the BTK inhibitor is the BTK inhibitor described in International Application WO 2015/079417.

[0758] In some embodiments, the kinase inhibitor is a PI3K inhibitor. PI3K is central to the PI3K/Akt/mTOR pathway involved in cell cycle regulation and lymphoma survival. Examples of a PI3K inhibitor include idelicib (a PI3Kδ inhibitor). In some embodiments, the additional agent is idelicib and rituximab.

[0759] In some embodiments, the implementation of the additional agent is an inhibitor of the target of rapamycin in mammalian cells (mTOR). In some embodiments, the kinase inhibitor is an mTOR inhibitor selected from temsirolimus; ridaphorolimus (also known as AP23573 and MK8669); everolimus (RAD001); rapamycin (AY22989); simapimoda; AZD8055; PF04691502; SF1126; and XL765. In some embodiments, the additional agent is a mitogen-activated protein kinase (MAPK) inhibitor, such as vemurafenib, dabrafenib, and trametinib.

[0760] In some embodiments, the implementation of the additional agent is an agent that regulates pro - or anti-apoptotic proteins. In some embodiments, the additional agent comprises an inhibitor of B-cell lymphoma 2 (BCL-2) (eg, venetoclax, also named ABT-199 or GDC-0199; or ABT-737). Venetoclax is a small molecule (4-(4-{[2-(4-chlorophenyl)-4,4-dimethyl-1-cyclohexen-1-yl]methyl}-1-piperazinyl)-N-({3-nitro- 4-[(Tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide), which inhibits the anti-apoptotic BCL-2 protein . Other pro- or anti-apoptotic protein modulating agents include the ABT-737 BCL-2 inhibitor, navitoclax (ABT-263); Mcl-1 siRNA or Mcl-1 inhibitor retinoid (4-hydroxyphenyl)N-retinamide (4-HPR) for maximum effectiveness. In some embodiments, the additional agent provides pro-apoptotic stimuli, such as a recombinant tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL), which can activate the apoptotic pathway by binding to tumor cell surface death receptors TRAIL DR-4 and DR-5 or TRAIL- R2 agonist antibodies.

[0761] In some embodiments, the additional agent includes a cytotoxic agent, e.g., CPX-351 (Cyclacel Pharmaceuticals), cytarabine, daunorubicin, vozaroxin (Sunesis Pharmaceuticals), sapacitabine (Cyclacel Pharmaceuticals), idarubicin, or mitoxantrone. In some embodiments, the additional agent comprises a hypomethylating agent, such as a DNA methyltransferase inhibitor, such as azacitidine or decitabine.

[0762] In another embodiment, the complementary therapy is transplantation, eg, an allogeneic stem cell transplant.

[0763] In some embodiments, the additional therapy is an antilymphocyte therapy. In some embodiments, lymphodepletion is performed on a patient, for example, before the introduction of genetically engineered cells, such as CAR-expressing cells. In some embodiments, lymphodepletion includes administration of one or more of melphalan, cytoxan, cyclophosphamide, and fludarabine. In some embodiments, anti-lymphocyte chemotherapy is administered to a patient before, simultaneously with, or after administration (eg, infusion) of engineered cells, eg, CAR-expressing cells. In an example, anti-lymphocyte chemotherapy is administered to a patient prior to the introduction of engineered cells, such as CAR-expressing cells.

[0764] In some embodiments, the additional agent is an oncolytic virus. In some embodiments, oncolytic viruses are capable of selectively replicating in and triggering the death or growth inhibition of cancer cells. In some cases, oncolytic viruses have no or minimal effect on non-cancerous cells. An oncolytic virus includes, but is not limited to, oncolytic adenovirus, oncolytic herpes simplex viruses, oncolytic retroviruses, oncolytic parvoviruses, oncolytic vaccinia viruses, oncolytic Sinbis viruses, oncolytic influenza viruses, or oncolytic RNA viruses (e.g., oncolytic reovirus, oncolytic reovirus (SARS) NDV), measles oncolytic virus, or oncolytic vesicular stomatitis virus (VSV)).

[0765] Other examples of combination therapies, treatments, and/or agents include antiallergenic agents, antiemetics, analgesics, and adjunctive therapies. In some embodiments, the additional agent includes cytoprotective agents such as neuroprotective agents, free radical scavengers, cardioprotective agents, anthracycline extravasation neutralizers, and nutrients.

[0766] In some embodiments, the antibody used as an additional agent is conjugated to or otherwise linked to a therapeutic agent, e.g., a chemotherapeutic agent (e.g., cytoxan, fludarabine, a histone deacetylase inhibitor, a demethylating agent, a peptide vaccine, antineoplastic antibiotic, a tyrosine kinase inhibitor, alkylating agent, antimicrotubule or antimitotic agent), antiallergic agent, antinausea (or antiemetic), analgesic or cytoprotective agent as described herein. In some embodiments, the additional agent is an antibody-drug conjugate.

[0767] Any of the additional agents described herein may be prepared and administered as a combination therapy described in the methods, uses, articles or compositions of the invention, for example, in pharmaceutical compositions comprising one or more combination therapy agents and a pharmaceutically acceptable carrier, such as any described here. In some embodiments, the combination therapy in the methods, uses, articles or compositions of the invention may be administered simultaneously, in parallel or sequentially, in any order, with additional agents, therapy or treatment, and such administration provides therapeutically effective levels of each of the agents in the patient's body . In some embodiments, an additional agent may be used in conjunction with combination therapy in the methods, uses, articles, or compositions of the invention, for example, as part of the same pharmaceutical composition or using the same delivery method. In some embodiments, the additional agent is administered concurrently with cell therapy, eg, a dose of engineered T cells (eg, CAR ⁺ T cells), but in separate compositions. In some embodiments, an additional agent is incubated with a genetically engineered cell, such as CAR expressing cells, prior to cell administration.

[0768] In some examples, one or more additional agents are administered after or before the introduction of cell therapy, for example, a dose of genetically engineered T cells (eg, CAR ⁺ T cells) at a selected time interval. In some examples, the time interval is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or 3 months. In some examples, one or more additional agents are administered multiple times. In some embodiments, an additional agent is administered prior to cell therapy, e.g., doses of engineered T cells (CAR ⁺ T cells) in the methods, uses, articles, or compositions of the invention, e.g., two weeks, 12 days, 10 days, 8 days, one week, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day prior to administration. In some embodiments, an additional agent is administered after cell therapy, e.g., doses of engineered T cells (e.g., CAR ⁺ T cells) in the methods, uses, articles, or compositions of the invention, e.g., two weeks, 12 days, 10 days, 8 days, one week, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day after administration.

[0769] The dose of the additional agent may be any therapeutically effective amount, for example, any amount of the dose described herein, and the appropriate dosage of the additional agent may depend on the type of disease being treated, the type, dose, and/or frequency of the binding molecule, recombinant receptor , cells and/or composition administered, severity and course of the disease, previous therapy, patient history and response to cell therapy, such as the dose of engineered T-cells (CAR ⁺ T-cells), and is at the discretion of the attending physician.

VI. PRODUCTS AND SETS

[0770] The invention also relates to articles and kits containing genetically engineered cells expressing the recombinant receptor, or compositions thereof, and optionally instructions for use, for example, instructions for administration, according to the methods of the invention.

[0771] In some embodiments, the invention relates to articles and/or kits comprising a composition comprising a therapeutically effective amount of any of the genetically engineered cells described herein and instructions for administration to a patient for the treatment of a disease or condition. In some embodiments, the instructions may define some or all of the elements of the methods of the invention. In some embodiments, the instructions define specific instructions for administering cells for cell therapy, such as dose, timing, selection and/or identification of patients to administer, and conditions for administering. In some embodiments, the articles and/or kits further include one or more additional agents for therapy, e.g., anti-lymphocyte therapy and/or combination therapy, such as any described herein, and optionally further includes instructions for administering the additional agent for therapy. In some embodiments, the articles and/or kits further comprise an anti-lymphocyte therapy agent, and optionally further include instructions for administering the anti-lymphocyte therapy. In some embodiments, instructions may be included as a label or package insert accompanying the administration compositions.

[0772] In some embodiments, the instructions define criteria for selecting or identifying patients for therapy. In some embodiments, the implementation of such criteria include patients with NHL, or highlight their subtypes and/or high risk of NHL. In some embodiments, the instructions define that patients being treated include patients having a disease or condition characterized or defined as aggressive NHL, diffuse large B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), T-cell/histocyte-rich large B-cell lymphoma (TCHRBCL), Burkitt's lymphoma, mantle cell lymphoma (MCL), and/or follicular lymphoma (FL). In some embodiments, the patient being treated includes patients with aggressive NHL, specifically J with diffuse large B-cell lymphoma (DLBCL), unspecified (NOS) and in some aspects including de novo and transformed from indolent). In some aspects, the patient or population being treated may include and/or additionally include patients with primary mediastinal B cell lymphoma (PMBCL) or grade 3B follicular lymphoma (FL3B). In some embodiments, the implementation of the patient or population that is treated includes patients with unsatisfactory status. In some aspects, the population being treated includes, for example, patients having an Eastern Cooperative Oncology Group Performance Status (ECOG) that is between 0-2. In other aspects of any of the embodiments, the patients being treated include ECOG 0-1 or do not include ECOG2 patients. In some embodiments of any of the embodiments, the patients being treated have failed two or more previous therapies. In some embodiments, the patient does not have DLBCL transformed from marginal zone lymphoma (MZL) and chronic lymphocytic leukemia (CLL; Richter) and/or has DLBCL characterized as de novo or transformed from indolent disease. In some embodiments, the patient has mantle cell lymphoma (MCL). In some embodiments, the instructions specify that administering cell therapy to a patient who is or has been identified as having double/triple hit lymphoma (or well-differentiated B-cell lymphoma with MYC and BCL2 and/or BCL6 permutations with DLBCL histology) has been identified as having chemo-refractory lymphoma (eg, chemorefractive DLBCL) and/or has not achieved complete remission (CR) in response to prior therapy.

[0773] In some embodiments, the instructions specify the dose of cells to be administered. For example, in some embodiments, the dose specified in the instructions includes total recombinant receptor (e.g., CAR) expressing cells in an amount from about 1x10 ⁶ to 3x10 ⁸ , for example, in the range from about 1x10 ⁷ to 2x10 ⁸ such cells, for example, 1x10 ⁷ , 5x10 ⁷ , 1x10 ⁸ , or 1.5x10 ⁸ such total cells, or a range between any two of the specified values. In some embodiments, the patient is administered multiple doses, and each of the doses or the total dose may be any of the indicated values.

[0774] In some embodiments, the article or kit includes a container, optionally a vial, comprising a plurality of CD4 ⁺ T cells expressing the recombinant receptor, and a container, optionally a vial, comprising a plurality of CD8 ⁺ T cells expressing the recombinant receptor. In some embodiments, the product or kit includes a container, optionally a vial, comprising a plurality of CD4 ⁺ T cells expressing the recombinant receptor and further comprising, in the same container, a plurality of CD8 ⁺ T cells expressing the recombinant receptor. In some embodiments, the cryoprotectant is included with the cells. In some aspects, the container is a bag.

[0775] In some embodiments, the container, such as a vial, includes more than or more than about 10x10 ⁶ T cells or expressing a recombinant T cell receptor, more than or more than about 15x10 ⁶ T cells or expressing a recombinant T-receptor. cells greater than or greater than about 25x10 ⁶ T cells, or expressing a recombinant T cell receptor. In some aspects, the vial contains from about 10 million cells per ml to about 70 million cells per ml, from about 10 million cells per ml to about 50 million cells per ml, from about 10 million cells per ml to about 25 million cells per ml, from about 10 million cells per ml to about 15 million cells per ml, from 15 million cells per ml to about 70 million cells per ml, from about 15 million cells per ml to about 50 million cells per ml, from about 15 million cells per ml to about 25 million cells per ml, from about 25 million cells per ml to about 70 million cells per ml, from about 25 million cells per ml to about 50 million cells per ml, and from about 50 million cells per ml to about 70 million cells per ml.

[0776] In some embodiments, the plurality of vesicles or the plurality of cells or unit doses of cells defined for administration collectively comprise a cell dose comprising from or about 1x10 ⁵ to 5x10 ⁸ total recombinant receptor-expressing T cells or total T cells, from 1x10 ⁵ to 1x10 ⁸ total recombinant receptor-expressing T cells or total T cells, from or from about 5x10 ⁵ to 1x10 ⁷ total recombinant receptor-expressing T cells or total T cells, or from or from about 1x10 ⁶ to 1x10 ⁷ total recombinant receptor-expressing T cells or total T cells, inclusive. In some aspects, the article of manufacture comprises one or more unit doses of CD4 ⁺ and CD8 ⁺ cells, or CD4 ⁺ receptor ⁺ cells and CD8 ⁺ receptor ⁺ cells, wherein a single dose comprises from or about 1x10 ⁷ to or up to about 2x10 ⁸ expressing the recombinant T receptor -cells, from or from about 5x10 ⁷ to or up to about 1.5x10 ⁸ expressing the recombinant receptor T cells, equals or approximately equals 5x10 ⁷ expressing the recombinant receptor T cells, equals or approximately equals 1x10 ⁸ expressing the recombinant receptor T cells , or up to or up to about 1.5x10 ⁸ expressing the recombinant receptor T cells, and optional information in the product specifies the introduction of one or multiple single doses and/or volume corresponding to such a dose or multiple single doses. In some instances, the article of manufacture comprises one or more single doses of CD8 ⁺ cells, the dose comprising from or from about 5x10 ⁶ to or up to about 1x10 ⁸ recombinant CD8 ⁺ receptor-expressing T cells, the dose comprising from or from about 1x10 ⁷ to or up to approximately 0.75x10 ⁸ recombinant CD8 ⁺ receptor-expressing T cells, dose is or approximately 2.5x10 ⁷ recombinant CD8+ receptor-expressing T cells, or dose is or approximately 5x10 ⁷ recombinant CD8 ⁺ receptor ^- expressing T cells, or dose is or approximately is 0.75x10 ⁸ expressing the recombinant CD8 ⁺ receptor T cells, and optional information in the product specifies the introduction of one or multiple single doses and/or volume corresponding to such a dose or multiple single doses. In some embodiments, the cells in the article, collectively, comprise a dose of cells comprising no more than 1x10 ⁸ total recombinant receptor-expressing T cells or total T cells, no more than 1x10 ⁷ total recombinant receptor-expressing T cells or total T cells , not more than 0.5x10 ⁷ total recombinant receptor-expressing T cells or total T cells, not more than 1x10 ⁶ total recombinant receptor-expressing T cells or total T cells, not more than 0.5x10 ⁶ total recombinant receptor-expressing T cells or common T cells.

[0777] In some embodiments, each vesicle or plurality of vesicles or plurality of cells or unit dose of cells defined for administration collectively includes a flat cell dose or a fixed cell dose such that the cell dose is not tied to or calculated based on surface area. or body weight of the patient.

[0778] In some embodiments, a single dose of cells is or includes a number or number of cells, such as engineered T cells, that can be administered to a person or patient in a single dose.

[0779] In some embodiments, each vesicle or plurality of vesicles or plurality of cells or single dose of cells defined for administration collectively includes a dose comprising less than about 5x10 ⁸ total recombinant receptor (e.g., CAR) expressing cells, T cells, or mononuclear peripheral blood cells (PBMC), for example, in the range from about 1x10 ⁶ to 5x10 ⁸ such cells, for example, 2x10 ⁶ , 5x10 ⁶ , 1x10 ⁷ , 5x10 ⁷ , 1x10 ⁸ or 5x10 ⁸ such total cells or a range between any two of these values.

[0780] In some embodiments, each vesicle or plurality of vesicles or plurality of cells or single dose of cells defined for administration collectively includes a dose of genetically engineered cells comprising from or from about 1x10 ⁵ to 5x10 ⁸ total CAR-expressing T cells, ^1x105 to 2.5x108 total CAR-expressing T cells ^{, 1x105} to 1x108 total CAR-expressing T ^cells , ^1x105 to ^5x107 total CAR-expressing T cells, ^1x105 to 2, 5x10 ⁷ total CAR expressing T cells, 1x10 ⁵ to 1x10 ⁷ total CAR expressing T cells, 1x10 ⁵ to 5x10 ⁶ total CAR expressing T cells, 1x10 ⁵ to 2.5x10 ⁶ total CAR expressing T cells, 1x10 ⁵ to 1x10 ⁶ total CAR-expressing T cells, 1x10 ⁶ to 5x10 ⁸ total CAR-expressing T cells, 1x10 ⁶ to 2.5x10 ⁸ total CAR-expressing T cells, 1x10 ⁶ to 1x10 ⁸ total CAR-expressing T cells, 1x10 ⁶ to 5x10 ⁷ total CAR-expressing T cells, 1x10 ⁶ to 2.5x10 ⁷ total CAR-expressing T cells, 1x10 ⁶ to 1x10 ⁷ total CARs ^-expressing T cells, ^1x106 to ^5x106 total CAR-expressing T-cells, ^1x106 to 2.5x106 total CAR-expressing T-cells, ^2.5x106 to ^5x108 total CAR-expressing T- cells, 2.5x10 ⁶ to 2.5x10 ⁸ total CAR-expressing T cells, 2.5x10 ⁶ to 1x10 ⁸ total CAR-expressing T cells, 2.5x10 ⁶ to 5x10 ⁷ total CAR-expressing T- cells, 2.5x10 ⁶ to 2.5x10 ⁷ total CAR-expressing T-cells, 2.5x10 ⁶ to 1x10 ⁷ total CAR-expressing T-cells, 2.5x10 ⁶ to 5x10 ⁶ total CAR-expressing T- cells, ^5x106 to ^5x108 total CAR-expressing ^T cells, ^5x106 to 2.5x108 total CAR-expressing T cells, ^5x106 to ^1x108 total CAR-expressing T cells, ^5x106 to 5x10 ⁷ total CAR-expressing T cells, 5x10 ⁶ to 2.5x10 ⁷ total CAR-expressing T cells, 5x10 ⁶ to 1x10 ⁷ total CAR-expressing T cells, 1x10 ⁷ to 5x10 ⁸ total CAR-expressing T cells, 1x10 ⁷ to 2.5x10 ⁸ total CAR-expressing T cells, 1x10 ⁷ to 1x10 ⁸ total CAR-expressing T cells, 1x10 ⁷ to 5x10 ⁷ total CAR-expressing T cells, 1x10 ⁷ to 2.5x10 ⁷ total CAR-expressing T cells, 2.5x10 ⁷ to 5x10 ⁸ total CAR-expressing T cells, 2.5x10 ⁷ to 2.5x10 ⁸ total CAR-expressing T cells, from 2 .5x10 ⁷ to 1x10 ⁸ total CAR-expressing T cells, 2.5x10 ⁷ to 5x10 ⁷ total CAR-expressing T cells, 5x10 ⁷ to 5x10 ⁸ total CAR-expressing T cells, 5x10 ⁷ to 2, 5x10 ⁸ total CAR-expressing T cells, 5x10 ⁷ to 1x10 ⁸ total CAR-expressing T cells, 1x10 ⁸ to 5x10 ⁸ total CAR-expressing T cells, 1x10 ⁸ to 2.5x10 ⁸ total CAR-expressing T cells or 2.5x10 ⁸ to 5x10 ⁸ total CAR-expressing T cells.

[0781] In some embodiments, each vesicle or plurality of vesicles or plurality of cells or single dose of cells defined for administration collectively includes a dose of genetically engineered cells comprising at least or at least about 1x10 ⁵ CAR-expressing cells, at least at least or at least about 2.5x10 ⁵ CAR-expressing cells, at least or at least about 5x10 ⁵ CAR-expressing cells, at least or at least about 1x10 ⁶ CAR-expressing cells, at least or at least about 2.5x10 ⁶ CAR expressing cells, at least or at least about 5x10 ⁶ CAR expressing cells, at least or at least about 1x10 ⁷ CAR expressing cells, at least or at least about 2.5x10 ⁷ CAR expressing cells, at least or at least about 5x10 ⁷ CAR expressing cells, at least or at least about 1x10 ⁸ CAR expressing cells, at least or at least about 2.5x10 ⁸ CAR expressing cells or at least or at least about 5x10 ⁸ CAR expressing cells.

[0782] In some embodiments, the instructions for administering a dose of genetically engineered cells define the administration of a number of cells from or from about 1x10 ⁵ to 5x10 ⁸ total recombinant receptor expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMC), from or from about 5x10 ⁵ to 1x10 ⁷ total recombinant receptor-expressing cells, total T-cells, or total peripheral blood mononuclear cells (PBMC) or from or from about 1x10 ⁶ to 1x10 ⁷ total recombinant receptor-expressing cells, total T-cells, or total mononuclear cells peripheral blood (PBMC), inclusive. In some embodiments, cell therapy comprises administering a dose of cells comprising at least or at least about 1x10 ⁵ total recombinant receptor expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMCs), e.g., at least or at least 1x10 ⁶ , at least or at least about 1x10 ⁷ , at least or at least about 1x10 ⁸ of these cells. In some embodiments, the number is in relation to the total number of CD3+ or CD8+, in some cases also expressing the recombinant receptor (eg, CAR+) cells. In some embodiments, the cell therapy comprises administering a dose comprising from or about 1x10 ⁵ to 5x10 ⁸ CD3+ or CD8+ total T cells, or CD3+ or CD8+ recombinant receptor-expressing cells, from or about 5x10 ⁵ to 1x10 ⁷ CD3+ or CD8+ total T cells or CD3+ or CD8+ recombinant receptor-expressing cells, or from or from about 1x10 ⁶ to 1x10 ⁷ CD3+ or CD8+ total T cells or CD3+ or CD8+ recombinant receptor-expressing cells, inclusive. In some embodiments, cell therapy comprises administering a dose comprising from or about 1x10 ⁵ to 5x10 ⁸ total CD3+/CAR+ or CD8+/CAR+ cells, from or about 5x10 ⁵ to 1x10 ⁷ total CD3+/CAR+ or CD8+/CAR+ cells or from or from about 1x10 ⁶ to 1x10 ⁷ total CD3+/CAR+ or CD8+/CAR+ cells, inclusive.

[0783] In some embodiments, the instructions may define a dosage regimen and timing of administration. For example, in some embodiments, the instructions may specify administering multiple doses to a patient, eg, two or more doses of cells. In some embodiments, the instructions specify the timing of multiple doses, e.g., the second dose administered at approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20 or 21 days after the first dose; and/or the amount in each dose.

[0784] In some embodiments, the product or kit includes a plurality of CD4 ⁺ T cells expressing the recombinant receptor and instructions for administering to a patient having a disease or condition all or part of the plurality of CD4 ⁺ T cells and additional administration of CD8 ⁺ T cells expressing the recombinant receptor. In some embodiments, the instructions define the introduction of CD4 ⁺ T cells prior to the introduction of CD8 ⁺ cells. In some cases, the instructions specify the introduction of CD8 ⁺ T cells before the introduction of CD4 ⁺ cells. In some embodiments, the article of manufacture or kit includes a plurality of CD8 ⁺ T cells expressing the recombinant receptor and instructions for administering to a patient having a disease or condition all or part of the plurality of CD8 ⁺ T cells and CD4 ⁺ T cells expressing the recombinant receptor. In some embodiments, the instructions define the mode of administration and the timing of the administration of the cells.

[0785] In some aspects, the instructions define the administration of all or part of the CD4 ⁺ T cells and all or part of the CD8 ⁺ T cells at intervals of 0 to 12 hours, at intervals of 0 to 6 hours, or at intervals of 0 to 2 hours. In some cases, the instructions specify administration of CD4 ⁺ T cells and CD8 ⁺ T cells at intervals of 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.

[0786] In some embodiments, the instructions define the dose or number of cells or cell type(s) and/or ratios of cell types, eg, individual populations or subtypes, such as the ratio of CD4 ⁺ to CD8 ⁺ . In some embodiments, cell populations or subtypes such as CD8 ⁺ and CD4 ⁺ T cells. For example, in some embodiments, the instructions specify that the cells are administered at or within the tolerable range of an output ratio of multiple cell populations or subtypes, such as CD4 ⁺ and CD8 ⁺ cells or subtypes, from or from about 5:1 to or up to about 5: 1 (or greater than about 1:5 and less than about 5:1), or from or from about 1:3 to or up to about 3:1 (or greater than about 1:3 and less than about 3:1), for example, from or from about 2:1 to or up to about 1:5 (or more than about 1:5 and less than about 2:1, for example, in a ratio of 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, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9: 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5). In some aspects, the tolerable difference is about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, approximately 45%, approximately 50% of the desired ratio, including any value in between these ranges.

[0787] In some embodiments, the articles and/or kits further include one or more additional agents for therapy, such as anti-lymphocyte therapy and/or combination therapy, as described herein, and optionally instructions for administering the additional agents. In some examples, the products may additionally contain one or more therapeutic agents. In some embodiments, the therapeutic agent is an immunomodulatory agent, a cytotoxic agent, an anticancer agent, or a radiotherapeutic agent.

[0788] In some embodiments, the articles and/or kits further comprise one or more therapeutic agents or agents to treat, prevent, delay, attenuate, or reduce the development or risk of toxicity, and/or instructions for administering the one or more agents or agents to treatment to treat, prevent, delay, attenuate or reduce the development or risk of developing toxicity in a patient. In some embodiments, the agent is or includes an anti-IL-6 antibody or an anti-IL-6 antibody receptor. For example, in some embodiments, the treatment agent or agent is or includes an agent selected from among tocilizumab, siltuximab, clasakizumab, sarilumab, olokizumab (CDP6038), elsilimomab, ALD518/BMS-945429, sirukumab (CNTO 136), CPSI-2634 , ARGX-109, FE301 and FM101. For example, in some embodiments, the agent or agent for treatment is or includes one or more steroids; antagonists or inhibitors of a cytokine receptor or a cytokine selected from IL-10, IL-10R, IL-6, IL-6 receptor, IFNγ, IFNGR, IL-2, IL-2R/CD25, MCP-1, CCR2, CCR4, MIP1β, CCR5, TNFalpha, TNFR1, IL-1 and IL-1Ralpha/IL-1beta; or an agent capable of preventing, blocking or attenuating the activity or function of microglial cells.

[0789] In some embodiments, an agent capable of preventing, blocking, or attenuating microglial cell activity or function is selected from an anti-inflammatory agent, a NADPH oxidase (NOX2) inhibitor, a calcium channel blocker, a sodium channel blocker, inhibits GM-CSF, inhibits CSF1R, binds specifically to CSF-1, binds specifically to IL-34, inhibits nuclear factor kappa-B (NF-κB) activation, activates the CB2 receptor and/or is a CB2 agonist, phosphodiesterase inhibitor, inhibits miRNA-155 ((miR-155) or up-regulates miRNA-124 ((miR-124). In some cases, the agent is selected from minocycline, naloxone, nimodipine, Riluzole, MOR103, lenalidomide, cannabinoid (optionally WIN55 or 212-2), intravenous immunoglobulin (IVIg), ibudilast, anti -miR-155 closed nucleic acid (LNA), MCS110, PLX-3397, PLX647, PLX108-D1, PLX7486, JNJ-40346527, JNJ28312141, ARRY-382, AC-708, DCC-3014, 5-(3-methoxy- 4-(4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine (GW2580), AZD6495, Ki20227, BLZ945, emaktuzumab, IMC-CS4, FPA008, LY-3022855, AMG-820 and TG-3003. In some embodiments, the agent is a colony stimulating factor 1 receptor (CSF1R) inhibitor. For example, the agent is PLX-3397, PLX647, PLX108-D1, PLX7486, JNJ-40346527, JNJ28312141, ARRY-382, AC-708, DCC-3014, 5-(3-methoxy-4-(4-methoxybenzyl)oxy )benzyl)pyrimidine-2,4-diamine (GW2580), AZD6495, Ki20227, BLZ945, or a pharmaceutical salt or prodrug thereof; emactuzumab, IMC-CS4, FPA008, LY-3022855, AMG-820 and TG-3003, or is an antigen-binding fragment or a combination of any of the preceding.

[0790] In some embodiments, the articles and/or kits further include one or more reagents for testing biological samples, such as biological samples from patients who are candidates for administration or who have received therapy, and optionally instructions for using the reagents or tests. In some embodiments, the biological sample is or is derived from a blood, plasma, or serum sample. In some embodiments, the reagents may be used prior to the administration of cell therapy or after the administration of cell therapy, for diagnostic purposes, to identify patients and/or evaluate treatments and/or toxicity. For example, in some embodiments, the product and/or kits further comprise reagents for measuring the level of certain biomarkers, such as cytokines or toxicity-associated analytes, and instructions for measuring. In some embodiments, the reagents include components for performing an in vitro test to measure biomarkers (eg, analytes), such as an immunoassay, an aptamer-based test, a histological or cytological test, or an mRNA expression level test. In some embodiments, the in vitro test is selected from an enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, flow cytometry test, surface plasmon resonance (SPR), chemiluminescence test, immunochromatographic assay, inhibition test, and avidity. In some aspects, the reagent is a binding reagent that specifically binds to biomarkers (eg, analytes). In some instances, the binding reagent is an antibody or an antigen-binding fragment thereof, an aptamer, or a nucleic acid probe.

[0791] In some embodiments, the articles and/or kits include one or more reagents capable of detecting one or more analytes and instructions for using the reagent to test a biological sample from a patient who is a candidate for treatment, where one or more analytes are selected. from LDH, ferritin, CRP, IL-6, IL-7, IL-8, IL-10, IL-15, IL-16, TNF-alpha, IFN-gamma, MCP-1, MIP-1beta, eotaxin, G -CSF, IL-1Ralpha, IL-1Rbeta, IP-10, perforin and D-dimer (fibrin breakdown product). In some embodiments, instructions are also included for testing the presence or absence, level, amount, or concentration of an analyte in a patient against an analyte threshold.

[0792] In some embodiments, instructions are included that determine if the level, amount, or concentration of an analyte in a sample is equal to or greater than a threshold for the analyte, administering to the patient an agent or other treatment capable of treating, preventing, delaying, attenuating, or reducing development or risk of development of toxicity (i) before, (ii) within one, two or three days, (iii) simultaneously with and/or (iv) at the first increase in temperature after the initiation of the introduction of cell therapy to the patient. In some cases, the instructions specify that if the level, amount, or concentration of an analyte in a sample is equal to or greater than the threshold value for the analyte, the cell therapy is administered to the patient at a reduced dose or at a dose not associated with a risk of developing toxicity or severe toxicity, or not associated with the risk of developing toxicity or severe toxicity in most patients and/or most patients with a disease or condition that the patient has or is suspected of having, after the introduction of cell therapy. In some instances, the instructions specify that if the level, amount, or concentration of the analyte in the sample is equal to or greater than the threshold value for the analyte, the cell therapy is administered on an inpatient basis and/or with admission to a hospital for one or more days, optionally the cell therapy must otherwise be administered to patients on an outpatient or non-hospital basis for one or more days.

[0793] In some embodiments, instructions for administering cell therapy determine if the level, amount, or concentration of an analyte in a sample is below a threshold, administering cell therapy to a patient, optionally at a non-reduced dose, optionally on an outpatient basis, or without hospital admission for one or more days. In some embodiments, the instructions for administering the cell therapy determine if the level, amount, or concentration of the analyte in the sample is below a threshold, before or simultaneously with the administration of the cell therapy and/or before the development of a sign or symptom of toxicity other than fever, an agent or agent for treatment, capable of treating, preventing, delaying or reducing the development of toxicity, is not administered to the patient. In some aspects, the instructions for administering the cell therapy specify that if the level, amount, or concentration of the analyte in the sample is below a threshold value, the cell therapy administration should or may be administered to the patient on an outpatient basis and/or without the patient being admitted to the hospital overnight or overnight. one or more days in a row and/or without admission of the patient to the hospital for one or more days.

[0794] Articles and/or kits may further include cell therapy and/or further include instructions for use with, before and/or in connection with cell therapy treatment. In some embodiments, instructions are included for administering the agent, and the instructions determine if the level, amount, or concentration of the analyte in the sample is equal to or above a threshold, administering the agent to the patient. In some aspects, the instructions further define the administration of the cell therapy to the patient, wherein the administration of the agent must be (i) prior to, (ii) within one, two, or three days, (iii) concurrently with, and/or (iv) at the first rise in temperature after initiating the administration of cell therapy to the patient.

[0795] Articles and/or kits may include a container and a label or leaflet on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container, in some embodiments, contains a composition that is alone or combined with another composition effective for treating, preventing, and/or diagnosing a condition. In some embodiments, the container has a sterile access port. Examples of containers include bags for intravenous solution, vials, including those with stoppers, pierced by an injection needle, or bottles or vials for orally administered agents. The label or package insert may indicate that the composition is used to treat a disease or condition. The article of manufacture may include (a) a first container with a composition contained therein, the composition comprising engineered cells expressing the recombinant receptor; and (b) a second container with the composition contained therein, the composition comprising a second agent. In some embodiments, the implementation of the product may include (a) the first container with the first composition contained therein, and the composition includes a subtype of genetically engineered cells expressing the recombinant receptor; and (b) a second container with a composition contained therein, the composition comprising a different subtype of engineered cells expressing the recombinant receptor. The article of manufacture may further include a package leaflet indicating that the compositions may be used to treat a particular condition. Also or additionally, the product may further include a different or the same container, including a pharmaceutically acceptable buffer. It may further include other materials such as other buffers, diluents, filters, needles and/or syringes.

VII. EXAMPLES OF EMBODIMENTS

[0796] Among the embodiments of the invention:

1. A method of treating a patient having or suspected of having a disease or condition, comprising administering to the patient a dose of CD4 ⁺ and CD8 ⁺ T cells, each of the CD4 ⁺ and CD8 ⁺ T cells, individually, includes a receptor that specifically binds to an antigen- a target expressed by a disease or condition, or a cell or tissue thereof, and/or which is associated with the disease or condition, wherein the administration comprises the administration of a plurality of individual compositions, the plurality of individual compositions comprising a first composition comprising one of CD4 ⁺ T cells and CD8 ⁺ T cells, and the introduction of a second composition, including the other of CD4 ⁺ T cells and CD8 ⁺ T cells.

2. The method according to embodiment 1, wherein the receptor contained in CD4 ⁺ T cells and/or the receptor contained in CD8 ⁺ T cells comprises a recombinant receptor and/or wherein CD4 ⁺ T cells and/or CD8 ⁺ T cells are genetically modified to express the receptor.

3. The method according to embodiment 1 or embodiment 2, wherein:

administration of the first composition and administration of the second composition are carried out on the same day, carried out at intervals of from about 0 to about 12 hours, at intervals of from about 0 to about 6 hours, or at intervals of from about 0 to 2 hours; or

initiation of administration of the first composition and initiation of administration of the second composition are carried out at intervals of from about 1 minute to about 1 hour, or at intervals of from about 5 minutes to about 30 minutes.

4. The method of any one of embodiments 1-3, wherein the first composition and the second composition are administered at intervals of 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.

5. The method of any one of embodiments 1-4, wherein the first composition comprises CD4 ⁺ T cells.

6. The method of any one of embodiments 1-5, wherein the first composition comprises CD8 ⁺ T cells.

7. The method of any one of embodiments 1-6, wherein the initiation of administration of the first composition is prior to the initiation of administration of the second composition.

8. A method according to any one of embodiments 1-7, wherein:

the dose of cells includes a certain ratio of CD4 ⁺ cells expressing the recombinant receptor to CD8 ⁺ cells expressing the recombinant receptor, and/or CD4+ cells to CD8+ cells, and this ratio is optionally or approximately 1:1 or from about 1:3 to about 3 :1; and/or

The CD4 ⁺ T cells comprising the receptor in one of the first and second compositions and the CD8 ⁺ T cells comprising the receptor in the other of the first and second compositions are present in a ratio, the ratio being optionally or approximately 1:1 or from about 1:3 and about 3:1; and/or

The CD4 ⁺ T cells comprising the receptor and the CD8 ⁺ T cells comprising the receptor administered in the first and second compositions are present in a ratio, the ratio being optionally or about 1:1 or from about 1:3 to about 3:1.

9. The method of Embodiment 8 wherein the specified ratio is or approximately 1:1.

10. The method of any one of embodiments 1-9, wherein the dose of T cells is administered to the patient 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.

11. The method of any one of embodiments 1-9 wherein the T cell dose is administered as a double dose comprising a first T cell dose and a subsequent T cell dose, wherein one or both of the first dose and the second dose comprises administering a plurality of compositions T cells.

12. The method of embodiment 11 wherein the subsequent dose is administered at a time point of at least or greater than about 7 days, or 14 days after and less than about 28 days after the initiation of the first dose of cells.

13. The method of any one of embodiments 1-12, wherein:

the cell dose comprises from or from about 1x10 ⁵ to or up to about 5x10 ⁸ total recombinant receptor-expressing T cells or total T cells, from or from about 1x10 ⁵ to or up to about 1x10 ⁸ total recombinant receptor-expressing T cells, or total T cells, from or from about 5x10 ⁵ to or up to about 1x10 ⁷ total recombinant receptor-expressing T cells or total T cells, or from or from about 1x10 ⁶ to or up to about 1x10 ⁷ total recombinant receptor-expressing T -cells or common T-cells, inclusive.

14. The method of any one of embodiments 1-13, wherein the dose of T cells comprises administering no more than 1x10 ⁸ total recombinant receptor-expressing T cells or total T cells, no more than 1x10 ⁷ total recombinant receptor-expressing T cells or total T cells not more than 0.5x10 ⁷ total recombinant receptor-expressing T cells or total T cells not more than 1x10 ⁶ total recombinant receptor-expressing T cells or total T cells not more than 0.5x10 ⁶ total recombinant receptor-expressing T cells or total T cells.

15. The method of any one of embodiments 1-14, wherein the dose of CD4 ⁺ and CD8 ⁺ T cells comprises:

from or from about 5x10 ⁷ expressing the recombinant receptor T cells to or up to about 1x10 ⁸ expressing the recombinant receptor T cells, inclusive;

from or from about 1x10 ⁷ to or up to about 2x10 ⁸ expressing the recombinant receptor T cells;

from or from about 5x10 ⁷ to or up to about 1.5x10 ⁸ recombinant receptor-expressing T cells

5x10 ⁷ or approximately 5x10 ⁷ expressing recombinant receptor T cells; or

1x10 ⁸ or approximately 1x10 ⁸ expressing recombinant receptor T cells; or

the dose includes 1.5x10 ⁸ or about 1.5x10 ⁸ expressing the recombinant receptor T cells;

from or from about 5x10 ⁶ to or up to about 1x10 ⁸ expressing the recombinant receptor CD8 ⁺ T cells;

from or from about 1x10 ⁷ to or up to about 0.75x10 ⁸ expressing the recombinant receptor CD8 ⁺ T cells;

2.5x10 ⁷ or approximately 2.5x10 ⁷ expressing recombinant CD8 ⁺ receptor T cells;

5x10 ⁷ or approximately 5x10 ⁷ expressing recombinant CD8 receptor ⁺ T cells; and/or

0.75x10 ⁸ or approximately 0.75x10 ⁸ expressing recombinant CD8 ⁺ receptor T cells.

16. The method of any one of embodiments 1-15, wherein the recombinant receptor specifically binds to an antigen associated with the disease or condition or expressed in cells in the lesion environment associated with the disease or condition.

17. The method of any one of embodiments 1-16 wherein the disease or condition is cancer.

18. The method of any one of embodiments 1-17, wherein the disease or condition is myeloma, leukemia, or lymphoma.

19. The method of any of embodiments 1-18 wherein the antigen is ROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2/neu (erbB2 receptor tyrosine kinase), L1-CAM, CD19 , CD20, CD22, mesothelin, CEA and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2 , erb-B2, erb-B3, erb-B4, erbB dimers, EGFR vIII, folate-binding protein (FBP), FCRL5, FCRH5, embryonic acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL -13R-alpha2, receptor containing kinase insertion domain (kdr), kappa light chain, Ley antigen, L1 cell adhesion molecule, (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE -A6, preferably expressed melanoma antigen (PRAME), survivin, TAG72, B7-H6, IL-13 receptor alpha 2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AI MAGE Al , HLA-A2 NY-ESO-1, PSCA, folic acid receptor alpha, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, embryonic AchR, NKG2D ligands, CD44v6, dual antigen, testicular cancer antigen , mesothelin, mouse CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), Her2/neu , estrogen receptor, progesterone receptor, ephrinB2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms tumor 1 (WT-1), cyclin, cyclin A2, CCL-1, CD138, 5D G protein-coupled receptor (GPCR5D) or pathogen-specific antigen.

20. The method of any of embodiments 1-19 wherein the antigen is CD19.

21. The method of any one of embodiments 1-20, wherein the disease or condition is a B-cell malignancy and/or is acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin's lymphoma (NHL), and diffuse large B-cell lymphoma (DLBCL).

22. The method of any one of embodiments 1-21 wherein the disease or condition is NHL and the NHL is selected from the group consisting of aggressive NHL, diffuse large B cell lymphoma (DLBCL), NOS (de novo and transformed from indolent) , primary mediastinal large B-cell lymphoma (PMBCL), T-cell/histocyte-rich large B-cell lymphoma (TCHRBCL), Burkitt's lymphoma, mantle cell lymphoma (MCL), and/or follicular lymphoma (FL), optionally stage 3B follicular lymphoma (FL3B).

23. The method of any one of embodiments 1-22, wherein the recombinant receptor is a chimeric antigen receptor (CAR), optionally the recombinant receptor is a chimeric antigen receptor (CAR), optionally the CAR comprises an extracellular antigen-recognition domain specifically binding to an antigen, and an intracellular signaling domain comprising ITAM, optionally the intracellular signaling domain comprising an intracellular domain of the CD3-zeta (CD3ζ) chain; and/or wherein the CAR further comprises a costimulatory signaling domain, optionally including a CD28 or 4-1BB signaling domain.

24. An article of manufacture comprising (1) a vial containing a composition comprising a plurality of CD4 ⁺ T cells expressing the recombinant receptor and (2) instructions for administration to a patient having a disease or condition:

a single dose of composition cells, wherein the single dose includes all or part of a plurality of CD4 ⁺ T cells, and a single dose of a composition comprising CD8 ⁺ T cells expressing the recombinant receptor, wherein the vial does not include a single dose of a composition comprising recombinant receptor.

25. An article of manufacture comprising (1) a vial containing a composition comprising a plurality of CD8 ⁺ T cells expressing the recombinant receptor and (2) instructions for administration to a patient having a disease or condition:

a single dose of composition cells, wherein the single dose includes all or part of a plurality of CD8 ⁺ T cells, and a single dose of a composition comprising CD4 ⁺ T cells expressing the recombinant receptor, wherein the vial does not include a single dose of a composition comprising recombinant receptor.

26. The product according to embodiment 24 or embodiment 25, wherein:

the recombinant receptor expressed by CD4 ⁺ cells and the recombinant receptor expressed by CD8 ⁺ T cells are the same;

the recombinant receptor expressed by CD4 ⁺ cells and the recombinant receptor expressed by CD8 ⁺ T cells are different; or

a recombinant receptor expressed by CD4 ⁺ cells and a recombinant receptor expressed by CD8 ⁺ T cells bind to the same antigen expressed or associated with a disease or condition, or a cell or tissue thereof.

27. The article of manufacture according to any one of embodiments 24-26, wherein the vial comprises more than or more than about 10x10 ⁶ T cells or recombinant receptor expressing T cells, more than or more than about 15x10 ⁶ T cells or recombinant receptor expressing T cells greater than or greater than about 25x10 ⁶ T cells or expressing a recombinant T cell receptor.

28. The article of any one of embodiments 24-27, wherein the vial comprises from about 10 million cells per ml to about 70 million cells per ml, from about 10 million cells per ml to about 50 million cells per ml, from about 10 million cells per ml to about 25 million cells per ml, from about 10 million cells per ml to about 15 million cells per ml, from 15 million cells per ml to about 70 million cells per ml, from about 15 million cells per ml to about 50 million cells per ml, from about 15 million cells per ml to about 25 million cells per ml, from about 25 million cells per ml to about 70 million cells per ml, from about 25 million cells per ml to about 50 million cells per ml, and from about 50 million cells per ml to about 70 million cells per ml.

29. The article of manufacture of any of embodiments 24-28, wherein the composition further comprises a cryoprotectant and/or the article further includes instructions for thawing the composition prior to administration to a patient.

30. An article of any one of embodiments 24-29, wherein:

the plurality of CD4 ⁺ cells expressing the recombinant receptor and the plurality of CD8 ⁺ cells expressing the recombinant receptor in the product and/or the CD4 ⁺ cells and CD8 ⁺ cells in the vesicle are present in a ratio that is optionally or approximately 1:1 or from about 1:3 to about 3:1; and/or

the product further includes information or instructions defining the introduction of a plurality of cells in a certain ratio of cells of a plurality of CD4 ⁺ cells expressing the recombinant receptor, and cells of a plurality of CD8 ⁺ cells expressing the recombinant receptor, and/or CD4 ⁺ cells and CD8 ⁺ cells, and this ratio is optional is or is about 1:1 or is from about 1:3 to about 3:1; and/or

the product further includes information or instructions specifying the administration of formulations in vials in a specific volume or weight ratio, which is optionally at or about 1:1 and/or from or from about 3:1 to or up to about 1:3, by volume. or weight ratio, and/or optionally corresponds to a ratio of CD4 ⁺ cells expressing the recombinant receptor to CD8 ⁺ cells expressing the recombinant receptor and/or CD4+ cells to CD8+ cells, which ratio is optionally or approximately 1:1 or from about 1:3 to about 3:1.

31. The article of Embodiment 30 wherein the specified ratio is or approximately 1:1.

32. The article of manufacture of any one of embodiments 24-31 wherein the plurality of vesicles or plurality of cells or single dose of cells defined for administration collectively comprise a dose of cells comprising from or about 1x10 ⁵ to 5x10 ⁸ total expressing the recombinant T receptor -cells or total T cells, from 1x10 ⁵ to 1x10 ⁸ total recombinant receptor-expressing T cells or total T cells, from or from about 5x10 ⁵ to 1x10 ⁷ total recombinant receptor-expressing T cells or total T cells, or from or from about 1x10 ⁶ to 1x10 ⁷ total recombinant receptor-expressing T cells or total T cells, inclusive;

the product includes one or more single doses of CD4 ⁺ and CD8 ⁺ cells or CD4 ⁺ receptor + cells and CD8 ⁺ receptor + cells, and a single dose includes from or from about 1x10 ⁷ to or up to about 2x10 ⁸ expressing the recombinant receptor T-cells, from or from about 5x10 ⁷ to or up to about 1.5x10 ⁸ expressing recombinant receptor T cells, 5x10 ⁷ or about 5x10 ⁷ expressing recombinant receptor T cells, 1x10 ⁸ or about 1x10 ⁸ expressing recombinant receptor T cells, or up to or up to about 1.5x10 ⁸ expressing the recombinant receptor T-cells, and optional information in the product specifies the introduction of one or multiple single doses and/or volume corresponding to such one or multiple single doses;

the product includes one or more single doses of CD8 ⁺ cells, and the dose includes from or from about 5x10 ⁶ to or up to about 1x10 ⁸ expressing recombinant CD8 ⁺ receptor T cells, the dose includes from or from about 1x10 ⁷ to or up to about 0 .75x10 ⁸ recombinant CD8 ⁺ receptor-expressing T cells, dose includes 2.5x10 ⁷ or approximately 2.5x10 ⁷ recombinant CD8 ⁺ receptor-expressing T cells, or dose includes 5x10 ⁷ or approximately 5x10 ⁷ recombinant CD8 ⁺ receptor-expressing T cells , or the dose comprises 0.75x10 ⁸ or about 0.75x10 ⁸ recombinant CD8 ⁺ receptor-expressing T cells, optionally the information in the article of manufacture specifies administration of one or multiple single doses and/or a volume corresponding to such one or multiple single doses.

33. The article of manufacture according to any one of embodiments 24-32, wherein the cells in the article, collectively, comprise a dose of cells comprising no more than 1x10 ⁸ total recombinant receptor-expressing T cells, or total T cells no more than 1x10 ⁷ total expressing not more than 0.5x10 ⁷ total recombinant receptor-expressing T cells or total T-cells, not more than 1x10 ⁶ total recombinant receptor-expressing T-cells or total T-cells, not more than more than 0.5x10 ⁶ total recombinant receptor-expressing T cells or total T cells.

34. An article of any one of embodiments 24-33 wherein the plurality of containers collectively comprise a dose of cells comprising:

from or from about 5x10 ⁷ expressing the recombinant receptor T cells to 1x10 ⁸ expressing the recombinant receptor T cells, inclusive;

from or from about 1x10 ⁷ to or up to about 2x10 ⁸ expressing the recombinant receptor T cells;

from or from about 5x10 ⁷ to or up to about 1.5x10 ⁸ expressing the recombinant receptor T cells;

5x10 ⁷ or approximately 5x10 ⁷ expressing recombinant receptor T cells;

1x10 ⁸ or approximately 1x10 ⁸ expressing recombinant receptor T cells;

1.5x10 ⁸ or approximately 1.5x10 ⁸ expressing recombinant receptor T cells;

from or from about 5x10 ⁶ to or up to about 1x10 ⁸ expressing the recombinant receptor CD8 ⁺ T cells;

from or from about 1x10 ⁷ to or up to about 0.75x10 ⁸ expressing the recombinant receptor CD8 ⁺ T cells;

2.5x10 ⁷ or approximately 2.5x10 ⁷ expressing recombinant CD8 ⁺ receptor T cells;

5x10 ⁷ or approximately 5x10 ⁷ expressing recombinant CD8 receptor ⁺ T cells; or

0.75x10 ⁸ or approximately 0.75x10 ⁸ expressing recombinant CD8 ⁺ receptor T cells.

35. The article of manufacture according to any one of embodiments 24-34, wherein the instructions specify the administration of all or part of the CD4 ⁺ T cells and all or part of the CD8 ⁺ T cells at intervals of 0 to 12 hours, at intervals of 0 to 6 hours, or at intervals of 0 to 2 hours.

36. An article of manufacture according to any one of embodiments 24-34, wherein the instructions specify administration of CD4 ⁺ T cells and CD8 ⁺ T cells at intervals of no more than 2 hours, no more than 1 hour, no more than 30 minutes, no more than 15 minutes, not more than 10 minutes, or not more than 5 minutes.

37. An article of manufacture as in any one of embodiments 24-36, wherein the instructions specify administration of CD4 ⁺ T cells prior to administration of CD8 ⁺ cells.

38. The article of manufacture of any one of embodiments 24-36, wherein the instructions specify administration of CD8 ⁺ T cells prior to administration of CD4 ⁺ cells.

39. The article of manufacture of any one of embodiments 24-38, wherein the recombinant receptor specifically binds to an antigen associated with the disease or condition or expressed in cells of the lesion environment associated with the disease or condition.

40. The article of any one of embodiments 24-39, wherein the disease or condition is cancer.

41. The article of any one of embodiments 24-40, wherein the disease or condition is myeloma, leukemia, or lymphoma.

42. The article of manufacture according to any one of embodiments 24-41, wherein the antigen is ROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2/neu (erbB2 receptor tyrosine kinase), L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2 , erb-B2, erb-B3, erb-B4, erbB dimers, EGFR vIII, folate-binding protein (FBP), FCRL5, FCRH5, embryonic acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL -13R-alpha2, receptor containing kinase insertion domain (kdr), kappa light chain, Ley antigen, L1 cell adhesion molecule, (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE -A6, preferably expressed melanoma antigen (PRAME), survivin, TAG72, B7-H6, IL-13 receptor alpha 2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AI MAGE Al , HLA-A2 NY-ESO-1, PSCA, folic acid receptor alpha, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, embryonic AchR, NKG2D ligands, CD44v6, dual antigen, testicular cancer antigen , mesothelin, mouse CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), Her2/neu , estrogen receptor, progesterone receptor, ephrinB2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms tumor 1 (WT-1), cyclin, cyclin A2, CCL-1, CD138, 5D G protein-coupled receptor (GPCR5D) or pathogen-specific antigen.

43. The article of manufacture of any one of embodiments 24-42, wherein the antigen is CD19.

44. The article of any one of embodiments 24-43, wherein the disease or condition is a B-cell malignancy and/or is acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin's lymphoma (NHL), and diffuse large B-cell lymphoma (DLBCL).

45. The article of manufacture of any one of embodiments 24-44, wherein the disease or condition is NHL and the NHL is selected from the group consisting of aggressive NHL, diffuse large B cell lymphoma (DLBCL), NOS (de novo and transformed from indolent) , primary mediastinal large B-cell lymphoma (PMBCL), T-cell/histocyte-rich large B-cell lymphoma (TCHRBCL), Burkitt's lymphoma, mantle cell lymphoma (MCL), and/or follicular lymphoma (FL), optionally stage 3B follicular lymphoma (FL3B).

46. The article of any one of embodiments 24-45, wherein the T cells are primary T cells obtained from a patient.

47. The article of manufacture of any one of embodiments 24-46, wherein the T cells are autologous to the patient.

48. The article of any one of embodiments 24-46, wherein the T cells are allogeneic to the patient.

49. A method of treating a patient with or suspected of having a B-cell malignant tumor, optionally non-Hodgkin's lymphoma (NHL), comprising administering to the patient a dose of T cells, including T cells expressing a chimeric antigen receptor (CAR) that specifically binds to an antigen- a target expressed by NHL, wherein:

the dose of T cells includes from or from about 2.5x10 ⁷ CAR-expressing T cells to 2x10 ⁸ CAR-expressing T cells, inclusive, optionally from or from about 5x10 ⁷ CAR-expressing T cells and up to or up to about 1x10 ⁸ CAR-expressing T cells, inclusive; And

the malignancy, optionally NHL, includes diffuse large B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), NOS (de novo or transformed from indolent lymphoma), or stage 3B follicular lymphoma, and wherein the patient is or has been identified as having an Eastern Cooperative Oncology Group Performance Status (ECOG) status of 0 or 1, optionally the method further comprising identifying the patient as having diffuse large B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), NOS ( de novo or transformed from indolent lymphoma) or stage 3B follicular lymphoma with an ECOG status of 0 or 1.

50. The method according to embodiment 49, wherein the dose of T cells comprises a specified ratio of CD4+ cells expressing CAR to CD8+ cells expressing CAR and/or CD4+ cells to CD8+ cells, the ratio being optionally about 1:1 or from about 1:3 to about 3:1.

51. A method of treating a patient with non-Hodgkin's lymphoma (NHL), comprising administering to the patient a dose of T cells, including T cells expressing a chimeric antigen receptor (CAR) that specifically binds to a target antigen expressed by NHL, the dose of T cells comprising defined ratio of CD4+ CAR-expressing cells to CAR-expressing CD8+ cells and/or CD4+ cells to CD8+ cells, the ratio being approximately or equal to 1:1, NHL including diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), NOS (de novo or transformed from indolent lymphoma), or stage 3B follicular lymphoma.

52. The method of embodiment 51, wherein the patient is or has been identified as having an Eastern Cooperative Oncology Group Performance Status (ECOG) status of 0, 1, or 2.

53. The method according to embodiment 51 or embodiment 52, wherein the patient is or has been identified as having an ECOG status of 0 or 1.

54. The method of any one of embodiments 49-53, wherein:

at least 35%, at least 40%, or at least 50% of patients treated according to the method achieve a complete response (CR), optionally CR being sustained or sustained in at least 60%, 70%, 80% , 90% or 95% of patients achieving CR within 3 months or more, or within 6 months or more; and/or

at least 60%, 70%, 80%, 90%, or 95% of patients who achieve CR at one month and/or three months remain in response, remain in CR, and/or are alive or alive without progression for 3 months or more and/or for 6 months or more and/or more than nine months; and/or

at least 50%, at least 60%, or at least 70% of patients treated according to the method achieve an objective response (OR), optionally the OR is prolonged or prolonged in at least 60%, 70%, 80% , 90% or 95% of patients achieving OR within 3 months or more, or within 6 months or more; and/or

at least 60%, 70%, 80%, 90%, or 95% of patients who achieve OR remain in response or survive for 3 months or more and/or for 6 months or more; and/or

at least 40%, at least 50%, at least 60%, at least 70% of patients who, at or before cell dosing, had or were identified as having double/triple hit lymphoma or relapse, optionally relapse within 12 months after the introduction of autologous stem cell transplant (ASCT) achieved OR, and optionally OR lasts for 3 months or more or for 6 months or more.

55. The method of any one of embodiments 49-54, wherein:

the cells are autologous to the patient and no minimum absolute lymphocyte count (ALC) for apheresis is required and/or determined to produce therapy; and/or

cells are obtained in a manner that at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of patients with a disease or condition, or selected population of patients, is able to generate a cellular product for administration and results according to the method.

56. The method of any one of embodiments 49-55, wherein:

more than or more than about 50%, about 60%, about 70%, or about 80% of patients treated according to the method do not exhibit grade 3 or greater cytokine release syndrome (CRS) and/or do not exhibit grade 3 or greater neurotoxicity, and /or more than 40% or 50% or 55% do not show neurotoxicity or CRS.

57. A method of treating a patient with non-Hodgkin's lymphoma (NHL), comprising administering to the patient a dose of T cells, including T cells expressing a chimeric antigen receptor (CAR) that specifically binds to a target antigen expressed by NHL, wherein:

the dose of T cells includes from or from about 5x10 ⁷ expressing the recombinant receptor T cells to 1x10 ⁸ expressing the recombinant receptor T cells, inclusive, and the indicated dose includes a certain ratio of CD4 ⁺ cells expressing the recombinant receptor to CD8 ⁺ cells expressing the recombinant receptor, and/or CD4+ cells to CD8+ cells, the ratio being approximately or equal to 1:1; And

the method results in (1) a complete response (CR) in at least 35%, at least 40%, or at least 50% of treated patients and/or an objective response (OR) in at least 50%, at least 60% or at least 70% of treated patients, and (2) results in no more than 50% of patients showing cytokine release syndrome (CRS) greater than Grade 2 and/or neurotoxicity greater than Grade 2.

58. The method of embodiment 57, wherein at least 40%, at least 50%, at least 60%, at least 70% of patients who, at or before the cell dose had or were identified as having double/triple hit lymphoma or relapse after autologous stem cell transplant (ASCT) administration, achieved an OR, optionally OR lasting for 3 months or more, or for 6 months or more.

59. The method according to embodiment 57, wherein:

CR or OR lasts for more than 3 months or more than 6 months; and/or

at least 20%, at least 25%, at least 35%, at least 40%, or at least 50% of patients treated according to the method achieve a CR that is long lasting; and/or

at least 60%, 70%, 80%, 90% or 95% of patients treated according to the method and who achieve CR remain in CR or remain in response or survive for 3 months or more or for 6 months or more, or for 9 months or more; and/or

at least 60%, 70%, 80%, 90%, or 95% of patients treated according to the method who achieve CR in one month and/or three months remain in response, remain in CR and/ or remains alive or remains alive without progression for 3 months or more and/or for 6 months or more and/or more than nine months; and/or

at least 50%, at least 60%, or at least 70% of patients treated according to the method achieve an objective response (OR), and optionally the OR is prolonged or prolonged in at least 60%, 70%, 80% , 90% or 95% of patients achieving OR within 3 months or more, or within 6 months or more; and/or

at least 60%, 70%, 80%, 90%, or 95% of patients treated according to the method and achieving an OR remain in response or survive for 3 months or more and/or for 6 months or more.

60. The method of any one of embodiments 49-59, wherein:

at or prior to administration of the cell dose, the patient is or has been identified as having lymphoma associated with or involving central nervous system (CNS) involvement; and/or

at least 70%, at least 80%, at least 90%, or at least 95% of patients treated according to the method who, at or prior to cell dosing, showed or were identified as showing lymphoma involving the CNS, achieved elimination of CNS disease.

61. A method of treating a patient comprising administering to a patient having lymphoma a dose of T cells comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds to a target antigen expressed by the lymphoma, wherein the patient's lymphoma is associated with or involves involvement central nervous system (CNS).

62. The method of Embodiment 60 or Embodiment 61 wherein, at or before the cell dose is administered, the patient has a brain lesion, optionally a temporal lobe lesion.

63. The method of embodiment 62 wherein the lymphoma is a B cell malignancy.

64. The method of Embodiment 61 or Embodiment 62, wherein the lymphoma is non-Hodgkin's lymphoma (NHL).

65. The method of any one of embodiments 60-64, wherein:

at least 35%, at least 40%, or at least 50% of patients treated according to the method achieve a complete response (CR) or remission of CNS disease, and optionally CR or remission of CNS disease is prolonged or prolonged in at least 60%, 70%, 80%, 90%, or 95% of patients achieving CR within 3 months or more, or within 6 months or more; and/or

at least 60%, 70%, 80%, 90%, or 95% of patients who achieve CR or CNS disease remission at one month and/or three months remain in response, remain in CR, and/or survive or remain alive without progression for 3 months or more and/or for 6 months or more and/or more than nine months; and/or

at least 50%, at least 60%, or at least 70% of patients treated according to the method achieve an objective response (OR) or remission of CNS disease, and optionally OR or remission of CNS disease is long-lasting or is long-lasting for at least at least 60%, 70%, 80%, 90%, or 95% of patients achieving OR within 3 months or more, or within 6 months or more; and/or

at least 60%, 70%, 80%, 90%, or 95% of patients who achieve OR or remission of CNS disease remain in response or survive for 3 months or more and/or for 6 months or more; and/or

the brain lesion is reduced in size or volume, optionally by more than or more than about 25%, 50%, 75%, or more; and/or

reduction or remission or clearance of CNS disease is achieved, optionally achieved in at least 35%, at least 40%, or at least 50% of patients treated according to the method.

66. The method of any one of embodiments 49-65, wherein:

more than or more than about 30%, 35%, 40%, or 50% of patients treated according to the method do not show any degree of cytokine release syndrome (CRS) or neurotoxicity; and/or

at least or about 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of patients treated according to the method do not experience early onset CRS or neurotoxicity and/or the onset of CRS is not observed earlier than 3 days after the initiation of administration, and/or the onset of neurotoxicity is not observed earlier than 5 days after the initiation of administration, and/or the average onset of neurotoxicity among patients receiving treatment according to the method is occurs at or after the mean peak, or the mean time to resolve CRS in patients treated according to the method, and/or the mean onset of neurotoxicity among patients treated according to the method is greater than or about 8, 9, 10, or 11 days.

67. The method of any one of embodiments 49-66, wherein:

prior to initiation of the dose of cells, the patient has not been administered a therapeutic agent or agent capable of treating, preventing, delaying, attenuating or reducing the development or risk of developing toxicity; and/or

the patient has not been administered an agent or agent to treat or prevent or reduce or attenuate neurotoxicity and/or cytokine release syndrome or the risk thereof, for a period of time post-dose, the time period being optionally or approximately 1, 2, 3, 4 , 5 days, or optionally 6, 7, 8, 9, 10, 11 days, or optionally 1, 2, 3, or 4 weeks; and/or

the patient has not been administered a treatment agent or agent to treat or prevent or reduce or attenuate neurotoxicity and/or cytokine release syndrome or the risk of it, post-dose, before or if the patient does not show signs or symptoms of toxicity and/or before or if the patient shows no signs or symptoms of toxicity other than fever, and optionally the fever is not a prolonged fever, or the fever is or has been reduced by or has been reduced by more than 1°C after treatment with an antipyretic agent; and/or

administration and any observation is on an outpatient basis and/or without admission of the patient to the hospital and/or without overnight stay in the hospital and/or without requiring admission or overnight stay in the hospital, optionally unless or until the patient shows prolonged fever or fever temperature, which is or has not been reduced by or has not been reduced by more than 1°C after treatment with an antipyretic.

68. The method of any one of embodiments 49-67, wherein:

prior to initiation of cell dose administration, the patient was not administered an anti-IL-6 or anti-IL-6R antibody, optionally tocilizumab or siltuximab, and/or was not administered a steroid, optionally dexamethasone; and/or

the patient has not received an anti-IL-6 or anti-IL-6R antibody, optionally tocilizumab or siltuximab, and/or has not received a steroid, optionally dexamethasone, for a period of time post-dose, the time period being optionally or approximately 1.2 , 3, 4, 5 days, or optionally 6, 7, 8, 9, 10, 11 days, or optionally 1, 2, 3, or 4 weeks; and/or

the patient has not received an anti-IL-6 or anti-IL-6R antibody, optionally tocilizumab or siltuximab, and/or has not received a steroid, optionally dexamethasone, following a cell dose, before or if the patient does not show signs or symptoms of toxicity, optionally neurotoxicity or CRS, and/or before or if the patient shows no signs or symptoms of toxicity, optionally neurotoxicity or CRS, other than fever, and optionally the fever is not a sustained fever, or the fever is or has been reduced by or has been reduced by more than than 1°C after antipyretic treatment; and/or

administration and any observation is on an outpatient basis and/or without admission of the patient to the hospital and/or without overnight stay in the hospital and/or without requiring admission or overnight stay in the hospital, optionally unless or until the patient shows prolonged fever or fever temperature, which is or has not been reduced by or has not been reduced by more than 1°C after treatment with an antipyretic.

69. The method of any one of embodiments 49-67, wherein:

the introduction is carried out on an outpatient basis and/or without requiring admission or overnight stay in the hospital; And

if the patient shows a prolonged fever or a fever that is or has not been reduced by or has not been reduced by more than 1°C after treatment with an antipyretic, the patient is admitted to hospital or overnight in the hospital and/or an agent or treatment is administered for treatment or prophylaxis or reducing or reducing neurotoxicity and/or cytokine release syndrome or the risk thereof.

70. The method of any one of embodiments 49-60 and 64-69 wherein the NHL is selected from the group consisting of aggressive NHL, diffuse large B cell lymphoma (DLBCL), NOS (de novo and transformed from indolent), primary mediastinal large B cell lymphoma (PMBCL), mantle cell lymphoma (MCL) and/or follicular lymphoma (FL), optionally stage 3B follicular lymphoma (FL3B).

71. The method of any one of embodiments 49-60 and 64-70 wherein the NHL includes diffuse large B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), NOS (de novo or transformed from indolent lymphoma ) or stage 3B follicular lymphoma.

72. The method of any one of embodiments 49-60 and 64-71, wherein the NHL includes DLBCL.

73. The method of any one of embodiments 49-60 and 70-72, wherein the DLBCL does not include DLBCL transformed from MZL and CLL (Richter) and/or the patient dosed with cells has DLBCL characterized as de novo or transformed from sluggish, and/or does not include DLBCL transformed from MZL and CLL.

74. The method of any one of embodiments 49-60 and 64-73, wherein the NHL does not include PMBCL and/or the cell dosed patient does not have PMBCL.

75. The method of any one of embodiments 57-73, wherein the patient is or has been identified as having an Eastern Cooperative Oncology Group Performance Status (ECOG) status of 0, 1, or 2.

76. The method of any one of embodiments 49-75, wherein the patient is or has been identified as having an ECOG status of 0 or 1.

77. The method of any one of embodiments 49-76 wherein, at or shortly before administration of the cell dose, the patient is relapsed after being in remission after treatment with or has become refractory to one or more prior NHL therapies, optionally one, two, or three prior therapies other than another dose of CAR-expressing cells.

78. The method of any one of embodiments 49-77, wherein, at or before the cell dose is administered:

the patient is or has been identified as having double/triple hit lymphoma; and/or

the patient is or has been identified as having a chemo-refractory lymphoma, optionally a chemo-refractory DLBCL; and/or

the patient has not achieved complete remission (CR) in response to prior therapy; and/or

the patient has a recurrence within 1 year or less than 1 year after receiving an autologous stem cell transplant (ASCT).

79. The method of any one of embodiments 49-78, comprising, prior to administration of the cell dose, identifying or selecting a patient for cell dose administration having:

double/triple hit lymphoma;

chemorefractive lymphoma, optionally chemorefractive DLBCL;

has not achieved complete remission (CR) in response to prior cancer therapy, optionally NHL; and/or

has a recurrence within 1 year or less than 1 year after receiving an autologous stem cell transplant (ASCT); and/or

has lymphoma associated with or involving the central nervous system (CNS).

80. The method of any one of embodiments 49-79, further comprising administering an additional therapeutic agent or therapy, optionally other than cell therapy, optionally other than CAR ⁺ T cell therapy.

81. The method of embodiment 80, wherein the additional therapeutic agent or therapy is for the treatment of NHL or cancer and/or increases the persistence, activity, and/or efficacy of a cell dose.

82. The method of Embodiment 80 or Embodiment 81 wherein the additional therapeutic agent or therapy is administered if the patient does not respond, optionally does not show a CR or OR, to cell therapy for 1 month, for 2 months, or for 3 months after a dose of cells.

83. The method according to embodiment 80 or embodiment 81, wherein the additional therapeutic agent or therapy is administered to the patient:

who is or has been identified as having stable or progressive disease (SD/PD) following treatment with prior therapy, optionally prior therapy with a chemotherapeutic agent;

which is or has been identified as having status 2 according to the Eastern Cooperative Oncology Group Performance Status (ECOG);

who is or has been identified as having transformed follicular lymphoma (tFL); or

which is or has been identified as having a DLBCL transformed from MZL and CLL.

84. The method of any one of embodiments 80-83, comprising, prior to administration of the cell dose or additional therapeutic agent or therapy, identifying or selecting a patient for cell dose administration having:

stable or progressive disease (SD/PD) following treatment with prior therapy, optionally prior therapy with a chemotherapeutic agent;

Eastern Cooperative Oncology Group Performance Status (ECOG) status 2;

transformed follicular lymphoma (tFL); or

DLBCL transformed from MZL and CLL.

85. The method of any one of embodiments 80-84, wherein the additional therapeutic agent or therapy is administered before, with, or simultaneously with and/or after initiation of cell dose administration.

86. The method of any one of embodiments 49-85, wherein:

CAR includes an scFv antigen-specific transmembrane domain, a cytoplasmic signaling domain derived from a co-stimulatory molecule, which optionally is or includes 4-1BB, and a cytoplasmic signaling domain derived from an ITAM-containing primary signaling molecule, which optionally is or includes CD3zeta signaling domain and optionally further includes a spacer between the transmembrane domain and scFv;

CAR includes, in that order, an scFv antigen-specific transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is or includes a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule , which is optionally the CD3zeta signaling domain; or

CAR includes, in that order, an antigen-specific scFv, a spacer, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which is optionally a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is or includes the CD3zeta signaling domain; and moreover:

the spacer is optionally a polypeptide spacer that (a) includes or consists of all or part of the hinge region of an immunoglobulin or a modified version thereof, or comprises about 15 amino acids or less and does not include the CD28 extracellular domain or the CD8 extracellular domain, (b) includes or consists of all or a portion of an immunoglobulin hinge region, optionally an IgG4 hinge or a modified version thereof, and/or comprises about 15 amino acids or less and does not include a CD28 extracellular domain or a CD8 extracellular domain, or (c) is 12 or about 12 amino acids long and/or includes or consists of from all or part of the hinge region of an immunoglobulin, optionally IgG4 or a modified version thereof; or (d) has or consists of the sequence 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 above, having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with it, or (e) includes or consists of the formula X ₁ PPX ₂ P, where X ₁ is glycine, cysteine or arginine, and X ₂ is cysteine or threonine; and/or

the co-stimulatory domain 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 with it; and/or

the primary signaling domain includes SEQ ID NO: 13 or 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 with it; and/or

scFv includes a CDRL1 RASQDISKYLN sequence (SEQ ID NO: 35), a CDRL2 SRLHSGV sequence (SEQ ID NO: 36), and/or a CDRL3 GNTLPYTFG sequence (SEQ ID NO: 37), and/or a CDRH1 DYGVS sequence (SEQ ID NO: 38 ), a VIWGSETTYYNSALKS CDRH2 sequence (SEQ ID NO: 39) and/or a YAMDYWG CDRH3 sequence (SEQ ID NO: 40), or wherein the scFv comprises an FMC63 heavy chain variable region and an FMC63 light chain variable region and/or an FMC63 CDRL1 sequence, a CDRL2 sequence FMC63, FMC63 CDRL3 sequence, FMC63 CDRH1 sequence, FMC63 CDRH2 sequence, and FMC63 CDRH3 sequence, either binds to the same epitope as or competes for binding to any of these, and optionally the scFv includes, in that order, a V _H , a linker , optionally including SEQ ID NO: 24, and V _L and/or scFv includes a flexible linker and/or includes the amino acid sequence shown as SEQ ID NO: 24.

87. The method of any one of embodiments 49-86, wherein the antigen is a B cell antigen, which is optionally CD19.

88. The method of any one of embodiments 1-23 and 49-87 wherein, prior to administration, the patient has been preconditioned using anti-lymphocyte therapy comprising administration of fludarabine and/or cyclophosphamide.

89. The method of any one of embodiments 1-23 and 49-88, further comprising, just prior to administration, administering anti-lymphocyte therapy to the patient, comprising administering fludarabine and/or cyclophosphamide.

90. The method according to embodiment 88 or embodiment 89 wherein the antilymphocyte therapy comprises administering cyclophosphamide in an amount of about 200-400 mg/m ² , optionally in an amount of or about 300 mg/m ² inclusive, and/or fludarabine in an amount of approximately 20-40 mg/m ² , optionally 30 mg/m ² , per day for 2-4 days, optionally for 3 days, or in which antilymphocyte therapy includes the administration of cyclophosphamide in an amount of approximately 500 mg/m ² .

91. The method of Embodiment 88 or Embodiment 89, wherein:

antilymphocyte therapy includes administration of cyclophosphamide at or about 300 mg/m ² and fludarabine at about 30 mg/m ² per day for 3 days; and/or

antilymphocyte therapy includes the administration of cyclophosphamide at or about 500 mg/m ² and fludarabine at about 30 mg/m ² per day for 3 days.

92. The method of any one of embodiments 49-91, wherein administration of the cell dose and/or antilymphocyte therapy is via outpatient delivery, optionally unless or until the patient exhibits a sustained fever or fever that is or has not been reduced by or not reduced by more than 1°C after treatment with an antipyretic.

93. The method of Embodiment 92, wherein if the patient shows a prolonged fever or a fever that is or has not been reduced by or has not been reduced by more than 1°C after treatment with an antipyretic agent, the patient is hospitalized or overnight in the hospital and/or administering a therapeutic agent or agent to treat or prevent or reduce or reduce neurotoxicity and/or cytokine release syndrome or the risk thereof.

94. The method of any one of embodiments 49-92 wherein the dose of cells is administered parenterally, optionally intravenously.

95. The method of any one of embodiments 49-94, wherein:

at least 40% or at least 50% of patients treated according to the method achieve complete remission (CR), progression-free survival (PFS) and/or overall survival (OS) of greater than or about 3 months, 6 months, or 12 months;

on average, patients treated according to the method show an average PFS or OS of more than or about 6 months, 12 months, or 18 months; and/or

the patient shows PFS or OS after therapy for at least or about 6, 12, 18 or more months.

96. The method of any one of embodiments 1-23 and 49-95, wherein at or about 14 or 28 days after initiation of cell dosing, the number of CAR ⁺ T cells, optionally CAR ⁺ CD8 ⁺ T cells, and /or CAR ⁺ CD4 ⁺ T cells detected in the blood of the patient or the majority of patients receiving treatment with the method is more than 1 cell per μl, more than 5 cells per μl or more than 10 cells per μl.

97. The method of any one of embodiments 1-23 and 49-96, wherein the T cells are primary T cells obtained from a patient.

98. The method of any of embodiments 1-23 and 49-97, wherein the T cells are autologous to the patient.

99. The method of any of embodiments 1-23 and 49-97, wherein the T cells are allogeneic to the patient.

100. The method of any one of embodiments 49-99, wherein the T cells comprise CAR-expressing CD4 ⁺ T cells and CAR-expressing CD8 ⁺ T cells, and administration comprises administering a plurality of distinct compositions, wherein said plurality of distinct compositions includes a first composition comprising one of CD4 ⁺ T cells and CD8 ⁺ T cells, and a second composition comprising the other of CD4 ⁺ T cells or CD8 ⁺ T cells.

101. The method according to embodiment 100, wherein:

the first composition and the second composition are administered at an interval of 0 to 12 hours, at an interval of 0 to 6 hours, or at an interval of 0 to 2 hours, or in which the administration of the first composition and the administration of the second composition are carried out on the same day, are carried out at an interval of about 0 to about 12 hours, in an interval of about 0 to about 6 hours, or in an interval of about 0 to about 2 hours; and/or

initiation of administration of the first composition and initiation of administration of the second composition are carried out at intervals of from about 1 minute to about 1 hour, or at intervals of from about 5 minutes to about 30 minutes.

102. The method according to embodiment 100 or embodiment 101, wherein the first composition and the second composition are administered at intervals of 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.

103. The method of any one of embodiments 100-102, wherein the first composition comprises CD4 ⁺ T cells.

104. The method of any one of embodiments 100-102 wherein the first composition comprises CD8 ⁺ T cells.

105. The method of any one of embodiments 100-104 wherein the first composition is administered prior to the second composition.

106. The method of any one of embodiments 49-105, wherein the dose of T cells is administered to the patient 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.

107. The method of any one of embodiments 49-106 wherein the dose of T cells is administered as a double dose comprising a first dose of T cells and a subsequent dose of T cells, wherein one or both of the first dose and the second dose comprises administering a plurality of compositions of T cells.

108. The method of embodiment 107 wherein the subsequent dose is administered at a time point of at least or greater than about 7 days, or 14 days after and less than about 28 days after the initiation of the first dose of cells.

109. A method for assessing the probability of response to cell therapy, including:

(a) assessing the level, amount, or concentration of one or more analytes in the biological sample, wherein the one or more analytes are selected from ferritin, LDH, CXCL10, G-CSF, and IL-10, wherein:

the biological sample is obtained from a patient who is a candidate for cell therapy treatment, said cell therapy comprising a dose of genetically engineered cells expressing the recombinant receptor; And

the biological sample is obtained from the patient before the introduction of cell therapy and/or the specified biological sample does not include the recombinant receptor and/or the specified genetically engineered cells; And

(b) comparing, individually, the level, amount, or concentration of the analyte in the sample to a threshold value, thereby determining the likelihood that the patient will achieve a response to cell therapy.

110. The method of embodiment 109, further comprising administering cell therapy to the patient if the patient is shown to be likely to achieve a response.

111. A method for selecting a patient for treatment, including:

(a) assessing the level, amount, or concentration of one or more analytes in the biological sample, wherein the one or more analytes are selected from ferritin, LDH, CXCL10, G-CSF, and IL-10, wherein:

the biological sample is obtained from a patient who is a candidate for cell therapy treatment, said cell therapy comprising a dose of genetically engineered cells expressing the recombinant receptor; And

the biological sample is obtained from the patient before the introduction of cell therapy and/or the specified biological sample does not include the recombinant receptor and/or the specified genetically engineered cells; And

(b) selecting a patient for whom a probability of response to treatment is determined based on the results of determining the probability that the patient will achieve a response to cell therapy by comparing, individually, the level, amount or concentration of the analyte in the sample with a threshold value.

112. The method of embodiment 111, further comprising administering cell therapy to the patient selected for treatment.

113. A method of treatment, including:

(a) selecting a patient for whom the probability of responding to cell therapy treatment is determined based on the results of determining the probability that the patient will achieve a response to cell therapy, by comparing, individually, the level, amount or concentration of one or more analytes in a biological sample, where one or more analytes are selected from ferritin, LDH, CXCL10, G-CSF, and IL-10, with a cut-off where:

the biological sample is obtained from a patient who is a candidate for cell therapy treatment, said cell therapy comprising a dose of genetically engineered cells expressing the recombinant receptor; And

the biological sample is obtained from the patient before the introduction of cell therapy and/or the specified biological sample does not include the recombinant receptor and/or the specified genetically engineered cells; And

(b) administering the cell therapy to the patient selected for treatment.

114. The method of any one of embodiments 109-113, wherein:

a patient is likely to achieve a response if the level, amount, or concentration of one or more analytes is below a threshold, and a patient is unlikely to achieve a response if the level, amount, or concentration of one or more analytes is above a threshold.

115. The method of any one of embodiments 109-114 wherein the threshold is within 25%, 20%, 15%, 10% or 5% and/or within a standard deviation below the median or mean level, amount or concentration, or equal to, or approximately equal to, the median, or mean level, amount, or concentration, of an analyte in a biological sample obtained from a group of patients prior to receiving cell therapy, with each of the patients in the group continuing to achieve a response after administration of the composition of cells expressing the recombinant receptor for the treatment of the same disease or states.

116. The method of any one of embodiments 109-114 wherein the threshold is within 25%, 20%, 15%, 10% or 5% and/or within a standard deviation above the median or mean level, amount or concentration of the analyte in a biological sample obtained from a group of patients prior to receiving cell therapy, wherein each of the patients in the group continued to show stable disease (SD) and/or progressive disease (PD) after administration of the composition of cells expressing the recombinant receptor to treat the same disease or condition.

117. The method of any one of embodiments 109-116, wherein the response includes an objective response.

118. The method of embodiment 117 wherein the objective response includes a complete response (CR) or a partial response (PR).

119. A method for assessing the likelihood of a long-term response to cell therapy, including:

(a) assessing the level, amount or concentration of one or more analytes in a biological sample, where one or more analytes are selected from LDH, ferritin, CRP, D-dimer, SAA-1, IL-6, IL-10, IL-15, IL-16, TNF-α, IFN-γ, MIP-1α, CXCL-10, IL-8, MCP-1 and MIP-1β, and:

the biological sample is obtained from a patient who is a candidate for cell therapy treatment, said cell therapy comprising a dose of genetically engineered cells expressing the recombinant receptor; And

the biological sample is obtained from the patient before the introduction of cell therapy and/or the specified biological sample does not include the recombinant receptor and/or the specified genetically engineered cells; And

(b) comparing, individually, the level, amount, or concentration of the analyte in the sample to a threshold value, thereby determining the likelihood that the patient will achieve a sustained response to cell therapy.

120. The method of embodiment 119, further comprising administering cell therapy to the patient if the patient is shown to be likely to achieve a response.

121. A method for selecting a patient for treatment, including:

(a) assessing the level, amount or concentration of one or more analytes in a biological sample, where one or more analytes are selected from LDH, ferritin, CRP, D-dimer, SAA-1, IL-6, IL-10, IL-15, IL-16, TNF-α, IFN-γ, MIP-1α, CXCL-10, IL-8, MCP-1 and MIP-1β, and:

the biological sample is obtained from a patient who is a candidate for cell therapy treatment, said cell therapy comprising a dose of genetically engineered cells expressing the recombinant receptor; And

the biological sample is obtained from the patient before the introduction of cell therapy and/or the specified biological sample does not include the recombinant receptor and/or the specified genetically engineered cells; And

(b) selecting a patient for whom the probability of response to treatment is determined based on the results of determining the probability that the patient will achieve a long-term response to cell therapy, by comparing, individually, the level, amount or concentration of the analyte in the sample with a threshold value.

122. The method of embodiment 121, further comprising administering cell therapy to the patient selected for treatment.

123. A method of treatment, including:

(a) selecting a patient for whom the probability of responding to cell therapy treatment is determined based on the results of determining the probability that the patient will achieve a sustained response to cell therapy, by comparing, individually, the level, amount or concentration of one or more analytes in a biological sample with a threshold value where one or more analytes are selected from LDH, ferritin, CRP, D-dimer, SAA-1, IL-6, IL-10, IL-15, IL-16, TNF-α, IFN-γ, MIP-1α, CXCL-10, IL-8, MCP-1 and MIP-1β, and:

the biological sample is obtained from a patient who is a candidate for cell therapy treatment, said cell therapy comprising a dose of genetically engineered cells expressing the recombinant receptor; And

the biological sample is obtained from the patient before the introduction of cell therapy and/or the specified biological sample does not include the recombinant receptor and/or the specified genetically engineered cells; And

(b) administering the cell therapy to the patient selected for treatment.

124. The method of any one of embodiments 120-123, wherein:

a patient is likely to achieve a sustained response if the level, amount, or concentration of one or more analytes is below a threshold, and a patient is unlikely to achieve a sustained response if the level, amount, or concentration of one or more analytes is above a threshold.

125. The method of any one of embodiments 120-124 wherein the threshold is within 25%, 20%, 15%, 10% or 5% and/or within a standard deviation below the median or mean level, amount or concentration, or equal to, or approximately equal to, the median, or mean level, amount, or concentration, of an analyte in a biological sample obtained from a group of patients prior to receiving cell therapy, with each of the patients in the group continuing to achieve sustained response after administration of the recombinant receptor-expressing cell composition for treatment of the same diseases or conditions.

126. The method of any one of embodiments 120-124 wherein the threshold is within 25%, 20%, 15%, 11% or 5% and/or within a standard deviation above the median or mean level, amount or concentration of the analyte in a biological sample obtained from a group of patients prior to receiving cell therapy, and each of the patients of the group did not achieve a long-term response after administration of the composition of cells expressing the recombinant receptor for the treatment of the same disease or condition.

127. The method of any one of embodiments 120-126, wherein the sustained response comprises a complete response (CR) or a partial response (PR) that lasts for 3 months, 4 months, 5 months, or 6 months or more.

128. The method of any one of embodiments 120-127 wherein the sustained response comprises a CR or PR that lasts for at least 3 months.

129. A method for assessing the risk of developing toxicity after the introduction of cell therapy, including:

(a) assessing the level, amount or concentration of one or more analytes in a biological sample from a patient or a volumetric parameter of a tumor mass in a patient, wherein one or more analytes are selected from LDH, ferritin, C-reactive protein (CRP), D-dimer (product fibrin breakdown), IL-6, IL-8, IL-10, IL-15, IL-16 TNF-α, IFN-α2, MCP-1, MIP-1α and MIP-1β, with:

the patient is a candidate for cell therapy treatment, said cell therapy optionally comprising a dose or composition of engineered cells expressing the recombinant receptor; And

the biological sample is obtained from the patient before the introduction of cell therapy and/or the specified biological sample does not include the recombinant receptor and/or the specified genetically engineered cells; And

(b) comparing, individually, the level, amount, or concentration of the analyte in the sample, or the volumetric parameter of the tumor mass, with a threshold value, thereby determining the risk of developing toxicity after administration of the cell therapy.

130. A method for identifying a patient, including:

(a) assessing the level, amount or concentration of one or more analytes in a biological sample from a patient or a volumetric parameter of a tumor mass in a patient, wherein one or more analytes are selected from LDH, ferritin, C-reactive protein (CRP), D-dimer (product fibrin breakdown), IL-6, IL-8, IL-10, IL-15, IL-16 TNF-α, IFN-α2, MCP-1, MIP-1α and MIP-1β, with:

the patient is a candidate for cell therapy treatment, said cell therapy optionally comprising a dose or composition of engineered cells expressing the recombinant receptor; And

the biological sample is obtained from the patient before the introduction of cell therapy and/or the specified biological sample does not include the recombinant receptor and/or the specified genetically engineered cells; And

(b) identifying a patient who is at risk of developing toxicity after administration of the cell therapy based on a comparison, individually, of the level, amount or concentration of the analyte in the sample or tumor mass volume parameter with a threshold value.

131. A method of treatment, including:

(a) assessing the level, amount or concentration of one or more analytes in a biological sample from a patient or a volumetric parameter of a tumor mass in a patient, wherein one or more analytes are selected from LDH, ferritin, C-reactive protein (CRP), D-dimer (product fibrin breakdown), IL-6, IL-8, IL-10, IL-15, IL-16 TNF-α, IFN-α2, MCP-1, MIP-1α and MIP-1β, with:

The patient is a candidate for cell therapy treatment, said cell therapy optionally comprising a dose or composition of engineered cells expressing the recombinant receptor; And

the biological sample is obtained from the patient before the introduction of cell therapy and/or the specified biological sample does not include the recombinant receptor and/or the specified genetically engineered cells; And; And

(b) comparing, individually, the level, amount or concentration of the analyte in the sample or the volumetric parameter of the tumor mass with a threshold value, thereby determining the risk of developing toxicity after the introduction of cell therapy; And

(c) following or based on the results of the evaluation, administering to the patient a cell therapy, and optionally a treatment agent or other agent capable of treating, preventing, delaying, attenuating or reducing the development or risk of developing toxicity.

132. The method of any one of embodiments 129-131, wherein the biological sample is a blood or plasma sample.

133. The method of any one of embodiments 129-132, wherein the tumor mass volumetric parameter is a sum of product dimensions (SPD) or is a volumetric measurement based on CT and/or MRI imaging or other body imaging.

134. The method according to embodiment 133, wherein the measurement of the volumetric parameter of the tumor mass is performed prior to treatment, prior to apheresis, or prior to production of a cell product.

135. The method of any one of embodiments 129-134, further comprising monitoring the patient for symptoms of toxicity if the patient is receiving cell therapy and is identified as being at risk of developing toxicity.

136. The method of any one of embodiments 129-135, wherein:

the patient is at risk of developing toxicity if the level, amount, or concentration of one or more analytes or the tumor mass volume parameter is above a threshold, and the patient is at low risk of developing toxicity if the level, amount, or concentration of one or more analytes or the tumor mass volume parameter is below a threshold values.

137. The method of any one of embodiments 129-136 wherein the threshold is within 25%, 20%, 15%, 10% or 5% and/or within a standard deviation above the median or mean level, amount or concentration, or equal to or approximately equal to the median or mean level, amount or concentration of the analyte or volumetric parameter of the tumor mass in a biological sample obtained from a group of patients before the introduction of cell therapy, and each of the patients of the group still did not develop any toxicity after receiving the composition cells expressing the recombinant receptor to treat the same disease or condition.

138. The method of any one of embodiments 129-137 wherein the threshold is within 25%, 20%, 15%, 10% or 5% and/or within a standard deviation below the median or mean level, amount or concentration of the analyte or volumetric parameter of the tumor mass in a biological sample obtained from a group of patients before receiving cell therapy, and each of the patients of the group continued to develop toxicity after receiving a composition of cells expressing the recombinant receptor for the treatment of the same disease or condition.

139. The method of any one of embodiments 129-138, wherein the toxicity is neurotoxicity or CRS.

140. The method of any one of embodiments 129-139, wherein the toxicity is neurotoxicity or CRS grade 1 or greater.

141. The method of any one of embodiments 129-140, wherein:

the toxicity is severe neurotoxicity or is grade 2 neurotoxicity or higher, grade 3 neurotoxicity or higher, at least sustained grade 3 neurotoxicity, or grade 4 or grade 5 neurotoxicity or higher; or

toxicity is severe CRS or includes CRS grade 2 or higher or grade 3 or higher.

142. The method of any one of embodiments 129-141 wherein the toxicity is neurotoxicity and the tumor bulk volume parameter is SPD and one or more analytes are selected from LDH, IL-10, IL-15, IL-16, TNF- α and MIP-1β.

143. The method of any one of embodiments 129-141 wherein the toxicity is neurotoxicity and one or more analytes are assessed and the analytes are selected from LDH, ferritin, CRP, IL-6, IL-8, IL-10, TNF-α , IFN-α2, MCP-1 and MIP-1β.

144. The method of any one of embodiments 129-141 wherein the toxicity is neurotoxicity and one or more analytes are evaluated and the analytes are selected from IL-8, IL-10 and CXCL10.

145. The method of embodiment 144 wherein the neurotoxicity is severe neurotoxicity or grade 3 or higher neurotoxicity.

146. The method of any one of embodiments 129-141, wherein the toxicity is CRS and one or more analytes or tumor mass volume parameter are selected from LDH, SPD, CRP, d-dimer, IL-6, IL-15, TNF -α and MIP-1α.

147. The method of embodiment 146 wherein the CRS is severe CRS or grade 3 or higher CRS.

148. The method of any one of embodiments 129-147, wherein if the patient is identified as being at risk of developing toxicity, administering to the patient:

(a) (1) a therapeutic agent or other agent capable of treating, preventing, delaying, attenuating or reducing the development or risk of developing toxicity, and (2) a cellular therapy, wherein administration of the agent must be administered (i) before, (ii ) within one, two or three days, (iii) simultaneously with and/or (iv) at the first temperature increase after the initiation of the administration of cell therapy to the patient; and/or

(b) cell therapy at a reduced dose or at a dose not associated with a risk of developing toxicity or severe toxicity, or not associated with a risk of developing toxicity or severe toxicity in the majority of patients and/or the majority of patients with a disease or condition that the patient has or suspected of having it after the introduction of cell therapy; and/or

(c) administering the cell therapy to the patient on an inpatient basis and/or with hospital admission for one or more days, optionally the cell therapy must otherwise be administered to patients on an outpatient basis or without hospital admission for one or more days.

149. The method of Embodiment 148 wherein the agent or other treatment is an anti-IL-6 or anti-IL6 receptor antibody.

150. The method of Embodiment 149 wherein the agent or other treatment is or comprises an agent selected from tocilizumab, siltuximab, clasakizumab, sarilumab, olokizumab (CDP6038), elsilimomab, ALD518/BMS-945429, sirukumab (CNTO 136) , CPSI-2634, ARGX-109, FE301 and FM101.

151. The method of embodiment 148 wherein the agent or other therapeutic agent is or includes a steroid, optionally dexamethasone.

152. The method of any one of embodiments 129-142 and 148-151 wherein the volume parameter is estimated and the volume parameter is SPD and the threshold value is either about 30 cm ² , or about 40 cm ² , or about 50 cm ² or approximately 60 cm ² , or is or approximately 70 cm ² .

153. The method of Embodiment 152 wherein the volumetric parameter is SPD and the threshold value is or is about 50 cm ² .

154. The method of any one of embodiments 129-151, wherein one or more analytes are or include LDH and the threshold is either about 300 units per liter, or about 400 units per liter, or about 500 units per liter or is or approximately is 600 units per litre.

155. The method of any one of embodiments 154, wherein the analyte is LDH and the threshold is or is about 500 units per liter.

156. The method of any one of embodiments 109-155, wherein the recombinant receptor specifically binds to an antigen associated with the disease or condition, or expressed in cells in the lesion environment associated with the disease or condition.

157. The method of any one of embodiments 109-156 wherein the disease or condition is cancer.

158. The method of any one of embodiments 109-157, wherein the disease or condition is myeloma, leukemia, or lymphoma.

159. The method of any one of embodiments 109-158, wherein the disease or condition is a B-cell malignancy and/or is acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin's lymphoma (NHL), and diffuse large B-cell lymphoma (DLBCL).

160. The method of any one of embodiments 109-159, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

161. The method of any one of embodiments 109-160, wherein the engineered cells include T cells, optionally CD4 ⁺ and/or CD8 ⁺ .

162. The method of embodiment 161 wherein the T cells are primary T cells obtained from the patient or are autologous to the patient.

163. A method for selecting a patient for treatment, including:

(a) contacting the biological sample with one or more reagents capable of detecting or which are specific for one or more analytes, wherein the one or more analytes are selected from LDH, ferritin, CRP, IL-6, IL-7, IL-8, IL-10, IL-15, IL-16, TNF-alpha, IFN-gamma, MCP-1, MIP-1beta, eotaxin, G-CSF, IL-1Ralpha, IL-1Rbeta, IP-10, perforin and D- dimer (fibrin breakdown product),

and:

the biological sample is obtained from a patient who is a candidate for cell therapy treatment, said cell therapy optionally comprising a dose or composition of engineered cells expressing the recombinant receptor; And

the biological sample is obtained from the patient before the introduction of cell therapy and/or the specified biological sample does not include the recombinant receptor and/or the specified genetically engineered cells; And

(b) selecting, a patient who:

(i) the level, amount or concentration of the analyte in the sample is equal to or greater than a threshold value, thus identifying a patient at risk of developing toxicity to the cell therapy; or

(ii) the level, amount or concentration of the analyte is below a threshold value.

164. The method of embodiment 163, wherein:

(a) the patient in (i) is selected to administer to the patient (1) a therapeutic agent or other agent capable of treating, preventing, delaying, attenuating, or reducing the development or risk of developing toxicity, and (2) cell therapy, wherein the administration of the agent must be administered (i) before, (ii) within one, two or three days, (iii) simultaneously with and/or (iv) at the first temperature increase after initiation of administration of cell therapy to the patient; and/or

(b) the patient in (i) is selected to administer the cell therapy to the patient at a reduced dose or at a dose not associated with a risk of developing toxicity or severe toxicity, or not associated with a risk of developing toxicity or severe toxicity in the majority of patients and/or the majority of patients, having a disease or condition that the patient has or is suspected of having, after the introduction of cell therapy; and/or

(c) the patient in (i) is selected to administer the cell therapy to the patient on an inpatient basis and/or with hospital admission for one or more days, optionally the cell therapy must otherwise be administered to patients on an outpatient basis or without hospital admission within one or more days.

165. The method according to embodiment 163 or embodiment 164 wherein the patient in (i) is selected and the method further comprises:

(a) administering to the patient (1) a therapeutic agent or other agent capable of treating, preventing, delaying, attenuating or reducing the development or risk of developing toxicity, and (2) cell therapy, wherein the agent is administered (i) before, (ii ) within one, two or three days, (iii) simultaneously with and/or (iv) at the first temperature rise after the initiation of the administration of cell therapy to the patient; and/or

(b) administering the cell therapy to the patient at a reduced dose or at a dose not associated with a risk of developing toxicity or severe toxicity, or not associated with a risk of developing toxicity or severe toxicity in the majority of patients and/or the majority of patients having a disease or condition that the patient has or is suspected of having, after the introduction of cell therapy; and/or

(c) administering to a patient a cell therapy or a dose of genetically engineered cells of a cell therapy not associated with a risk of developing toxicity or severe toxicity, or not associated with a risk of developing toxicity or severe toxicity in the majority of patients and / or the majority of patients with a disease or condition, which the patient has or is suspected of having, after the introduction of cell therapy; and/or

(d) administering cell therapy to the patient on an inpatient basis and/or with hospital admission for one or more days, optionally the cell therapy must otherwise be administered to patients on an outpatient basis or without hospital admission for one or more days.

166. The method according to embodiment 163 or embodiment 164, wherein:

(a) the patient in (ii) is selected to administer the cell therapy to the patient, optionally at an unreduced dose, optionally on an outpatient basis or without hospital admission for one or more days;

(b) the patient in (ii) is selected to administer the cell therapy to the patient, wherein the cell therapy does not include administering, prior to or simultaneously with the administration of the cell therapy and/or prior to the development of a sign or symptom of toxicity other than fever, a treatment agent or agent capable of treating, preventing, delaying or reducing the development of toxicity; and/or

(c) the patient in (ii) is selected to receive cell therapy on an outpatient basis and/or without admission of the patient to the hospital overnight or for one or more consecutive days and/or without admission of the patient to the hospital for one or more days.

167. The method of embodiment 163, 164, or 166, wherein the patient in (ii) is selected, and the method further comprises administering cell therapy to the patient, optionally at a non-reduced dose, optionally on an outpatient basis, or without hospital admission for one or more days .

168. The method of any one of embodiments 163, 164, 166, and 167, wherein the patient in (ii) is selected, and the method further comprises administering cell therapy to the patient, wherein:

administering cell therapy does not include administering, prior to or simultaneously with administration of cell therapy and/or prior to the development of a sign or symptom of toxicity other than fever, a therapeutic agent or agent capable of treating, preventing, delaying or reducing the development of toxicity; and/or

administration of the cell therapy should or may be administered to the patient on an outpatient basis and/or without admission of the patient to the hospital overnight or for one or more consecutive days and/or without admission of the patient to the hospital for one or more days.

169. A method of treatment, including:

(a) testing a biological sample for a level, amount, or concentration of one or more analytes, wherein the biological sample is obtained from a patient who is a candidate for treatment, optionally by cell therapy, said cell therapy optionally comprising a dose or composition of genetically engineered cells expressing a recombinant receptor, for the treatment of a disease or condition, wherein one or more analytes are selected from LDH, ferritin, CRP, IL-6, IL-7, IL-8, IL-10, IL-15, IL-16, TNF-alpha, IFN-gamma, MCP-1, MIP-1beta, eotaxin, G-CSF, IL-1Ralpha, IL-1Rbeta, IP-10, perforin, and D-dimer (fibrin breakdown product); And

(b) following or based on the test results, administering to the patient a cell therapy, and optionally a treatment agent or other agent capable of treating, preventing, delaying, attenuating or reducing the development or risk of developing toxicity.

170. A method of treating a patient with or suspected of having a disease or condition, including:

(1) administering to a patient a cell therapy comprising a dose or composition of engineered cells expressing the recombinant receptor for treatment or that specifically recognizes a disease or condition, and optionally (2) further administering to the patient a treatment agent or other agent capable of treating, preventing, delaying, attenuating or reducing the development or risk of developing toxicity, and:

the administration is carried out after or is carried out on the basis of the results of an assessment of one or more parameters of the preliminary treatment, and:

(a) one or more pre-treatment parameters include a parameter associated with or correlated with or marker for the severity of the disease or condition in the patient, which parameters optionally include one or more analytes and/or one or more volumetric or other body measurements,

(b) evaluating one or more pre-treatment parameters includes assessing the amount or level in a biological sample that is or originates from a sample obtained from the patient, the level, amount or concentration of one or more analytes obtained from the biological sample from the patient prior to the introduction of cell therapy wherein the sample is obtained from a patient prior to administration of cell therapy and optionally prior to apheresis or prior to obtaining cells for administration, and/or one or more analytes are associated with disease severity and/or one or more analytes are selected from LDH, ferritin, CRP, IL -6, IL-7, IL-8, IL-10, IL-15, IL-16, TNF-alpha, IFN-gamma, MCP-1, MIP-1beta, eotaxin, G-CSF, IL-1Ralpha, IL -1Rbeta, IP-10, perforin and D-dimer (fibrin degradation product); and/or

(c) evaluating one or more pre-treatment parameters, optionally including evaluating a volumetric measurement of a disease or condition in a patient, which is not necessarily a measurement obtained by imaging, optionally the sum of product dimensions (SPD), or other volumetric measurement(s), optionally based on CT and/or MRI imaging or other body imaging performed prior to treatment and optionally prior to apheresis or cell retrieval.

171. The method according to embodiment 170, wherein said evaluation of one or more pre-treatment parameters comprises testing one or more analytes, including detection, optionally including contacting a reagent capable of directly or indirectly detecting the analyte(s) with a biological sample and determining the level , quantity or concentration of the analyte(s) in the biological sample.

172. The method according to embodiment 170 or embodiment 171, wherein the evaluation of one or more pre-treatment parameters comprises (A) evaluation in a biological sample that is or is derived from a sample obtained from a patient prior to treatment and/or prior to apheresis or processing cells, level, amount or concentration of one or more analytes, optionally associated with the severity of the disease, and (B) an assessment of the volumetric parameter of the severity of the disease, and the measurement of the severity of the disease is not necessarily the result of the sum of product sizes (SPD) or other volumetric measurement(s), optionally based on CT and/or MRI imaging or other imaging of the body and/or it is performed prior to treatment, prior to apheresis, or prior to obtaining a cell product.

173. The method of any one of embodiments 169-172, wherein if the level, amount, or concentration and/or measurement, or a combination thereof, resulting from or obtained through a pre-treatment evaluation, is equal to or greater than a threshold:

administration includes additional administration to a patient of a treatment agent or other agent capable of treating, preventing, delaying, attenuating or reducing the development or risk of developing toxicity, optionally said additional administration is carried out (i) before, (ii) within one, two or three days, or within four, five or six days, (iii) simultaneously with and/or (iv) at the first rise in temperature after the initiation of the introduction of cell therapy to the patient; and/or

the cell therapy administered to the patient is administered (A) at a reduced dose compared to if the level, amount, or concentration and/or measurement, or a combination thereof, is below a threshold value, or (B) at a dose not associated with a risk of developing toxicity, or severe toxicity, or not associated with a risk of developing toxicity or severe toxicity in most patients and/or most patients with a disease or condition that the patient has or is suspected of having, after the introduction of cell therapy, or (C) at a dose reduced by compared with the maximum tolerated dose, or the approved dose, or the maximum approved dose, for use in patients with a disease or condition or for the treatment of a disease or condition; and/or

administering cell therapy to a patient is or is determined to require inpatient treatment and/or hospital admission for one or more days, optionally cell therapy otherwise if the level, amount, concentration, measurement, or combination is below a threshold or has not been evaluated , should be administered to patients on an outpatient or non-hospital basis for one or more days or overnight.

174. The method of any one of embodiments 169-173, wherein if the level, amount, or concentration and/or measurement, or a combination thereof, results from or is obtained through a pre-treatment evaluation, is below a threshold:

administering cell therapy does not include administering, prior to or simultaneously with administration of cell therapy and/or prior to the development of a sign or symptom of toxicity other than fever, a therapeutic agent or agent capable of treating, preventing, delaying or reducing the development of toxicity; and/or

the administration of cell therapy should be, or may be, administered to the patient on an outpatient basis and/or without admission of the patient to the hospital overnight or for one or more consecutive days and/or without admission of the patient to the hospital for one or more days; and/or

cell therapy administered to the patient is administered (A) at a dose, optionally a number of T cells or genetically engineered T cells or T cells of the specified phenotype, which is higher than that which would be administered if the level, amount or the concentration and/or measurement, or combination thereof, were equal to or greater than a threshold value, or (B) at a dose, optionally a number of T cells or engineered T cells or T cells of the indicated phenotype, that is equal to or approximately equal to the maximum tolerated dose, or the maximum dose approved for use in the treatment of patients with a disease or condition.

175. A method of prophylactic treatment, comprising administering to a patient an agent or other treatment agent capable of treating, preventing, delaying, attenuating or reducing the development or risk of developing toxicity, wherein:

the patient is a candidate for treatment with optional cell therapy, said cell therapy optionally comprising a dose or composition of engineered cells expressing a recombinant receptor for treating a disease or condition; And

the patient has been identified as at risk of developing toxicity after or based on an assessment of one or more pre-treatment parameters, where:

(a) one or more pre-treatment parameters include a parameter associated with or correlated with, or a marker for, the severity of the disease or condition in the patient, which parameters optionally include one or more analytes and/or one or more volumetric or other body measurements, or

(b) evaluating one or more pre-treatment parameters includes assessing the amount or level in a biological sample that is or originates from a sample obtained from a patient, the level, amount or concentration of one or more analytes in a biological sample obtained from or from a sample from of the patient, in relation to the level, amount or concentration of one or more analytes, and the specified biological sample was obtained from the patient before the introduction of cell therapy and / or the specified biological sample does not include the recombinant receptor and / or the specified genetically engineered cells, and:

(i) one or more pre-treatment parameters include a parameter associated with or correlated with, or a marker for, the severity of the disease or condition in the patient, which parameters optionally include one or more analytes and/or one or more volumetric or other body measurements,

(ii) the assessment of one or more pre-treatment parameters includes assessing the amount or level in a biological sample that is or originates from a sample obtained from a patient, the level, amount or concentration of one or more analytes obtained from a biological sample from a patient prior to the introduction of cell therapy wherein the sample is obtained from a patient prior to administration of cell therapy and optionally prior to apheresis or prior to obtaining cells for administration, and/or one or more analytes are associated with disease severity and/or one or more analytes are selected from LDH, ferritin, CRP, IL -6, IL-7, IL-8, IL-10, IL-15, IL-16, TNF-alpha, IFN-gamma, MCP-1, MIP-1beta, eotaxin, G-CSF, IL-1Ralpha, IL -1Rbeta, IP-10, perforin and D-dimer (fibrin degradation product); and/or

(ii) evaluating one or more pre-treatment parameters optionally includes evaluating a volumetric measurement of a disease or condition in a patient, which is optionally a measurement obtained by imaging, optionally the result of a sum of product dimensions (SPD), or other volumetric measurement(s), optionally based on CT and/or MRI imaging or other body imaging performed prior to treatment and optionally prior to apheresis or cell retrieval; or

wherein one or more analytes are selected from LDH, ferritin, CRP, IL-6, IL-7, IL-8, IL-10, IL-15, IL-16, TNF-alpha, IFN-gamma, MCP-1, MIP -1beta, eotaxin, G-CSF, IL-1Ralpha, IL-1Rbeta, IP-10, perforin and D-dimer (fibrin degradation product).

176. The method according to embodiment 175, wherein said testing comprises detection, optionally comprising contacting a reagent capable of direct or indirect detection of an analyte with a biological sample, and determining the level, amount, or concentration of the analyte in the biological sample, and/or in which the agent is administered to a patient if the level, amount, or concentration of the analyte in the sample is equal to or above a threshold value, and optionally, the agent is administered (i) before, (ii) within one, two, or three days, (iii) simultaneously with and/or (iv ) at the first rise in temperature after initiation of the administration of cell therapy to the patient.

177. The method according to embodiment 175 or embodiment 176 wherein the evaluation of one or more pre-treatment parameters comprises (A) evaluation in a biological sample that is or is derived from a sample obtained from a patient prior to treatment and/or prior to apheresis or processing cells, level, amount or concentration of one or more analytes, optionally associated with the severity of the disease, and (B) an assessment of the volumetric parameter of the severity of the disease, and the assessment of the severity of the disease is not necessarily the result of the sum of product sizes (SPD) or other volumetric measurement(s), optionally based on CT and/or MRI imaging or other body imaging, and/or it is performed prior to treatment, prior to apheresis, or prior to obtaining a cell product.

178. The method of any one of embodiments 169-177, wherein the threshold is within 25%, 20%, 15%, 10%, or 5% of the mean level, amount, or concentration, or magnitude, and/or within a standard deviation of the average level, amount or concentration or value, analyte or parameter in a biological sample obtained from a group of patients prior to obtaining a therapeutic composition of cells expressing the recombinant receptor, and each of the patients of the group continued to develop toxicity after receiving the composition of cells expressing the recombinant receptor for treatment the same disease or condition.

179. The method of any one of embodiments 169-178, wherein the reagent is a binding molecule that specifically binds to the analyte.

180. The method of any one of embodiments 169-179, wherein the reagent is an antibody or an antigen-binding fragment thereof.

181. The method of any one of embodiments 169-180, wherein the biological sample is or is derived from a blood, plasma, or serum sample.

182. The method of any one of embodiments 169-181, wherein the testing or evaluation of the analyte cells comprises an immunoassay.

183. The method of any one of embodiments 169-182, wherein the toxicity includes neurotoxicity or cytokine release syndrome (CRS), optionally neurotoxicity or CRS grade 1 or greater.

184. The method of any one of embodiments 169-183, wherein:

toxicity includes severe neurotoxicity and/or includes grade 2 or higher neurotoxicity, grade 3 or higher neurotoxicity, at least prolonged grade 3 neurotoxicity, or grade 4 or grade 5 neurotoxicity or higher; and/or

toxicity includes severe CRS and/or includes CRS grade 2 or higher or grade 3 or higher.

185. The method of any of embodiments 169-184 wherein the toxicity is associated with cerebral edema.

186. The method of any one of embodiments 169-185, wherein the agent or other therapeutic agent is or includes one or more steroids; antagonists or inhibitors of a cytokine or cytokine receptor selected from IL-10, IL-10R, IL-6, IL-6 receptor, IFNγ, IFNGR, IL-2, IL-2R/CD25, MCP-1, CCR2, CCR4, MIP1β, CCR5, TNFalpha, TNFR1, IL-1, and IL-1Ralpha/IL-1beta; or an agent capable of preventing, blocking or attenuating the activity or function of microglial cells.

187. The method of embodiment 186 wherein the antagonist or inhibitor is or includes an agent selected from an antibody or antigen-binding fragment, a small molecule, a protein or peptide, and a nucleic acid.

188. The method of any one of embodiments 169-187 wherein the agent or other therapeutic agent is an anti-IL-6 or anti-IL6 receptor antibody.

189. The method of any one of embodiments 169-188, wherein the agent or other therapeutic agent is or comprises an agent selected from tocilizumab, siltuximab, clasakizumab, sarilumab, olokizumab (CDP6038), elsilimomab, ALD518/BMS-945429, sirukumab (CNTO 136), CPSI-2634, ARGX-109, FE301 and FM101.

190. The method of any one of embodiments 169-189 wherein the agent or other treatment is or includes tocilizumab.

191. The method of any one of embodiments 169-190 wherein the agent or other treatment is or includes siltuximab.

192. The method of embodiment 186 wherein the steroid is or includes dexamethasone.

193. The method of Embodiment 186, wherein the agent capable of preventing, blocking, or attenuating microglial cell activity or function is selected from an anti-inflammatory agent, a NADPH oxidase (NOX2) inhibitor, a calcium channel blocker, a sodium channel blocker, inhibits GM-CSF, inhibits CSF1R, binds specifically to CSF-1, binds specifically to IL-34, inhibits nuclear factor kappa-B (NF-κB) activation, activates the CB2 receptor and/or is a CB2 agonist, phosphodiesterase inhibitor, inhibits microRNA-155 ((miR -155) or upregulates microRNA-124 ((miR-124.

194. The method of embodiment 193, wherein the agent capable of preventing, blocking, or attenuating microglial cell activation or function is a small molecule, peptide, protein, antibody, or antigen-binding fragment thereof, antibody mimetic, aptamer, or nucleic acid molecule.

195. The method according to embodiment 193 or embodiment 194 wherein the agent is selected from minocycline, naloxone, nimodipine, riluzole, MOR103, lenalidomide, cannabinoid (optionally WIN55 or 212-2), intravenous immunoglobulin (IVIg), ibudilast, anti-miR -155 closed nucleic acid (LNA), MCS110, PLX-3397, PLX647, PLX108-D1, PLX7486, JNJ-40346527, JNJ28312141, ARRY-382, AC-708, DCC-3014, 5-(3-methoxy-4- (4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine (GW2580), AZD6495, Ki20227, BLZ945, emactuzumab, IMC-CS4, FPA008, LY-3022855, AMG-820 and TG-3003.

196. The method of any one of embodiments 193-195 wherein the agent is a colony stimulating factor 1 receptor (CSF1R) inhibitor.

197. The method of any one of embodiments 193-196 wherein the agent is selected from:

PLX-3397, PLX647, PLX108-D1, PLX7486, JNJ-40346527, JNJ28312141, ARRY-382, AC-708, DCC-3014, 5-(3-methoxy-4-(4-methoxybenzyl)oxy)benzyl)pyrimidine- 2,4-diamine (GW2580), AZD6495, Ki20227, BLZ945 or a pharmaceutical salt or prodrug thereof;

emaktuzumab, IMC-CS4, FPA008, LY-3022855, AMG-820 and TG-3003, or an antigen-binding fragment thereof; or a combination of any of the above.

198. The method of any of embodiments 193-197 wherein the inhibitor is PLX-3397.

199. The method of any one of embodiments 1-23 and 109-198, wherein the recombinant receptor specifically binds to an antigen associated with the disease or condition, or expressed in cells of the lesion environment associated with the disease or condition.

200. The method of any one of embodiments 1-23 and 109-199, wherein the disease or condition is cancer.

201. The method of any one of embodiments 1-23 and 109-200, wherein the disease or condition is myeloma, leukemia, or lymphoma.

202. The method of any one of embodiments 1-23 and 109-201, wherein the disease or condition is a B-cell malignancy and/or is acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin's lymphoma (NHL) and diffuse large B-cell lymphoma (DLBCL).

203. The method of any of embodiments 1-23 and 109-202 wherein the antigen is ROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2/neu (erbB2 receptor tyrosine kinase), L1 -CAM, CD19, CD20, CD22, mesothelin, CEA and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG- 40), EPHa2, erb-B2, erb-B3, erb-B4, erbB dimers, EGFR vIII, folate binding protein (FBP), FCRL5, FCRH5, embryonic acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R -alpha, IL-13R-alpha2, receptor containing kinase insertion domain (kdr), kappa light chain, Ley antigen, L1 cell adhesion molecule, (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE -A3, MAGE-A6, preferably expressed melanoma antigen (PRAME), survivin, TAG72, B7-H6, IL-13 receptor alpha 2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA -AI MAGE Al, HLA-A2 NY-ESO-1, PSCA, folic acid receptor alpha, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, embryonic AchR, NKG2D ligands, CD44v6, dual antigen , testicular cancer antigen, mesothelin, mouse CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA) , Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms tumor 1 (WT-1), cyclin, cyclin A2, CCL-1 , CD138, 5D G protein-coupled receptor (GPCR5D) or pathogen-specific antigen.

204. The method of any one of embodiments 1-23 and 109-203, wherein the recombinant receptor is a T cell receptor or a functional non-T cell receptor.

205. The method of any one of embodiments 1-23 and 109-204, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

206. The method of embodiment 205, wherein the CAR comprises an extracellular antigen-recognition domain specifically binding to an antigen and an intracellular signaling domain comprising ITAM, optionally the intracellular signaling domain comprising an intracellular domain of the CD3-zeta (CD3ζ) chain; and/or wherein the CAR further comprises a costimulatory signaling domain, optionally including a CD28 or 4-1BB signaling domain.

207. The method of any one of embodiments 1-23 and 109-206, wherein the engineered cells comprise T cells, optionally CD4 ⁺ and/or CD8 ⁺ .

208. The method of embodiment 207 wherein the T cells are primary T cells obtained from a patient.

209. The method of any one of embodiments 1-23 and 109-208, wherein the cell therapy comprises administering from or about 1x10 ⁵ to 1x10 ⁸ total recombinant receptor-expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMCs) , from or from about 5x10 ⁵ to 1x10 ⁷ total recombinant receptor-expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMCs), or from or from about 1x10 ⁶ to 1x10 ⁷ total recombinant receptor-expressing cells, total T cells, or common peripheral blood mononuclear cells (PBMC), inclusive.

210. The method of any one of embodiments 1-23 and 109-209, wherein the cell therapy comprises administering no more than 1x10 ⁸ total recombinant receptor-expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMCs), no more than 1x10 ⁷ total recombinant receptor-expressing cells, total T-cells or total peripheral blood mononuclear cells (PBMC), not more than 0.5x10 ⁷ total recombinant receptor-expressing cells, total T-cells or total peripheral blood mononuclear cells (PBMC), not more than 1x10 ⁶ total recombinant receptor-expressing cells, total T-cells or total peripheral blood mononuclear cells (PBMC), not more than 0.5x10 ⁶ total recombinant receptor-expressing cells, total T-cells or total peripheral blood mononuclear cells (PBMC) .

211. The method of any one of embodiments 109-210, wherein the dose not associated with a risk of toxicity or severe toxicity is or includes less than or less than about 5x10 ⁷ total recombinant receptor expressing cells, optionally CAR ⁺ cells, total T cells or total peripheral blood mononuclear cells (PBMC), e.g., less than or less than about 2.5x10 ⁷ , less than or less than about 1.0x10 ⁷ , less than or less than about 5.0x10 ⁶ , less than or less than about 1.0x10 ⁶ , less than or less than about 5.0x10 ⁵ , or less than or less than about 1x10 ⁵ total recombinant receptor-expressing cells, optionally CAR ⁺ cells, total T cells, or total peripheral blood mononuclear cells (PBMC).

212. The method of any one of embodiments 109-211 wherein the dose not associated with a risk of toxicity or severe toxicity is or comprises from or about 1x10 ⁵ to 5x10 ⁷ total recombinant receptor expressing cells, optionally CAR ⁺ cells, total T ^{cells or} total ^peripheral blood ^mononuclear cells ( ^{PBMC ) , e.g.} , from 1.0x10 ⁵ to 5.0x10 ⁵ , from 5.0x10 ⁵ to 5x10 ⁷ , from 5x10 ⁵ to 2.5x10 ⁷ , from 5x10 ⁵ to 1.0x10 ⁷ , from 5x10 ⁵ to 5.0x10 ⁶ , from 5x10 ⁵ to 1.0x10 ⁶ , 1.0x10 ⁶ to 5x10 ⁷ , 1x10 ⁶ to 2.5x10 ⁷ , 1x10 ⁶ to 1.0x10 ⁷ , 1x10 ⁶ to 5.0x10 ⁶ , 5.0x10 ⁶ to 5x10 ⁷ , 5x10 ⁶ to 2.5x10 ⁷ , 5x10 ⁶ to 1.0x10 ⁷ , 1.0x10 ⁷ to 5x10 7 , 1x10 ⁷ to 2.5x10 ⁷ , or ^{2.5x10 7 to} 5x10 ⁷ total expressing recombinant receptor cells, optionally CAR ⁺ cells, total T cells, or total peripheral blood mononuclear cells (PBMC).

213. The method of any one of embodiments 109-212, wherein the genetically engineered cells are autologous to the patient.

214. The method of any one of embodiments 109-213, wherein the engineered cells are allogeneic to the patient.

215. The method of any one of embodiments 109-214, wherein the reagent is labeled such that it can be detected, optionally fluorescently labeled.

216. The method of any one of embodiments 163-215 wherein the one or more analytes are LDH, ferritin, CRP, IL-6, IL-8, IL-10, TNF-alpha, IFN-alpha2, MCP-1, and MCP -1beta.

217. The method of any one of embodiments 163-215, wherein the one or more analytes are selected from LDH, ferritin, CRP, IL-6, IL-8, IL-10, TNF-α, MCP-1, and MIP-1beta, and toxicity is neurotoxicity.

218. The method of any one of embodiments 163-215 wherein one or more analytes are selected from IL-8 and IL-10 and the toxicity is neurotoxicity, optionally severe neurotoxicity, or grade 3 neurotoxicity or higher.

219. The method of any one of embodiments 163-215 wherein the one or more analytes are selected from LDH, ferritin, C-reactive protein (CRP), D-dimer (fibrin breakdown product), IL-6, IL-10, IL -15, IL-16 TNF-α, MIP-1α and MIP-1β, and the toxicity is CRS or neurotoxicity.

220. The method of any one of embodiments 109-219 wherein one or more analytes are or include LDH.

221. An article of manufacture comprising one or more doses of cell therapy, each dose comprising cells expressing a chimeric antigen receptor (CAR) and instructions for administering the cell therapy, the instructions specifying that:

the cell dose is intended to be administered to a patient having or identified as having non-Hodgkin's lymphoma (NHL), NHL selected from diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma (PMBCL), NOS (de novo or transformed from indolent lymphoma) or stage 3B follicular lymphoma, and the patient is or has been identified as having status 0 or 1 according to the Eastern Cooperative Oncology Group Performance Status (ECOG); And

the instructions specify the administration of a number of T cells or cells of the specified phenotype, or specify the administration of an amount or volume of one or more formulations corresponding to or containing the specified specified number of cells, the specified number of cells being from or from about 5x10 ⁷ CAR-expressing T cells to 1x10 ⁸ CAR-expressing T cells, inclusive, from or from about 5x10 ⁷ CAR-expressing T cells to or up to about 1.5x10 ⁸ CAR-expressing T cells, 5x10 ⁷ or about 5x10 ⁷ CAR-expressing T cells, 1x10 ⁸ or approximately 1x10 ⁸ CAR-expressing T cells, or ^up to or up to approximately ^{1.5x10 8} CAR-expressing T cells, from or from approximately 5x10 ⁷ CD8 ⁺ CAR-expressing T cells, inclusive, from or from about 2.5x10 ⁷ CAR-expressing T cells and up to or up to about 0.75x10 ⁸ CD8 ⁺ CAR-expressing T cells, or up to or up to about 2.5x10 ⁷ CD8 ⁺ CAR-expressing T cells, 5x10 ⁷ or about 5x10 ⁷ CAR-expressing T cells, or up to or up to about 0.75x10 ⁸ CD8 ⁺ CAR-expressing T cells, or a viable population of any of these .

222. The article of manufacture according to embodiment 221, wherein the instructions specify administering cell therapy at a specified ratio of CAR-expressing CD4 ⁺ cells to CD8 ⁺ cells, or specify administering a number of volumes of formulations corresponding to such a specified ratio, or include a formulation having cells in such a specific ratio. ratio, or includes cells in such a ratio, expressing CAR and/or CD4+ cells to CD8+ cells, and this ratio is not necessarily about 1:1 or from about 1:3 to about 3:1.

223. A product comprising cell therapy or one of a variety of cell therapy compositions, including a dose or composition of genetically engineered cells expressing a chimeric antigen receptor (CAR), and instructions for administering cell therapy, and the instructions define:

the dose of T cells is intended to be administered in a specific ratio of CAR-expressing CD4+ cells to CAR-expressing CD8+ cells and/or CD4+ cells to CD8+ cells, wherein the ratio is approximately or is 1:1; And

the cell dose is intended to be administered to a patient having or identified as having non-Hodgkin's lymphoma (NHL), NHL selected from diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma (PMBCL), NOS (de novo or transformed from indolent lymphoma) or stage 3B follicular lymphoma.

224. A product comprising cell therapy or one of a variety of cell therapy compositions, including a dose or composition of genetically engineered cells expressing a chimeric antigen receptor (CAR), and instructions for administering cell therapy, the instructions specifying:

the cell dose is intended to be administered to a patient having or identified as having non-Hodgkin's lymphoma (NHL), optionally NHL selected from aggressive NHL, diffuse large B cell lymphoma (DLBCL), NOS (de novo and transformed from indolent), primary mediastinal large B cell -cell lymphoma (PMBCL), mantle cell lymphoma (MCL) and/or follicular lymphoma (FL), optionally stage 3B follicular lymphoma (FL3B);

the dose of T cells to be administered includes from or from about 5x10 ⁷ CAR-expressing T cells to 1x10 ⁸ CAR-expressing T cells, inclusive; And

the dose of T cells is intended to be administered in a specific ratio of CAR-expressing CD4+ cells to CAR-expressing CD8+ cells and/or CD4+ cells to CD8+ cells, the ratio being approximately or equal to 1:1.

225. The article of manufacture according to embodiment 222 or embodiment 223, wherein the instructions further specify that the cell therapy is to be administered to a patient who is or has been identified as having an Eastern Cooperative Oncology Group Performance Status (ECOG) status of 0, 1, or 2, and/or which is or has been identified as having an ECOG status of 0 or 1.

226. The article of any one of embodiments 221-225, wherein:

the instructions specify that administering to a patient not receiving, immediately prior to cell dose administration or at or about 1 month prior to cell dose, a treatment agent or agent capable of treating, preventing, delaying, attenuating, or reducing the development or risk of developing toxicity; and/or

the instructions do not specify that administering to a patient who, prior to initiating cell dosing, was administered an anti-IL-6 or anti-IL-6R antibody, optionally tocilizumab or siltuximab, and/or no steroid, optionally dexamethasone; and/or

the instructions do not specify administration of an agent that is an anti-IL-6 or anti-IL-6R antibody, optionally tocilizumab or siltuximab, a steroid, optionally dexamethasone, and/or an agent for the purpose of prophylactically reducing the risk or treating CRS or neurotoxicity, and/or do not specify administering said agent within a period of time following administration of the cell dose, wherein the period of time is optionally or approximately 1, 2, 3, 4, 5 days, or optionally or approximately 6, 7, 8, 9, 10, 11 days, or optionally 1, 2, 3, or 4 weeks, and/or do not determine administration of said agent before the patient shows, or if the patient does not show, a sign or symptom of neurotoxicity and/or CRS, optionally other than fever, optionally fever is not a prolonged fever, or the fever is or has been reduced by or has been reduced by more than 1°C after treatment with an antipyretic; and/or

the instructions specify the administration of a dose of cells on an outpatient basis and/or without admission of the patient to the hospital and/or without overnight stay in the hospital and/or not requiring admission or overnight stay in the hospital, optionally if the risk factor defined for the patient or the patient's cells, not otherwise identified in the instructions or information in the product, or optionally unless or until the patient shows a prolonged fever or fever that is or has not been reduced by or has not been reduced by more than 1°C after treatment with an antipyretic agent.

227. The article of manufacture according to embodiment 226, wherein the instructions further specify that if, after administration of the cell dose on an outpatient basis without requiring admission or overnight stay in the hospital, the patient exhibits a prolonged fever or fever that is or has not been reduced by or not reduced by more than 1°C after treatment with an antipyretic, the patient must be hospitalized or overnight in the hospital and/or requires the administration of a treatment agent or agent to treat or prevent or reduce or alleviate the neurotoxicity and/or syndrome release of cytokines or the risk of it.

228. The article of manufacture according to embodiment 226 or embodiment 227, wherein the agent is or includes an anti-IL-6 or anti-IL-6R antibody, optionally tocilizumab or siltuximab, and/or a steroid, optionally dexamethasone.

229. An article of manufacture according to any one of embodiments 221-228, wherein the instructions specify that the patient has DLBCL characterized as de novo or transformed from an indolent disease and/or DLBCL not transformed from MZL and CLL (Richter).

230. The article of manufacture of any one of embodiments 221-229, wherein the instructions specify that the patient does not have primary mediastinal large B cell lymphoma (PMBCL).

231. The article of manufacture of any one of embodiments 221-230, wherein the instruction specifies that the administration of the cells is to a patient who is or has been identified as having a lymphoma associated with or involving the central nervous system (CNS).

232. The article of any one of embodiments 221-231, wherein the instructions specify that administering cell therapy is administered to a patient who is or has been identified as having double/triple hit lymphoma, is or has been identified as having chemo-refractory lymphoma, optionally chemo-refractory DLBCL; did not achieve complete remission (CR) in response to previous therapy; and/or had a relapse within 1 year or less than 1 year after receiving an autologous stem cell transplant (ASCT).

233. The article of manufacture of any one of embodiments 221-232, wherein the instructions specify further administration to the patient of an additional therapeutic agent or other, optionally other than cell therapy, optionally other than CAR ⁺ T cell therapy.

234. The article of manufacture according to embodiment 233, wherein the additional therapeutic agent or therapy is an agent for the treatment of NHL or cancer and/or increases the persistence, activity, and/or efficacy of a cell dose.

235. The article of manufacture according to embodiment 233 or embodiment 234, wherein the instructions specify that administration of an additional therapeutic agent or therapy is administered to a patient not showing a response, optionally not showing a CR or OR, to cell therapy within 1 month, within 2 months or within 3 months after a dose of cells.

236. The article of any one of embodiments 233-235, wherein the instructions specify that the administration of the additional therapeutic agent or therapy is administered to the patient:

who is or has been identified as having stable or progressive disease (SD/PD) following treatment with prior therapy, optionally prior therapy with a chemotherapeutic agent;

which is or has been identified as having status 2 according to the Eastern Cooperative Oncology Group Performance Status (ECOG);

who is or has been identified as having transformed follicular lymphoma (tFL); or

which is or has been identified as having a DLBCL transformed from MZL and CLL.

237. The article of any one of embodiments 233-236, wherein the instructions specify that the additional therapeutic agent or therapy is to be administered before, with, or simultaneously with and/or after the initiation of a cell dose.

238. The article of manufacture of any one of embodiments 221-237, wherein the CAR comprises an scFv antigen-specific transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which is optionally 4-1BB, and a cytoplasmic signaling domain derived from a primary a signal ITAM-containing molecule, which is optionally a CD3zeta.

239. The article of any one of embodiments 221-238, wherein the antigen is a B cell antigen, which is optionally CD19.

240. The article of any one of embodiments 221-239, further comprising instructions for use with, after, or in connection with antilymphocyte therapy, the antilymphocyte therapy optionally including fludarabine and/or cyclophosphamide.

241. The product according to embodiment 240, wherein the antilymphocyte therapy comprises administering cyclophosphamide in an amount of about 200-400 mg/m ² , optionally in an amount that is or is about 300 mg/m ² inclusive, and/or fludarabine in an amount of about 20 -40 mg/m ² , optionally 30 mg/m ² per day for 2-4 days, optionally for 3 days.

242. The article of manufacture according to embodiment 240 or embodiment 241, wherein the antilymphocyte therapy comprises administering cyclophosphamide at or about 300 mg/m ² and fludarabine at about 30 mg/m ² per day for 3 days.

243. The article of any one of embodiments 221-242, wherein the instructions further specify that the administration of the cell therapy is to be or may be administered to the patient on an outpatient basis and/or without the patient being admitted to the hospital overnight or for one or more consecutive days, and /or without admission of the patient to the hospital for one or more days, optionally unless or until the patient shows a prolonged fever or a fever that is or has not been reduced by or has not been reduced by more than 1 ° C after treatment with an antipyretic agent.

244. The article of embodiment 243, wherein if the patient exhibits a prolonged fever or fever that is or has not been reduced by or has not been reduced by more than 1°C after treatment with an antipyretic, the instructions further specify that the patient should be hospitalized or left overnight in the hospital and/or to be administered a treatment agent or agent to treat or prevent or reduce or reduce neurotoxicity and/or cytokine release syndrome or the risk thereof.

245. The article of manufacture of any one of embodiments 221-244, wherein the instructions further specify that the cell therapy is for parenteral administration, optionally intravenous administration.

246. The article of manufacture of any one of embodiments 221-245, wherein the cell therapy comprises primary T cells obtained from a patient.

247. The article of any one of embodiments 221-246, wherein the T cells are autologous to the patient.

248. The article of any one of embodiments 221-246 wherein the T cells are allogeneic to the patient.

249. The article of manufacture of any one of embodiments 221-248, wherein the article comprises one of a plurality of cell therapy compositions comprising a first composition of genetically engineered cells comprising CD4 ⁺ T cells or CD8 ⁺ T cells, wherein the instructions specify that the first the composition is for use with a second composition comprising others of CD4 ⁺ T cells or CD8 ⁺ T cells, optionally the cells of the first composition and the cells of the same composition originate from the same patient.

250. An article of manufacture according to embodiment 249, wherein the instructions specify that the first composition and the second composition are intended to administer a specific ratio of CD4+ cells expressing the recombinant receptor to CD8+ cells expressing the recombinant receptor and/or CD4+ cells to CD8+ cells, wherein this ratio is optionally about 1:1 or about 1:3 to about 3:1.

251. An article of manufacture according to embodiment 250 wherein the specified ratio is, or is approximately, 1:1.

252. The article of manufacture of any one of embodiments 221-251, wherein the composition further comprises a cryoprotectant and/or the article further includes instructions for thawing the composition prior to administration to a patient.

253. An article of manufacture according to any one of embodiments 249-252, wherein the instructions specify administering a composition comprising CD4 ⁺ T cells and a composition comprising CD8 ⁺ T cells at intervals of 0 to 12 hours, at intervals of 0 to 6 hours or at intervals of 0 to 2 hours.

254. An article of manufacture according to any one of embodiments 249-253, wherein the instructions specify administering a composition comprising CD4 ⁺ T cells and a composition comprising CD8 ⁺ T cells at intervals of not more than 2 hours, not more than 1 hour, not more than more than 30 minutes, no more than 15 minutes, no more than 10 minutes, or no more than 5 minutes.

255. The article of manufacture of any one of embodiments 249-254, wherein the instructions define administration of the composition comprising CD4 ⁺ T cells prior to administration of the composition comprising CD8 ⁺ cells.

256. The article of manufacture of any one of embodiments 249-254, wherein the instructions specify administering a composition comprising CD8 ⁺ T cells prior to administering a composition comprising CD4 ⁺ cells.

257. An article of manufacture comprising one or more reagents capable of detecting one or more analytes and instructions for using the reagent to test a biological sample from a patient who is a candidate for treatment, optionally by cell therapy, said cell therapy optionally including a dose or composition of a gene -engineered cells expressing the recombinant receptor, wherein one or more analytes are selected from LDH, ferritin, CRP, IL-6, IL-7, IL-8, IL-10, IL-15, IL-16, TNF-alpha, IFN -gamma, MCP-1, MIP-1beta, eotaxin, G-CSF, IL-1Ralpha, IL-1Rbeta, IP-10, perforin and D-dimer (fibrin breakdown product).

258. The article of manufacture according to embodiment 257 further comprising cell therapy and/or further comprising instructions for use with, before and/or in connection with cell therapy treatment.

259. The article of manufacture according to embodiment 257 or embodiment 258, further comprising one or more therapeutic agents or agents to treat, prevent, delay, attenuate, or reduce the development or risk of toxicity, and/or instructions for administering one or more agents or agents to treatment to treat, prevent, delay, attenuate or reduce the development or risk of developing toxicity in a patient.

260. The device of any one of embodiments 257-259, wherein the instructions further determine if the level, amount, or concentration of the analyte in the sample is equal to or greater than the threshold for the analyte:

administering to a patient a treatment agent or other agent capable of treating, preventing, delaying, attenuating or reducing the development or risk of developing toxicity (i) up to, (ii) within one, two or three days, (iii) concurrently with and/or (iv) at the first temperature increase after the initiation of the administration of cell therapy to the patient; and/or

administering cell therapy to a patient at a reduced dose or at a dose not associated with a risk of developing toxicity or severe toxicity, or not associated with a risk of developing toxicity or severe toxicity in most patients and / or most patients with a disease or condition that the patient has or is suspected of in its presence, after the introduction of cell therapy; and/or

administering cell therapy to a patient on an inpatient basis and/or with hospital admission for one or more days, optionally the cell therapy must otherwise be administered to patients on an outpatient basis or without hospital admission for one or more days.

261. The device of any one of embodiments 257-259, wherein the instructions further specify if the analyte level, amount, or concentration is below an analyte threshold, administering cell therapy to the patient, optionally at a non-reduced dose, optionally on an outpatient basis, or without hospital admission within one or more days.

262. The article of any one of embodiments 257-261, wherein the instructions further specify the administration of cell therapy to the patient, and wherein the instructions further specify if the level, amount, or concentration of the analyte is below a threshold:

administering cell therapy does not include administering, prior to or simultaneously with administration of cell therapy and/or prior to the development of a sign of a symptom of toxicity other than fever, a treatment agent or agent capable of treating, preventing, delaying or reducing the development of toxicity; and/or

administration of the cell therapy should or may be administered to the patient on an outpatient basis and/or without admission of the patient to the hospital overnight or for one or more consecutive days and/or without admission of the patient to the hospital for one or more days.

263. An article of any one of embodiments 257-262, wherein the threshold is within 25%, 20%, 15%, 10%, or 5% of the average level, amount, or concentration and/or within a standard deviation of the average level, amount or the concentration of an analyte in a biological sample obtained from a group of patients prior to the production of a therapeutic composition of cells expressing the recombinant receptor, and each of the patients of the group continued to develop toxicity after receiving the composition of cells expressing the recombinant receptor for the treatment of the same disease or condition.

264. Product, including:

cell therapy, said cell therapy optionally comprising a dose or composition of engineered cells expressing the recombinant receptor, and

instructions for administering cell therapy after or based on the results of an assessment in a biological sample of the level or amount or concentration of one or more analytes in the biological sample, wherein said biological sample is obtained from a patient prior to administration of cell therapy, and/or said biological sample does not include a recombinant receptor and/or said genetically engineered cells, wherein one or more analytes are selected from LDH, ferritin, CRP, IL-6, IL-7, IL-8, IL-10, IL-15, IL-16, TNF-alpha, IFN-gamma, MCP-1, MIP-1beta, eotaxin, G-CSF, IL-1Ralpha, IL-1Rbeta, IP-10, perforin, and D-dimer (fibrin breakdown product).

265. The article of manufacture according to embodiment 264, wherein said evaluation includes detection, optionally including contacting a reagent capable of direct or indirect detection of an analyte with a biological sample and determining the level, amount, or concentration of the analyte in the biological sample.

266. The article of manufacture according to embodiment 265 further comprising a reagent and/or further including instructions for use with, before and/or in connection with an analyte detection reagent.

267. The article of any one of embodiments 264-266, further comprising one or more agents or treatments to treat, prevent, delay, attenuate, or reduce the development or risk of developing toxicity, and/or instructions for administering one or more agents or agents to treatment to treat, prevent, delay, attenuate or reduce the development or risk of developing toxicity in a patient.

268. The device of any one of embodiments 264-267, wherein the instructions for administering the cell therapy determine if the level, amount, or concentration of the analyte in the sample is equal to or greater than a threshold:

administering to a patient a therapeutic agent or other agent capable of treating, preventing, delaying, attenuating or reducing the development or risk of developing toxicity (i) up to, (ii) within one, two or three days, (iii) concurrently with and/or (iv) at the first rise in temperature after initiation of administration of the administration of the therapeutic composition of the cell or genetically engineered cells; and/or

administering cell therapy to a patient at a reduced dose or at a dose not associated with a risk of developing toxicity or severe toxicity, or not associated with a risk of developing toxicity or severe toxicity in most patients and / or most patients with a disease or condition that the patient has or is suspected of in its presence, after the introduction of cell therapy; and/or

administering cell therapy to a patient on an inpatient basis and/or with hospital admission for one or more days, optionally the cell therapy must otherwise be administered to patients on an outpatient basis or without hospital admission for one or more days.

269. The article of manufacture of any one of embodiments 264-268, wherein the instructions for administering cell therapy determine if the level, amount, or concentration of the analyte in the sample is below a threshold, administering cell therapy to the patient, optionally at a non-reduced dose, optionally on an outpatient basis, or without admission to the hospital for one or more days.

270. The article of any one of embodiments 264-269, wherein the instructions further specify the administration of cell therapy to the patient, and wherein the instructions further specify if the level, amount, or concentration of the analyte is below a threshold:

not administering, prior to or concurrently with the administration of the cell therapy and/or prior to the development of a sign or symptom of toxicity other than fever, a treatment agent or agent capable of treating, preventing, delaying or reducing the development of toxicity; and/or

administration of the cell therapy should or may be administered to the patient on an outpatient basis and/or without admission of the patient to the hospital overnight or for one or more consecutive days and/or without admission of the patient to the hospital for one or more days.

271. An article of any one of embodiments 264-270 wherein the threshold is within 25%, 20%, 15%, 10% or 5% of the mean level, amount or concentration and/or within a standard deviation of the mean level, amount or the concentration of an analyte in a biological sample obtained from a group of patients prior to the production of a therapeutic composition of cells expressing the recombinant receptor, and each of the patients of the group continued to develop toxicity after receiving the composition of cells expressing the recombinant receptor for the treatment of the same disease or condition.

272. A device comprising an agent capable of treating, preventing, delaying, attenuating or reducing the development or risk of developing toxicity, and instructions for administering the agent after or based on the results of an assessment in a biological sample of the level, amount or concentration of one or more analytes in a biological sample where one or more analytes are selected from LDH, ferritin, CRP, IL-6, IL-7, IL-8, IL-10, IL-15, IL-16, TNF-alpha, IFN-gamma, MCP-1, MIP-1beta, eotaxin, G-CSF, IL-1Ralpha, IL-1Rbeta, IP-10, perforin, and D-dimer (fibrin breakdown product).

273. The article of manufacture according to embodiment 272, wherein said evaluation includes detection, optionally including contacting a reagent capable of direct or indirect detection of an analyte with a biological sample, and determining the level, amount, or concentration of the analyte in the biological sample.

274. An article of manufacture according to embodiment 272 or embodiment 273, wherein the instructions specify that the agent is to be administered i) before, (ii) within one, two, or three days, (iii) simultaneously with and/or (iv) at the first rise in temperature following initiation of the administration of cell therapy to the patient and/or further includes instructions for use with, before and/or in connection with cell therapy treatment.

275. The article of any one of embodiments 272-274, wherein said biological sample is obtained from a patient prior to administration of the cell therapy or agent.

276. The article of any one of embodiments 257-275, wherein the reagent is a binding molecule that specifically binds to the analyte.

277. The article of manufacture of any one of embodiments 257-276, wherein the reagent is an antibody or an antigen-binding fragment thereof.

278. The article of any one of embodiments 257-277, wherein the biological sample is either derived from a blood, plasma, or serum sample.

279. The article of manufacture of any one of embodiments 272-278 further comprising an analyte detection reagent and/or further comprising instructions for use with, before and/or in connection with the analyte detection reagent.

280. The article of any one of embodiments 272-279 further comprising cell therapy and/or further comprising instructions for use with, before and/or in connection with cell therapy treatment.

281. The article of any one of embodiments 272-280, wherein the instructions for administering the agent determine if the level, amount, or concentration of the analyte in the sample is equal to or greater than a threshold, administering the agent to the patient.

282. The article of manufacture according to embodiment 281, wherein the instruction further specifies the administration of cell therapy to the patient, wherein the administration of the agent must be (i) before, (ii) within one, two, or three days, (iii) simultaneously with and/or ( iv) at the first rise in temperature after the initiation of the administration of cell therapy to the patient.

283. The article of any one of embodiments 272-282, wherein the instructions for administering the agent determine if the level, amount, or concentration is below a threshold, administering the cell therapy to the patient, optionally the instructions specifying that the cell therapy should or may be administered to the patient on an outpatient basis and / or without admission of the patient to the hospital for the night or for one or more days in a row and / or without the admission of the patient to the hospital for one or more days.

284. An article of any one of embodiments 272-283, wherein the threshold is within 25%, 20%, 15%, 10%, or 5% of the average level, amount, or concentration and/or within a standard deviation of the average level, amount or the concentration of an analyte in a biological sample obtained from a group of patients prior to the production of a therapeutic composition of cells expressing the recombinant receptor, and each of the patients of the group continued to develop toxicity after receiving the composition of cells expressing the recombinant receptor for the treatment of the same disease or condition.

285. The article of manufacture of any one of embodiments 257-284, wherein testing or evaluation of cells for an analyte is performed by an immunological assay.

286. The article of any one of embodiments 257-285, wherein the toxicity includes neurotoxicity or cytokine release syndrome (CRS), optionally neurotoxicity or CRS grade 1 or higher.

287. The product according to any of the embodiments of 257-286, and:

toxicity includes severe neurotoxicity and/or includes grade 2 or higher neurotoxicity, grade 3 or higher neurotoxicity, at least prolonged grade 3 neurotoxicity, or grade 4 or grade 5 neurotoxicity or higher; and/or

toxicity includes severe CRS and/or includes CRS grade 2 or higher or grade 3 or higher.

288. The article of any one of embodiments 257-287, wherein the toxicity is associated with cerebral edema.

289. The article of any one of embodiments 257-288, wherein the agent or other therapeutic agent is or includes one or more steroids; antagonists or inhibitors of a cytokine receptor or a cytokine selected from IL-10, IL-10R, IL-6, IL-6 receptor, IFNγ, IFNGR, IL-2, IL-2R/CD25, MCP-1, CCR2, CCR4, MIP1β, CCR5, TNFalpha, TNFR1, IL-1, and IL-1Ralpha/IL-1beta; or agents capable of preventing, blocking or attenuating the activity or function of microglial cells.

290. The article of manufacture according to embodiment 289, wherein the antagonist or inhibitor is or includes an agent selected from an antibody or antigen-binding fragment, a small molecule, a protein or peptide, and a nucleic acid.

291. The article of manufacture of any one of embodiments 257-290, wherein the agent or other treatment is an anti-IL-6 antibody or an IL6 receptor antibody.

292. The article of any one of embodiments 257-291, wherein the agent or other therapeutic agent is or includes an agent selected from tocilizumab, siltuximab, clasakizumab, sarilumab, olokizumab (CDP6038), elsilimomab, ALD518/BMS-945429, sirukumab ( CNTO 136), CPSI-2634, ARGX-109, FE301 and FM101.

293. The article of any one of embodiments 257-292, wherein the agent or other treatment is or includes tocilizumab.

294. The article of manufacture of any one of embodiments 257-293, wherein the agent or other treatment is or includes siltuximab.

295. The article of manufacture according to embodiment 289, wherein the steroid is or includes dexamethasone.

296. The article of manufacture according to embodiment 289, wherein the agent capable of preventing, blocking, or attenuating microglial cell activity or function is selected from an anti-inflammatory agent, a NADPH oxidase (NOX2) inhibitor, a calcium channel blocker, a sodium channel blocker, inhibits GM-CSF, inhibits CSF1R, binds specifically to CSF-1, binds specifically to IL-34, inhibits nuclear factor kappa-B (NF-κB) activation, activates the CB2 receptor and/or is a CB2 agonist, phosphodiesterase inhibitor, inhibits miRNA-155 ((miR- 155) or upregulates miRNA-124 ((miR-124.

297. The article of manufacture according to embodiment 296, wherein the agent capable of preventing, blocking, or attenuating microglial cell activation or function is a small molecule, peptide, protein, antibody, or antigen-binding fragment thereof, antibody mimetic, aptamer, or nucleic acid molecule.

298. The article of manufacture according to embodiment 296 or embodiment 297, wherein the agent is selected from minocycline, naloxone, nimodipine, Riluzole, MOR103, lenalidomide, cannabinoid, optionally WIN55 or 212-2, intravenous immunoglobulin (IVIg), ibudilast, anti-miR-155 closed nucleic acid (LNA), MCS110, PLX-3297, PLX647, PLX108-D1, PLX7486, JNJ-40346527, JNJ28312141, ARRY-382, AC-708, DCC-3014, 5-(3-methoxy-4-(4 -methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine (GW2580), AZD6495, Ki20227, BLZ945, emaktuzumab, IMC-CS4, FPA008, LY-3022855, AMG-820 and TG-3003.

299. The article of any one of embodiments 296-298, wherein the agent is a colony stimulating factor 1 receptor (CSF1R) inhibitor.

300. The article of any one of embodiments 296-299, wherein the inhibitor is selected from:

PLX-3397, PLX647, PLX108-D1, PLX7486, JNJ-40346527, JNJ28312141, ARRY-382, AC-708, DCC-3014, 5-(3-methoxy-4-(4-methoxybenzyl)oxy)benzyl)pyrimidine- 2,4-diamine (GW2580), AZD6495, Ki20227, BLZ945, or a pharmaceutical salt or prodrug thereof;

emaktuzumab, IMC-CS4, FPA008, LY-3022855, AMG-820 and TG-3003, or an antigen-binding fragment thereof; or a combination of any of the preceding.

301. The article of any one of embodiments 296-300, wherein the inhibitor is PLX-3397.

302. The article of manufacture of any one of embodiments 24-48 and 257-301, wherein the recombinant receptor specifically binds to an antigen associated with a disease or condition, or expressed in cells in a lesion environment associated with the disease or condition.

303. The article of any one of embodiments 24-48 and 257-302, wherein the disease or condition is cancer.

304. The article of any one of embodiments 24-48 and 257-303, wherein the disease or condition is myeloma, leukemia, or lymphoma.

305. The article of any one of embodiments 24-48 and 257-304, wherein the disease or condition is B-cell malignancy and/or acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin's lymphoma ( NHL) and diffuse large B-cell lymphoma (DLBCL).

306. The article of manufacture of any one of embodiments 302-305, wherein the antigen is ROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2/neu (erbB2 receptor tyrosine kinase), L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2 , erb-B2, erb-B3, erb-B4, erbB dimers, EGFR vIII, folate-binding protein (FBP), FCRL5, FCRH5, embryonic acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL -13R-alpha2, receptor containing kinase insertion domain (kdr), kappa light chain, Ley antigen, L1 cell adhesion molecule, (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE -A6, preferably expressed melanoma antigen (PRAME), survivin, TAG72, B7-H6, IL-13 receptor alpha 2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AI MAGE Al , HLA-A2 NY-ESO-1, PSCA, folic acid receptor alpha, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, embryonic AchR, NKG2D ligands, CD44v6, dual antigen, testicular cancer antigen , mesothelin, mouse CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), Her2/neu , estrogen receptor, progesterone receptor, ephrinB2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms tumor 1 (WT-1), cyclin, cyclin A2, CCL-1, CD138, 5D G protein-coupled receptor (GPCR5D) or pathogen-specific antigen.

307. The article of manufacture of any one of embodiments 24-48 and 257-306, wherein the recombinant receptor is a T cell receptor or a functional non-T cell receptor.

308. The article of manufacture of any one of embodiments 24-48 and 257-307, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

309. The article of manufacture according to embodiment 308, wherein CAR includes an extracellular antigen recognition domain specifically binding to an antigen and an intracellular signaling domain including ITAM, optionally the intracellular signaling domain including an intracellular CD3-zeta (CD3ζ) chain domain; and/or wherein the CAR further comprises a costimulatory signaling domain, optionally including a CD28 or 4-1BB signaling domain.

310. The article of manufacture of any one of embodiments 24-48 and 257-309, wherein the engineered cells include T cells, optionally CD4 ⁺ and/or CD8 ⁺ .

311. An article of manufacture according to embodiment 310, wherein the T cells are primary T cells obtained from a patient.

312. An article of manufacture according to any one of embodiments 257-311, wherein the dose not associated with a risk of toxicity or severe toxicity is or includes less than or less than about 5x10 ⁷ total recombinant receptor expressing cells, optionally CAR ⁺ cells, total T -cells or total peripheral blood mononuclear cells (PBMC), e.g., less than or less than about 2.5x10 ⁷ , less than or less than about 1.0x10 ⁷ , less than or less than about 5.0x10 ⁶ , less than or less than about 1.0x10 ⁶ , less than or less than about 5.0x10 ⁵ , or less than or less than about 1x10 ⁵ total recombinant receptor-expressing cells, optionally CAR ⁺ cells, total T cells, or total peripheral blood mononuclear cells (PBMCs) ).

313. An article of manufacture according to any one of embodiments 257-312, wherein the dose not associated with a risk of toxicity or severe toxicity is or includes from or about 1x10 ⁵ to 5x10 ⁷ total recombinant receptor expressing cells, optionally CAR ⁺ cells, total T ^{-cells or} total ^peripheral blood mononuclear ^{cells ( PBMC} ) ^{, e.g.} from 1.0x10 ⁵ to 5.0x10 ⁵ , from 5.0x10 ⁵ to 5x10 ⁷ , from 5x10 ⁵ to 2.5x10 ⁷ , from 5x10 ⁵ to 1.0x10 ⁷ , from 5x10 ⁵ to 5.0x10 ⁶ , from 5x10 ⁵ to 1.0x10 ⁶ , from 1.0x10 ⁶ to 5x10 ⁷ , from 1x10 ⁶ to 2.5x10 ⁷ , from 1x10 ⁶ to 1.0x10 ⁷ , from 1x10 ⁶ to 5.0x10 ⁶ , from 5.0x10 ⁶ to 5x10 ⁷ , 5x10 ⁶ to 2.5x10 ⁷ , 5x10 ⁶ to 1.0x10 ⁷ , 1.0x10 ⁷ to 5x10 7 , 1x10 ⁷ to 2.5x10 ⁷ , or ^{2.5x10 7 to} 5x10 ⁷ total recombinant receptor-expressing cells , optionally CAR ⁺ cells, total T cells, or total peripheral blood mononuclear cells (PBMC).

314. The article of any one of embodiments 257-313, wherein the reagent is labeled such that it can be detected, optionally fluorescently labeled.

315. The article of manufacture of any one of embodiments 257-314, wherein the one or more analytes are LDH, ferritin, CRP, IL-6, IL-8, IL-10, TNF-alpha, IFN-alpha2, MCP-1, and MCP -1beta.

316. The article of manufacture of any one of embodiments 257-315, wherein one or more analytes is or includes LDH.

317. The product according to any of the embodiments 221-316, and:

CAR includes an scFv antigen-specific transmembrane domain, a cytoplasmic signaling domain derived from a co-stimulatory molecule, which optionally is or includes 4-1BB, and a cytoplasmic signaling domain derived from an ITAM-containing primary signaling molecule, which optionally is or includes CD3zeta signaling domain and optionally further includes a spacer between the transmembrane domain and scFv;

CAR includes, in that order, an scFv antigen-specific transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is or includes a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule , which is optionally the CD3zeta signaling domain; or

CAR includes, in that order, an antigen-specific scFv, a spacer, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which is optionally a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is or includes the CD3zeta signaling domain; and moreover:

the spacer is optionally a polypeptide spacer that (a) includes or consists of all or part of the hinge region of an immunoglobulin or a modified version thereof, or comprises about 15 amino acids or less and does not include the CD28 extracellular domain or the CD8 extracellular domain, (b) includes or consists of all or a portion of an immunoglobulin hinge region, optionally an IgG4 hinge or a modified version thereof, and/or comprises about 15 amino acids or less and does not include a CD28 extracellular domain or a CD8 extracellular domain, or (c) is 12 or about 12 amino acids long and/or includes or consists of from all or part of the hinge region of an immunoglobulin, optionally IgG4 or a modified version thereof; or (d) has or consists of the sequence 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 preceding having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or more sequence identity with it, or (e) includes or consists of the formula X ₁ PPX ₂ P, where X ₁ is glycine, cysteine or arginine, and X ₂ is cysteine or threonine; and/or

the costimulatory region 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 with it; and/or

the primary signaling domain includes SEQ ID NO: 13 or 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 with it; and/or

scFv includes a CDRL1 RASQDISKYLN sequence (SEQ ID NO: 35), a CDRL2 SRLHSGV sequence (SEQ ID NO: 36) and/or a CDRL3 GNTLPYTFG sequence (SEQ ID NO: 37), and/or a CDRH1 DYGVS sequence (SEQ ID NO: 38) , VIWGSETTYYNSALKS CDRH2 sequence (SEQ ID NO: 39) and/or YAMDYWG CDRH3 sequence (SEQ ID NO: 40), or wherein the scFv comprises an FMC63 heavy chain variable region and an FMC63 light chain variable region and/or an FMC63 CDRL1 sequence, an FMC63 CDRL2 sequence , FMC63 CDRL3 sequence, FMC63 CDRH1 sequence, FMC63 CDRH2 sequence, and FMC63 CDRH3 sequence either binds to the same epitope or competes for binding to any of the preceding ones, and optionally the scFv includes, in that order, V _H , a linker, optionally including SEQ ID NO: 24 and V _L and/or scFv includes a flexible linker and/or includes the amino acid sequence shown as SEQ ID NO: 24.

318. The product according to any one of paragraphs. 221-317, in which the instructions provide information on a threshold value, individually for each one or more analytes, which is an indicator of the likelihood that a patient will demonstrate a response to cell therapy treatment.

319. The product according to any one of paragraphs. 221-317, in which the instructions provide information on a threshold value, individually for each one or more analytes, which is an indicator of the likelihood that the patient will demonstrate a long-term response after the introduction of cell therapy.

320. The product according to any one of paragraphs. 221-317, in which the instructions provide information on a threshold value, individually for each one or more analytes, which is an indicator of the likelihood that the patient will demonstrate toxicity after the introduction of cell therapy.

VIII. Definitions

[0797] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides, including receptors of the invention and other polypeptides, such as linkers or peptides, may include amino acid residues including natural and/or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, and phosphorylation. In some aspects, the polypeptides may contain modifications relative to the native or natural sequence, provided that the protein maintains the desired activity. These modifications may be intentional, such as through site-directed mutagenesis, or may be accidental, such as through mutations in protein-producing hosts, or due to errors in PCR amplification.

[0798] As used herein, a "patient" is a mammal, such as a human or other animal, and is typically a human. In some embodiments, the individual, eg, the patient, to whom the agent or agents, cells, cell populations, or compositions are administered is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or a great ape. The patient may be male or female and may be of any suitable age, including infant, adolescent, adolescent, adult, and geriatric patients. In some embodiments, the patient is a non-primate mammal, such as a rodent.

[0799] For the purposes of the invention, "treatment" (and grammatical variations of the term, such as "treating" or "treating") refers to the total or partial alleviation or reduction of a disease or condition, or disorder, or symptom, adverse effect, or result, or phenotype associated with them. The desired effects of treatment include, but are not limited to, prevention of the onset or recurrence of the disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, improvement or temporary relief of the disease state, and remission or improved prognosis. The terms do not imply complete cure of a disease or complete elimination of any symptom or effect(s) on all symptoms or results.

[0800] As used herein, “delaying the development of a disease” means to delay, prevent, slow down, delay, stabilize, suppress, and/or delay the development of a disease (such as cancer). This delay may have variable lengths of time, depending on the history of the disease and/or the person receiving treatment. In some embodiments, a sufficient or significant delay may actually encompass prophylaxis in which the individual does not develop the disease. For example, advanced stage cancer, such as metastasis, may be delayed.

[0801] "Prevention", within the scope of the invention, includes providing prophylaxis regarding the occurrence or recurrence of a disease in a patient who may be predisposed to the disease, but who has not yet been diagnosed with the disease. In some embodiments, cells and compositions of the invention are used to delay the development of a disease or slow the progression of a disease.

[0802] In the context of the invention, to "suppress" a function or activity means to reduce the function or activity compared to the same states except for the state or parameter of interest, or alternatively, compared to another state. For example, cells that inhibit tumor growth reduce the rate of tumor growth compared to the rate of tumor growth in the absence of cells.

[0803] An “effective amount” of an agent, e.g., pharmaceutical formulation, cell, or composition, in the context of administration, refers to an effective amount, in dosages/amounts, and for periods of time necessary to achieve a desired result, such as a therapeutic or prophylactic result.

[0804] A "therapeutically effective amount" of an agent, such as a pharmaceutical formulation or cells, refers to an effective amount, in dosages and for periods of time, necessary to achieve a desired therapeutic result, such as treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of treatment. The therapeutically effective amount may vary depending on such factors as the disease state, the age, sex and body weight of the patient, and the population of cells administered. In some embodiments, the methods of the invention include administering the cells and/or compositions in effective amounts, eg, therapeutically effective amounts.

[0805] "Prophylactically effective amount" refers to an effective amount, in dosages and for periods of time necessary to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in patients before or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount. In the context of a lower tumor burden, a prophylactically effective amount will in some aspects be higher than a therapeutically effective amount.

[0806] The term "approximately" as used herein refers to the usual error range for the corresponding value, easily understood by a person skilled in the art. Reference to an “approximate” value or parameter herein includes (and describes) embodiments directed to that value or parameter per se.

[0807] For the purposes of the invention, singular forms include plural forms unless the context clearly dictates otherwise. For example, "one" means "at least one" or "one or more".

[0808] For the purposes of this disclosure, various aspects of the claimed invention are presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed invention. Accordingly, a description of a range is to be understood to specifically disclose all possible subranges as well as individual numerals within that range. For example, if a range of values is given, it is understood that every value between the upper and lower limits of that range, and any other value specified or referred to within that specified range, is covered by the claimed invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also covered by the claimed invention according to any specifically excluded limit within the stated range. If the stated range includes one or both of the limit values, ranges excluding one or both of these included limit values are also included within the scope of the claimed invention. This applies regardless of the range width.

[0809] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0810] For the purposes of the invention, "enrichment" with respect to one or more specific cell types or cell populations refers to an increase in the number or percentage of that cell type or population, for example, compared to the total number of cells or composition volume, or relative to other cell types. , such as selected by positive selection based on markers expressed by the population or cell, or negative selection based on marker not present on the population of cells or cell to be depleted. The term does not require the complete removal of other cells, type or population of cells from the composition and does not require that the cells thus enriched be present in an amount or even about 100% in the enriched composition.

[0811] Within the scope of the invention, a statement that a cell or population of cells is "positive" for a particular marker refers to the detectable presence on or in a cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the presence of surface expression detectable by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, the staining being detectable by flow cytometry at a level substantially higher than the staining detectable by performing the same procedures with isotype-matched control or fluorescence minus one (FMO) gating control under otherwise identical conditions and/or at a level substantially similar to that of a cell known to be positive for the marker and/or at a level substantially higher than this is for a cell known to be negative for the marker.

[0812] Within the scope of the invention, a statement that a cell or population of cells is "negative" for a particular marker refers to the absence of a significant detectable presence on or in the cell of a particular marker, typically a surface marker. 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 specifically binding to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected by performing the same a procedure with an isotype-matched control or a fluorescence control with a combination of detectable labels without one (FMO) under otherwise identical conditions, and/or at a level significantly lower than for a cell known to be positive for the marker, and/or at a level substantially similar to that of a cell known to be negative for the marker.

[0813] The term "vector", as used herein, refers to a nucleic acid molecule capable of replicating another nucleic acid to which it is linked. The term includes a vector as a self-replicating nucleic acid structure, as well as a vector incorporated into the genome of the host cell into which it has been introduced. Certain vectors are capable of directing the expression of the nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

[0814] Unless otherwise indicated, all prior art terms, notes, and other technical and scientific terms or terminology used herein have the same meaning as is commonly understood in the field to which the claimed invention pertains. In some cases, terms with commonly understood meanings are defined here for clarity and/or for quick reference, and the inclusion of such definitions in the present specification should not necessarily be construed to represent significant differences from what is commonly understood.

[0815] All publications, including patent documents, scientific articles and databases referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If the definition set forth here is inconsistent with or otherwise inconsistent with the definition set forth in patents, applications, publications of applications and other publications, which are incorporated into the present description by reference, the definition set forth here takes precedence over the definition included in the present description by reference.

[0816] The section headings used here are for organizational purposes only and should not be construed as limiting the invention described.

IX. Examples

[0817] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1 Administration of Anti-CD19 CAR Expressing Cells to Patients with Relapsed and Refractory Non-Hodgkin's Lymphoma (NHL)

A. Patients and Treatment

[0818] Therapeutic compositions of CAR ⁺ T cells containing autologous T cells expressing a chimeric antigen receptor (CAR) specific for CD19 are administered to patients with B-cell malignancies.

Example 1.A.1

[0819] This Example 1.A.1 describes the results of a point-in-time evaluation (1.A.1) in an ongoing clinical study in which such therapy was administered to patients with B-cell malignancies. Specifically, a cohort (full cohort) (at this point in time, fifty-five (55)) of adults with relapsed or refractory aggressive non-Hodgkin's lymphoma (NHL) (R/R), including diffuse large B-cell lymphoma (DLBCL), de novo or transformed from indolent lymphoma (NOS), well-differentiated B-cell lymphoma, with MYC and BCL2 and/or BCL6 permutations with DLBCL (double/triple hit) histology, DLBCL transformed from chronic lymphocytic leukemia (CLL) or marginal zone lymphoma (MZL), primary mediastinal large B cell lymphoma (PMBCL) and grade 3b follicular lymphoma (FL3B) after failure of 2 lines of therapy. Patients receiving treatment included those with an Eastern Cooperative Oncology Group (ECOG) score of 0 to 2 (mean follow-up 3.2 months). The full cohort did not include patients with mantle cell lymphoma (MCL). None of the patients were excluded based on prior allogeneic stem cell transplantation (SCT), secondary central nervous system (CNS) involvement, or ECOG score 2, and there was no minimum absolute lymphocyte count (ALC) for required apheresis.

[0820] Outcomes were separately assessed for the core subset of patients in the full cohort (patients in the full cohort, excluding patients with unsatisfactory status (ECOG 2), DLBCL transformed from marginal zone lymphoma (MZL) and/or chronic lymphocytic leukemia (CLL, Richter) , and patients with primary mediastinal large B-cell lymphoma (PMBCL) and stage 3B follicular lymphoma (FL3B) (main population)). The core population includes patients with DLBCL, NOS and transformed follicular lymphoma (tFL) or high-stage B-cell lymphoma (double/triple hit) or well-differentiated B-cell lymphoma, with MYC and BCL2 and/or BCL6 permutations with DLBCL histology (double/ triple hit) and with an Eastern Cooperative Oncology Group Performance Status (ECOG PS) of 0 or 1. At the time point in Example 1.A.1, outcomes were evaluated for 44 patients in this core cohort.

[0821] Demographic and baseline characteristics of the total and core population of patients at the time point in Example 1.A.1 are presented in Table E1.

Table E1. Demographic and basic characteristics Characteristic FULL
N=55 BASIC
N=44 Mean Age, years (range) 61(29-82) 61(29-82) ≥ 65 years, n (%) 22 (40) 17 (39) Male/Female, n (%) 38/17 (69/31) 28/16 (64/36) Months since diagnosis, median (range) 17 (3-259) 20 (8-259) Subtype B-NHL, n (%) DLBCL, NOS 40 (73) 35 (80) Transformed DLBCL 14 (26) 8 (18) Follicular, Stage 3B 12) 12) Molecular Subtype, n (%) Double/triple hit 15 (27) 12 (27) double expressor 6 (11) 4 (9) Patient characteristics, n (%) Chemorefractive† 42 (76) 34 (77) ECOG 0-1 48 (87) 44 (100) ECOG2 7(13) 0 Previous lines of therapy, median (range) 3 (1-11) 3 (1-8) < 5 lines of therapy 44 (80) 37 (84) Any HSCT 27 (49) 22 (50) Allogeneic 4(7) 3 (7) autologous 24 (44) 20 (45) ^* SD or PD at last chemotherapy or relapse <12 months after autologous SCT

[0822] Administered therapeutic T cell compositions were prepared by a method involving immunoaffinity (eg, immunomagnetic selection) enrichment of CD4 ⁺ and CD8 ⁺ cells from leukapheresis samples from a patient being treated. The isolated CD4 ⁺ and CD8 ⁺ T cells were separately activated and independently transformed with a viral vector (eg, lentiviral vector) encoding the anti-CD19 CAR, followed by separate growth and low volume cryopreservation of the genetically engineered cell population. The CARs contained an anti-CD19 scFv derived from a mouse antibody (variable region derived from FMC63, orientation V _L -linker-V _H ), an immunoglobulin derived spacer, a transmembrane domain derived from CD28, a costimulatory region derived from 4-1BB, and CD3-zeta intracellular signaling domain. The viral vector further contained sequences encoding a truncated receptor that served as a surrogate marker for CAR expression; separated from the CAR sequence by the T2A ribosome skipping sequence.

[0823] Freeze-preserved cell compositions were thawed prior to intravenous administration. The therapeutic dose of T cells was administered as a defined cell composition by administering a pooled population of CD4 ⁺ CAR ⁺ cells and a pooled population of CD8 ⁺ CAR ⁺ cells administered in a target ratio of approximately 1:1. Patients were administered a single or double dose of CAR-expressing T cells (each single dose via separate infusions of CD4 ⁺ CAR-expressing T cells and CD8 ⁺ CAR-expressing T cells, respectively) as follows: single dose level 1 (DL-1 ) dose containing 5x10 ⁷ total CAR-expressing T cells (n=30 for patients evaluated in Example 1.A.1), a double dose of DL1, in which each dose was administered approximately fourteen (14) days apart ( n=6 for patients evaluated in Example 1.A.1 administered on day 1 and day 14, including one patient who inadvertently received two doses of DL2 over a two-dose schedule due to administration error), or a single dose of level 2 (DL -2) dose containing 1x10 ⁸ total CAR-expressing T cells (n=18 for patients evaluated in Example 1.A.1). The target dose level and the number of T cell subpopulations for the administered compositions are shown in Table E2.

Table E2. Target Dose Levels and Number of T Cell Subpopulations for Cell Compositions Containing Anti-CD19 CAR T Cells Dose level Dose of helper T cells (T _H )
(CD4 ⁺ CAR ⁺ ) The dose of cytotoxic T cells (T _C )
(CD8 ⁺ CAR ⁺ ) Total dose of T cells
(CD3 ⁺ CAR ⁺ ) 1 ^{25x106 25x106 50x106} 2 ^{50x106 50x106 100x106}

[0824] From the time before the infusion of CAR ⁺ T cells, patients received antilymphocyte chemotherapy using fludarabine (flu, 30 mg/m ² ) and cyclophosphamide (Cy, 300 mg/m ² ) for three (3) days. Patients received CAR-expressing T cells 2-7 days after this lymphodepletion.

Example 1.A.2

[0825] In Example 1.A.2, at the next time point in the clinical study described in this Example 1 above, the results were analyzed. At this point in time in Example 1.A.2, 74 patients were treated (51 men, 23 women). Patients included sixty-nine (69) patients in the full DLBCL cohort (including 67 NOS DLBCL (45 de novo, 14 FL-transformed, 8 CLL or MZL-transformed), 1 Grade 3B FL, and 1 PMBCL) and 5 patients in the MCL cohort. Among patients in the full (DLBCL) cohort, mean age was 61 years (range 26, 82), mean number of prior therapies was 3 (range 1, 12), 46 (67%) were chemo-refractory, 32 (46%) had any prior transplantation , and at least 16 (23%) patients with double/triple hit lymphoma. Forty-nine (49) patients in the core cohort were evaluated at this time point in 1.A.2.

B. Security

[0826] Were assessed the presence or absence of adverse events that occurred during the course of treatment (TEAE) after the introduction of cell therapy CAR-T. Patients were also assessed and monitored for neurotoxicity (neurological complications, including symptoms of confusion, aphasia, encephalopathy, myoclonic seizures, convulsions, lethargy, and/or altered mental status), classified on a scale of 1-5, according to the National Cancer Institute-Common Toxicity scale. Criteria (CTCAE), Version 4.03 (NCI-CTCAE v4.03). Common Toxicity Criteria (CTCAE) v4.03 (NCI-CTCAE v4.03). See Common Terminology for Adverse Events (CTCAE) Version 4, USDepartment of Health and Human Services, published: May 28, 2009 (v4.03: June 14, 2010); and Guido Cavaletti & Paola Marmiroli Nature Reviews Neurology 6, 657-666 (December 2010). Cytokine release syndrome (CRS) has also been defined and tested, classified based on severity. See Lee et al, Blood. 2014;124(2):188-95. In some cases, adverse events were reported and data were collected from lymphodepletion up to 90 days after CAR ⁺ T cell administration.

Example 1.B,1

[0827] Example 1.B.1 describes the results based on the analysis in Example 1.A.1.

[0828] FIG. 1 depicts the percentage of such patients who were observed to experience laboratory pathologies and TEAEs that occurred in ≥20% of patients. In addition to the TEAEs shown in FIG. 1, the following terms of events were observed at Level 3-4 in ≥5% of patients: white blood cell count decreased by (13.6%), encephalopathy (12%), hypertension (7%). The degree of toxicity observed corresponded to dose levels 1 to 2.

[0829] In 84% of the total patient cohort in Example 1.B,1, severe (grade 3 or higher) cytokine release syndrome (CRS) and severe neurotoxicity were not observed. In addition, it was observed that 60% of patients in the full cohort did not develop any CRS or neurotoxicity. No differences in CRS level, neurotoxicity (NT), sCRS, or severe neurotoxicity (sNT) were observed between dose levels. Table E3 summarizes the rate of cytokine release syndrome (CRS) adverse events and neurotoxicity in patients 28 days after receiving at least one dose of CAR-T cells. As shown in Table E3, no sCRS (Grade 3-4) was observed in any patients who received a single dose of DL2 or a double dose of DL1. Severe neurotoxicity or severe CRS (grade 3-4) was observed in 16% (9/55) of the total patient cohort and in 18% (8/44) of patients in the core subset. 11% (n=6) of patients received tocilizumab, 24% (n=13) of patients received dexamethasone. Among ECOG2 patients in the full cohort, the observed incidence of CRS and neurotoxicity was 71% and 29%, respectively.

Table E3. Evaluation of the presence or absence of CRS adverse events and neurotoxicity, Example 1.B,1 FULL BASIC ALL dose levels DL1S DL2S DL1D ^† Safety, N 55 thirty 19 6 44 sCRS or sNT, n (%) 9 (16) 6 (20) 2 (11) 1 (17) 8 (18) CRS or NT, n (%) 22 (40) 12 (40) 7 (37) 3 (50) 15 (34) CRS Grade 1-2, n (%) 18 (33) 10 (33) 5 (26) 3 (50) 12 (27) Grade 3-4, n (%) 12) 13) 0 0 12) Neurotoxicity Grade 1-2, n (%) 3 (6) 13) 2 (11) 0 2(5) Grade 3-4, n (%) 9 (16) 6 (20) 2 (11) 1 (17) 8 (18) ^† Incl ^† Including one patient treated with Rechyme 2 doses of DL2 due to administration error

[0830] FIG. 2 shows a Kaplan-Meier curve depicting the observed time to onset of CRS and/or neurotoxicity for the analysis in 1.B,1. As shown, the observed median times to onset of CRS and to onset of neurotoxicity were 5 and 11 days, respectively, with only 11% of patients experiencing onset of CRS less than 72 hours after initiation of cell therapy administration. The median time to resolution of CRS and neurotoxicity for Grade 1 or better was 5 and 7 days, respectively. The mean time to complete clearance of CRS and neurotoxicity was 5 and 11 days, respectively. The results were consistent with the conclusion that there was a low percentage of early onset CRS or neurotoxicity in patients.

Example 1.B,2

[0831] Example 1.B,2 describes the evaluation at a point in time in Example 1.B,2. Until this point in time, data on adverse events (AE) were collected from the time of lymphodepletion (LD) to 90 days after the introduction of CAR-expressing T cells. At the second time point, 69 patients in the DLBCL cohort (full cohort) were assessed for safety, 38 received a single dose of DL1, 25 received a single dose of DL2, and 6 received a double dose of DL1. The most common TEAEs other than CRS or NT included neutropenia (41%, 28/69), fatigue (30%, 21/69), thrombocytopenia (30%, 21/69) and anemia (26%, 18/69). One case of TEAE was observed, which was grade 5 diffuse alveolar damage.

[0832] No acute infusion toxicity was observed, and the majority of patients in the entire cohort, 64% (44/69), were observed to have no CRS or NT, indicating that outpatient delivery of CAR-expressing T cells may be possible. . The frequency of CAR T cell related toxicity, including CRS and NT, did not differ between dose levels. The safety profile was observed to be comparable across dose levels and populations. Among the 25 patients in the full cohort (36%) who experienced some degree of CRS or NT, 21 (30%) had CRS and 14 (20%) had NT. None of the patients had Grade 3 CRS and only one (1%, 1/69) had Grade 4 CRS and required an ICU; the other 29% (20/69) had Grade 1-2 CRS. Of the 20% of patients with NT, 6% (4/69) had Grades 1-2 and 14% (10/69) had Grades 3-4; 2 (3%) had seizures. No Grade 5 CRS or Grade 5 NT was observed. No incidence of cerebral edema was observed. All CRS and NT events were eliminated except for one Grade 1 tremor that continued during the analysis. The median time to onset of first CRS and NT was 5 days (range 2.12) and 10 days (range 5.23), respectively. In the first 72 hours after infusion, none of the patients were observed to have NT and only 10% (7/69) were observed to have CRS (all Grade 1); NT preceded CRS in >70% of patients. Overall, thirteen (13) patients (19%) required intervention for CRS or NT therapy using an anticytokine (tocilizumab individually 1 (1%), dexamethasone individually 6 (9%), or both 6 (9%)) and only one required vasopressor support. The mean doses of tocilizumab and dexamethasone were 1 and 6, respectively. The mean duration of CRS and NT was 5 days and 11 days, respectively. Analysis of the main cohort (n=49) also showed a similar frequency of CRS and NT.

[0833] In this evaluation, low incidence and late onset of CRS and/or NT were observed at both dose levels, confirming the feasibility of outpatient infusion, for example, with hospitalization at the first symptom of fever or fever lasting longer than a certain period of time. No Grade 5 CRS or Grade 5 NT was observed, and all severe CRS and severe NT were eliminated. Further, approximately 2 out of 3 patients had no CRS or NT, which confirms that the cells can be administered on an outpatient basis. At the time of evaluation in 1.B,2, four patients were treated on an outpatient basis. Further, no significant differences in toxicity were observed in patients receiving DL1 or DL2, indicating that a higher response rate was achieved without the risk of increasing toxicity or safety concerns.

C. Post-treatment response outcomes

[0834] Patients were monitored for response, including assessment of tumor mass at 1, 3, 6, 7, 12, 18, and 24 months after administration of CAR ⁺ T cells.

Example 1.C,1

[0835] Example 1.C,1 describes the results based on the analysis in Example 1.A.1 and 1.B,1.

[0836] Response rates are listed in Table E4. High sustained response rates have been observed in a cohort of patients who have received significant prior treatment or patients with poor prognosis and/or relapsed or refractory disease. For patients treated at all dose levels in the Core (n=44) cohort, the observed overall response rate (ORR) was 86% and the observed complete response rate (CR) was 59%. At three months for the core population, the overall response rate (ORR) was 66%; the three-month CR rate was 50% among the main contingent. In the core cohort, the 3-month RRR was 58% (11/19) at the 1st dose level and 78% at the 2nd dose level; The 3-month CR rate was 42% (8/19) for dose level 1 and 56% (5/9) for dose level 2, consistent with the proposed dose-response effect on treatment outcomes. In addition, the results were consistent with the relationship between dose and duration of response.

Table E4. Answer FULL BASIC ALL dose levels DL1S DL2S DL1D ^c ALL dose levels DL1S DL2S DL1D ^a Best complete answer, N ^a 54 thirty 18 6 44 25 15 4 ORR,%
(95% CI) 76
(62, 87) 80
(61, 92) 72
(47, 90) 67
(23, 96) 86
(73, 95) 84
(64, 95) 87
(60, 98) 100
(40, 100) CR%
(95% CI) 52
(38, 66) 53
(34, 72) 50
(26, 74) 50
(12, 88) 59
(43, 74) 56
(35, 76) 60
(32, 84) 75
(19, 99) ≥ 3 mos f/u, ^nb 41 24 eleven 6 32 19 9 4 3 mo ORR,%
(95% CI) 51
(35, 67) 46
(26, 67) 64
(31, 89) 50
(12, 88) 66
(47, 81) 58
(34, 80) 78
(40, 97) 75
(19, 99) 3mo CR,%
(95% CI) 39
(24, 56) 33
(16, 55) 46
(17, 77) 50
(12, 88) 50
(32, 68) 42
(20, 67) 56
(21, 86) 75
(19, 99) DL1S: schedule with 1 dose of DL1; DL2S: schedule with 1 dose of DL2; DL1D: DL1 schedule with 2 doses;
^a Including patients with a PD event, death, or 28-day follow-up. Patients treated 28 days before the data snapshot were not included.
^b The denominator is the number of patients treated with CAR
T cell therapy ≥ 3 months prior to the date of the assessment snapshot
efficacy at Month 3 or until evaluation of PD or death.

^c Includes one patient treated with 2 doses of DL2 due to administration error

[0837] The overall response rates among various patient subgroups in the overall and core populations are shown in FIG. 3A and 3B, respectively. In the low-risk DLBCL subgroup, response rates were generally high. ORR greater than 50% was seen at 3 months in patients with the double/triple hit molecular subtype who had primary refractory or chemorefractory DLBCL or who had never previously achieved CR. Complete elimination of CNS involvement in lymphoma was observed in 2 patients.

[0838] Among patients treated for six months or more prior to the assessment time point, ten (10) patients who were responsive at three months, 9 (90%) remained responsive at six months. At the time of evaluation, 97% of patients in the core subpopulation who responded were alive and had a mean follow-up time of 3.2 months.

[0839] Duration of response and overall survival results (grouped by best overall response (non-responders, CR/PR, CR and/or PR)) are shown for the complete and core patient populations in FIG. 4A and 4B, respectively. As shown, long-term survival was observed in responding patients with extended response duration in patients with CRs. All patients responding at three months remained alive at the time of evaluation, despite the fact that 5/6 patients with unsatisfactory status (ECOG 2) died.

[0840] FIG. 8 shows a graph showing time without progression (months) for individual patients in the full and main populations. Each tick represents a single patient. Hatching indicates the best complete response (in each case, unless otherwise indicated, achieved at 1 month); the texture indicates the dose (solid shading = dose level 1, single dose; oblique shading, dose level 2, single dose; vertical shading = dose level 1, two doses). Horizontal arrows indicate an ongoing response. Some individual patients were initially assessed (eg, at 1 month) as showing stable disease (SD) or partial response (PR), and were later observed to achieve PR (eg, SD to PR conversion) or CR. In such cases, the shading of the individual patient's mark is noted to indicate the best overall response, and the dots (same hatching correspondence to the achieved response) along each individual patient's mark indicate when each of the SD, PR, and/or CR was observed to occur in the patient. .

[0841] Complete elimination of CNS involvement in lymphoma was observed in two patients. CAR ⁺ cells in one patient were observed to show growth after biopsy after relapse. The patient who showed growth after biopsy had chemorefractory transformed DLBCL (germinal center subtype with BCL2 rearrangement and multiple copies of MYC and BCL6). The patient was injected with CAR ⁺ T cells in DL-1, and the numbers of CD3 ⁺ /CAR ⁺ , CD4 ⁺ /CAR ⁺ , CD8 ⁺ /CAR ⁺ T cells in peripheral blood measured at certain time points are shown in FIG. 6A. The patient had previously received, and was found to be refractory to, five previous lines of therapy including individually dosed etoposide, doxorubicin and cyclophosphamide with vincristine and prednisone plus rituximab (DA-EPOCH-R) and moderately intensive allogeneic stem cell transplantation from 8/8 HLA- matched unrelated donor. After allogeneic stem cell transplantation and prior to receiving CAR ⁺ T cells, the patient showed 100% donor chimerism in all blood lines, stopped receiving immunosuppressive therapy, and had no graft-versus-host response (GVHD). Prior to administration of CAR ⁺ T cells, the patient had a periauricular mass and temporal lobe lesion observed using positron emission tomography and computed tomography (PET-CT) (FIG. 6B) and confirmed by magnetic resonance imaging (MRI) (FIG. 6D).

[0842] After receiving anti-CD19 treatment with CAR-T cells, the patient achieved CR 28 days post-infusion as shown by PET-CT (FIG. 6C) and brain MRI (FIG. 6E), with no observable signs of neurotoxicity or CRS. Three months after infusion of CAR-T cells, this patient had a recurrence of the periauricular mass (FIG. 6F) and an incisional biopsy was performed. As shown in FIG. 6A, after biopsy, the visible tumor receded without further therapy. Pharmacokinetic analysis showed a marked re-growth of CAR ⁺ T cells in peripheral blood (to a level higher than the initial observed increase, with peak levels observed approximately 113 days after infusion), which coincided with tumor regression. The patient then achieved a second CR confirmed by repeat PET-CT one month after biopsy (FIG. 6G) and remained in CR 6 months after CAR-T cell infusion. Further evaluation of the patient indicated that the CNS response was sustained and the patient remained in CR for 12 months.

[0843] The results in a patient who received a biopsy accompanied by observed regrowth of CAR ⁺ T cells were consistent with the conclusion that regrowth and activation of CAR ⁺ T cells can be initiated in vivo after the reduction or loss of functional or active CAR ⁺ T cells and /or relapse after antitumor oedema on CAR-T cell therapy. Further, after re-growth in vivo after the initial infusion of CAR ⁺ T cells, CAR ⁺ T cells can again show antitumor activity. This result confirms that regrowth and activation of CAR ⁺ T cells can be induced in vivo and that methods for reactivating CAR ⁺ T cells can further increase their effectiveness.

[0844] Complete responses in two DLBCL patients involving the CNS were observed without developing any degree of neurotoxicity. These results were consistent with the observation that CAR ⁺ T cells according to embodiments of the invention are able to enter the CNS and exert an effector function to reduce or eliminate CNS tumors without increasing or significantly increasing the risk of toxicity such as neurotoxicity. In other studies, among patients receiving anti-CD19 treatment with CAR T cells, no clear correlation was observed between the level of neurotoxicity and the presence of CNS leukemia in the brain (which has been observed to respond to such CAR T cell therapy). Thus, while neurotoxicity may occur in some contexts following treatment with CAR-T therapies, such neurotoxicity is not necessarily the result of targeted brain expression or CAR T cell activity in the CNS, and may not be due to toxicity at the target of CAR ⁺ T cells.

Example 1.C,2

[0845] Example 1.C.2 describes the results based on the analysis in Example 1.A.2 and 1.B.2.

[0846] Up to the time point in Example 1.C,2, 68 patients in the full DLBCL cohort were assessed for response. For complete or objective response (OR), the 3-month and 6-month objective response rates were 75% (51/68), 49% (27/55), and 40% (14/35), respectively. The complete response (CR) rate, 3-month CR rate and 6-month CR rate, were 56% (38/68), 40% (22/55) and 37% (13/35), respectively. A trend toward improved response rates at 3 months was observed in patients treated in DL2 compared to DL1: 63% (12/19; 95% CI 38, 84) versus 40% (12/30; 95% CI 23 , 59) for OPPA with p=0.148, and 58% (11/19; 95% CI 34.80) compared with 27% (8/30; 95% CI: 12.46) for CR with p=0. 0385. Among 16 patients with double/triple hit lymphoma, the ORR was 81% and the 3-month CR rate was 60%.

[0847] In the main population (n=49 for time point in Example 1.C,2), OR, 3-month and 6-month OR frequencies were 84% (41/49), 65% (26/40) and 57% (13/23), respectively. The CR rate, 3-month CR rate and 6-month CR rate were 61% (30/49), 53% (21/40) and 52% (12/23), respectively. There was a similar trend towards improved long-term OPPE and CR at 3 months at higher doses. Specifically, for patients in the core population treated with DL2, the 3-month RRR was 80% (12/15; 95% CI 52,96) and the 3-month CR was 73% (11/15; 95% CI 45 , 92), compared with 3-month RRR and CR of 52% (11/21; 95% CI 30, 74) and 33% (7/21; 95% CI 15, 57) in the core DL1-treated population, with p=0.159 and p=0.0409, respectively. Among patients in the main cohort treated with DL2 and with 3-month follow-up (n=15), the 3-month RRR was 80% and the 3-month CR was 73%.

[0848] The average DOR in the full population and the main population at this time point in 1.C.2 was 5.0 and 9.2 months, respectively; the mean duration of CR was 9.2 months in the full cohort. The average duration of CR was not achieved in the core cohort. The median overall survival (OS) was 13.7 months in the full cohort and was not achieved in the core cohort. The 6-month OS was 75% in the full cohort with a mean follow-up of 5.8 months. 6-month OS was 88% in the general population, with a median follow-up of 5.6 months.

D. Assessment of CAR ⁺ T cells in blood

[0849] Based on data from the time point described in Example 1.A.1, 1.B.1 and 1.C.1, a pharmacokinetic analysis was performed to estimate the numbers of CAR ⁺ T cells in peripheral blood at various time points after treatment. Fifty-five (55) patients assessed at the time point in Example 1.A.1 in the DLBCL cohort and four (4) patients (evaluated at the same time point) in the mantle cell lymphoma (MCL) cohort were analyzed as described. in Example 2 below. Pharmacokinetic (PK) measurements were performed using validated flow cytometry to detect a marker expressed in the CAR construct and quantitative PCR-based tests to detect integration of the CAR construct. B cell aplasia was assessed by flow cytometry with anti-CD19 antibodies. As shown in FIG. 5A, CD4 ⁺ and CD8 ⁺ CAR-expressing cells, as measured by cell count/µl blood (mean ± quartiles) on a logarithmic scale, were detected throughout the evaluation course at both administered dose levels. Patients receiving DL2 relative to DL1 had higher mean AUC _0-28 and mean AUC _0-28 for subsets of CD3 ⁺ /CAR ⁺ , CD4 ⁺ /CAR ⁺ and CD8 ⁺ /CAR ⁺ T cells in peripheral blood (AUC _0-28) . ₂₈ : DL2 vs. DL1 was 1836 vs. 461, 350 vs. 182, and 1628 vs. 114, for CD3 ⁺ , CD4 ^+, and CD8 ⁺ , respectively; p<0.05 for CD8 ⁺ ; AUC _{0 -28} : DL2 versus DL1 was 99.8 versus 27.9, 15.1 versus 5.2, and 73.1 versus 5.5 cells/µl, respectively). The median time to maximum growth of CD3 ⁺ CAR ⁺ T cells was 15 days (range 8-29) and did not differ between dose levels. CD4 ⁺ and CD8 ⁺ CAR-expressing T cells located in the bone marrow at relatively similar levels.

[0850] An increased mean area under the curve (AUC) (numbers of CD8 ⁺ CAR ⁺ T cells over time in the blood) was observed among patients who were administered a higher dose level compared to a lower dose level, with no observed increase in toxicity. Higher peaks in CD8 ⁺ /CAR ⁺ T cell exposure were observed in responding patients (CR/PR) than in non-responding patients (PD); cell persistence over evaluation times, including up to 3 and 6 months, was observed even in patients whose disease progressed (FIG. 5B). The mean AUC _0-28 and mean AUC _0-28 of CD8 ⁺ CAR ⁺ T cells were higher in responders and sustained response at month 3 (CD8 ⁺ C _max median = 20.8 vs. 5.5; CD8 ⁺ AUC _{0 -28} median = 235 vs. 55 in CR/PR in month 3 vs. PD in month 3). Among patients assessed for CAR T cell persistence, 90% and 93% of 29 patients had detectable CD8 ⁺ and CD4 ⁺ CAR ⁺ T cells, respectively, at month 3; 63% and 58% of 19 patients had detectable CD8 ⁺ and CD4 ⁺ CAR ⁺ T cells, respectively, at month 6. At months 3 and 6, no statistically significant differences in CAR ⁺ T cell persistence were observed between patients with long-term response or relapse. CAR ⁺ T cells could be detected during relapse in 89% of 11 PK patients, even though B-cell aplasia (<1 cell/µl) was demonstrated in almost all patients 97% (34/35) in month 3, and 100% (24/24) in month 6.

[0851] Higher AUC _0-28 and AUC _0-28 in DL2 compared to DL1 were not observed to be associated with increased CRS or NT. For either NT or > Grade 2 CRS, the mean CD4 ⁺ /CAR ⁺ and CD8 ⁺ /CAR ⁺ T cell AUCs were 5-10-fold and 3-5-fold higher, respectively, than the mean AUC for DL2. Higher disease severity and baseline levels of inflammatory cytokines have been observed to be associated with higher peak CAR ⁺ T cell levels, higher peak cytokine levels, and higher levels of CRS and NT. The results were consistent with the conclusion that higher AUC _0-28 and mean AUC _0-28 in DL2 did not increase CRS or NT.

[0852] The results were consistent with the conclusion that the treatment resulted in long-term exposure and persistence of engineered cells even in patients with poor responses. In some embodiments, combined approaches are used, such as administering an immune checkpoint modulator or other immune modulating agent, e.g., after disease recurrence or progression, at a point in time at which genetically engineered cells are retained in the patient, e.g., by measuring cell levels in peripheral blood. In some aspects, cells that persist for an extended period regrow or become activated and/or exhibit anti-tumor function following administration of another treatment agent or agent. Higher average numbers of CD4 ⁺ and CD8 ⁺ CAR ⁺ T cells were generally observed over time in the blood of patients who developed neurotoxicity (FIG. 5C). The results showed that CAR ⁺ T cells showed growth and persistence, a response duration of 3 months, increased at higher dose levels without increased toxicity. Results were observed that were consistent with the suggestion that high peak blood levels of CAR ⁺ T cells and cytokines may be associated with NT and CRS and may be influenced by underlying patient factors. It was observed that CAR ⁺ T cells were present at the time of relapse, indicating that combined approaches or retreatment may provide certain benefits.

E. Blood Analytes and Neurotoxicity, CRS and Response

[0853] Various post-treatment blood analytes, including cytokines, were measured in the sera of patients (assessed at the time point in Example 1.A.1) prior to administration of CAR ⁺ T cells. Cytokines were measured using the multiplex cytokine assay. Potential correlations to the risk of developing neurotoxicity were assessed using statistical analysis based on one-way non-parametric analyses.

[0854] FIG. 7 shows the mean levels of analytes evaluated in units (LDH, U/L; ferritin, ng/mL; CRP, mg/L; cytokines, pg/mL) in patients who did not develop neurotoxicity compared to patients who did develop neurotoxicity. after CAR ⁺ T-cell therapy. Levels of several blood analytes, including LDH, Ferritin, CRP, IL-6, IL-8, IL-10, TNF-α, IFN-α2, MCP-1 and MIP-1β, have been observed to be associated with the level of risk of developing neurotoxicity (Wilcoxon p values <0.05 excluding multiplicity comparison). In particular, the results were consistent with the conclusion that post-treatment LDH levels, which in some embodiments is a surrogate for disease severity, may be useful for potential neurotoxicity risk assessment and/or risk-adapted dosing or treatment management for certain patients. In addition, tumor burden measured prior to administration of the CAR-T cell composition correlated (Spearman p values <0.05) with the risk of developing neurotoxicity. In some aspects, LDH levels may be assessed individually and/or in combination with another pre-treatment parameter, such as another measure or indicator of disease severity, such as a tumor volume measurement, such as sum product dimensions (SPD) or other CT-based or MRI is a volumetric measurement of disease severity. In some aspects, one or more parameters representative of the severity of the disease were evaluated, and in some contexts may indicate the presence, absence or level of risk of developing neurotoxicity after T-cell therapy. In some aspects, one or more parameters include LDH and/or tumor volume measurement.

[0855] In an additional analysis, fifty-five (55) patients in the DLBCL cohort at the time point in Example 1.A.1 and four (4) patients with mantle cell lymphoma (MCL) described in Example 2 below were included in the analysis for correlation with safety ratings. Of the 59 patients assessed for safety, CRS was observed in 32% (30% Grade 1-2, 0% Grade 3, 2% Grade 4); NT was observed in 20% (5% Grade 1-2, 10% Grade 3, 5% Grade 4). The dose level did not correlate with CRS or NT (p=0.565 and p=1.00, respectively). Patient factors correlated with any grade of CRS and NT were poor general condition (e.g., ECOG Status 2) (p=0.03) and higher disease severity (p<0.05), as measured by the sum of product diameters (SPD ) based on display results. Pre-CAR ⁺ T cell infusion clinical laboratory parameters and pre-CAR ⁺ T cell infusion cytokine measurements observed to be associated with the occurrence of any grade of NT included higher serum LDH, ferritin, and CRP, and higher plasma IL -6, IL-8, IL-10, TNF-α, IFN-α2, MCP-1 and MIP-1β (p<0.05 for each). Higher plasma levels of IL-8, IL-10 and CXCL10 prior to CAR ⁺ T cell infusion were also associated with Grade 3-4 NT (p<0.05 for each).

[0856] Of the 54 patients in the DLBCL cohort assessed for response, higher ECOG and DLBCL scores transformed from CLL or MZL correlated with lower duration of response at month 3 (p=0.02 for both). Pre-CAR ⁺ T cell infusion parameters associated with better ORPM included lower ferritin, LDH, CXCL10, G-CSF, and IL-10, and those associated with a sustained response of 3 months included lower ferritin, CRP, LDH, CXCL10, IL-8, IL-10, IL-15, MCP-1, MIP-1β, TNF-α, and higher hemoglobin and albumin before CAR ⁺ T infusion (p<0.05 for each).

[0857] In some cases, the apheresis sample and the composition of CAR ⁺ T cells for injection were evaluated and correlated with clinical results. The results indicated that memory T cell subpopulations and T cell functionality may be correlated with certain clinical outcomes.

[0858] The results indicated that several initial patient characteristics, including an inflammatory state and a high pre-treatment tumor burden, may be useful in identifying patients at risk for increased toxicity following administration of CAR-expressing T cells. A low tumor burden and a low inflammatory state have been observed to be associated with an improved toxicity profile and a better duration of response. These results confirm that treating patients earlier in the course of therapy and/or evaluating a group of clinical and laboratory biomarkers for risk stratifies patients for potential early intervention, may reduce the risk of toxicity, and enhance duration of response.

Example 2 Administration of CAR Expressing Anti-CD19 Cells to Patients with Mantle Cell Lymphoma (MCL)

[0859] Therapeutic compositions of CAR ⁺ T cells containing autologous T cells expressing a chimeric antigen receptor (CAR) specific for CD19, generated as described in Example 1, were administered to four (4) individuals with mantle cell lymphoma (MCL), in whom first-line therapy failed. The frozen cell compositions were thawed prior to intravenous administration. The T cell therapeutic composition is administered as a defined composite cell product with engineered populations of CD4 ⁺ and CD8 ⁺ CAR ⁺ T cells derived from the same patient, administered at a target ratio of approximately 1:1. Patients were dosed with CAR expressing T cells (as a divided dose of CD4 ⁺ and CD8 ⁺ CAR expressing T cells) in a single dose level 1 (DL1) dose containing 5x10 ⁷ CAR expressing T cells. Beginning on the third (3) day prior to CAR ⁺ T cell infusion, patients received anti-lymphocyte chemotherapy with fludarabine (flu, 30 mg/m ² ) and cyclophosphamide (Cy, 300 mg/m ² ).

[0860] Patients were tested for response and toxicity as described in Example 1. No CRS or neurotoxicity was observed in any of the patients. Of the 4 patients receiving treatment, two (2) patients achieved PR (non-long term) and two (2) patients had progressive disease.

Example 3: Further evaluation of response, safety, pharmacokinetics, pharmacodynamics and blood analytes in patients with relapsed and refractory non-Hodgkin's lymphoma (NHL) after administration of Anti-CD19 CAR-expressing cells

[0861] Response outcomes, safety outcomes, pharmacokinetic and pharmacodynamic parameters, and blood analytes were evaluated in patients at a subsequent time point in the clinical study described in Example 1 above.

A. Patients and Treatment

[0862] The analysis at this point in time presented in this example is based on the evaluation of a total of 91 patients in the full DLBCL cohort (88 (65 of the main cohort) assessed for response and 91 (67 of the main cohort) assessed for response regarding safety) administered with anti-CD19 CAR-expressing cells. The full cohort included DLBCL, NOS de novo, and transformed from any indolent lymphoma, ECOG 0-2; The primary analysis population included patients with DLBCL, NOS, and transformed from follicular lymphoma (tFL) or high-grade B-cell lymphoma and with an Eastern Cooperative Oncology Group Performance Status (ECOG PS) of 0 or 1. Approximately 90% of treated patients in the general population had at least 1 low-risk disease predictive of a short mean overall survival (OS) of 3-6 months, such as double/triple-hit expressors, primary refractory disease, refractory to 2 or more lines of therapy, never achieved CR, or never received autologous stem cell transplantation (ASCT). In some embodiments, a cohort of patients having diffuse large B-cell lymphoma (DLBCL) not otherwise characterized (NOS; de novo and transformed from follicular lymphoma tFL)), or well-differentiated B-cell lymphoma, with MYC and BCL2 permutations and/ or BCL6 with DLBCL histology, and excluding patients with an ECOG score of 2 or patients who had received prior hematopoietic stem cell transplantation (HSCT), CAR-T compositions were administered as described here. In some embodiments, the mainstream patients were administered anti-CD19 CAR ⁺ T cells in a single dose of DL2 (1x10 ⁸ total CAR-expressing T cells).

[0863] At this point in time, a total of 140 patients were subjected to leukapheresis, of which 10 were waiting for composition preparation, 2 withdrew from the study before composition was ready, and for 2 compositions were not available. Of the 18 patients whose products were available, 4 were waiting for treatment, 4 dropped out of the study, and 10 developed progressive disease or died. A total of 108 patients were injected with anti-CD19 CAR-expressing cells, of which 6 were not evaluated and 11 received inappropriate anti-CD19 CAR-expressing cells (compositions did not always meet certain specifications, but were considered safe to administer). Patients received DL1 (n=45), a double dose of DL1 (n=6), or DL2 (n=40). Six (6) patients with mantle cell lymphoma (MCL) were injected with CAR ⁺ cells in DL1 (five received an appropriate product, one received an inappropriate product), and five (5) completed the full 28 days of follow-up. One MCL patient developed CRS and none received tocilizumab or dexamethasone. The product was available to 98% of apheresis patients (126/128) in the DLBCL cohort.

[0864] Patients at this time point included 5 patients treated on an outpatient basis (including four (4) patients treated with DL1, one (1) treated with DL2; four (4) of which were included in the main cohort). For patients treated on an outpatient basis, the mean age was 57 years (range 26-61), 3 had DLBCL, NOS, 1 had tFL, and 1 had PMBCL. All five (5) patients had ECOG scores of 0 or 1. Outpatient outcome data included outcomes for three (3) additional outpatients (total of eight (8) patients) whose data became available after the time point for analysis in this example.

[0865] Demographic and baseline characteristics of the total and core population of patients at a point in time are presented in Table E5 .

[0866]

Table E5. Patient characteristics: DLBCL population Characteristic FULL
(n=91) BASIC
(n=67) Mean Age, years (range) 61 (20-82) 60 (20-82) ≥ 65 years, n (%) 34 (37) 24 (36) Male/Female, n (%) 61/30 (67/33) 46/21 (69/31) Subtype B-NHL, n (%) DLBCL, NOS de novo 59 (65) 51 (76) Transformed from FL (tFL) 19 (21) 16 (24) Transformed from MZL (tMZL) /CLL (tCLL) 6 (7) / 4 (4) 0 Follicular, Stage 3B/PMBCL 1 (1) / 2 (2) 0 Molecular subtype, n (%) Double/triple hit [high-stage B-cell lymphoma] ^a 18 (20) 16 (24) Patient characteristics, n (%) Chemorefractive ^b 61 (67) 44 (66) ECOG PS 0-1/2 (pre-LD) 81 (89) / 10 (11) 67 (100) / 0 IPI 3-5/Disease Stage 3-4 38 (42) / 70 (77) 24 (36) / 49 (73) CNS involvement 2(2) 2(3) Prior lines of therapy, median (range) 3 (1-12) 3 (1-8) Didn't reach CR 47 (52) 34 (51) Any HSCT 39 (43) 28 (42) Previous autologous 36 (40) 28 (42) Previous allogeneic 5 (5) 0 HSCT, hematopoietic stem cell transplantation; LD, lymphodepletion.
^a At trial initiation, included in DLBCL, NOS histology; based on new WHO criteria (Swerdlow et al., (2016) Blood 127(20):2375-2390), are now considered “well-differentiated B-cell lymphoma, with MYC and BCL2 and/or BCL6 permutations with DLBCL histology (double/triple hit).
^b SD or PD for last chemotherapy regimen or relapse < 12 months after autologous SCT.

B. Post-Treatment Safety and Response Results

[0867] As shown in Table E6 , the objective response rate (ORR) was 74%, including 52% of patients who showed a complete response (CR). The rate of any degree of cytokine release syndrome (CRS) was 35% with 1% severe CRS; and the rate of any degree of neurotoxicity (NT) was 19% with 1% severe NT.

Table E6. Response and safety after CAR administration ⁺ cells FULL BASIC All dose levels All dose levels ^a DL1S DL2S Best Complete Response (BOR), n ^b 88 65 34 27 ORR,% (95% CI) 74 (63.83) 80(68, 89) 77 (59.89) 82 (62, 94) CR% (95% CI) 52 (41.63) 55(43, 68) 47 (30, 65) 63 (42, 81) Security, n ^c 91 67 34 29 Any CRS, % (95% CI) 35 (25, 46) 36 (24, 48) 41 (25, 59) 24 (10, 44) sCRS(degrees 3-4),% (95% CI) 1 (0, 6) 1 (0.8) 38 (0.15) 0 Any NT,% (95% CI) 19 (11.28) 21 (12, 33) 24 (11, 41) 17 (6, 36) sNT (degrees 3-4),% (95% CI) 12 (6, 21) 15 (7, 26) 21 (9, 38) 7 (1, 23)

^a Four patients treated with DL1D (dose level 1, two-dose regimen) with similar results.

^b Includes patients with a PD event, death, or 28-day follow-up. One patient had no repeat studies.

^c Includes all patients who received at least one dose of a CAR-expressing cell product within 28 days prior to the date of data collection or who died.

[0868] As shown in Table E7 , high response rates and low - severe toxicity were observed in the total DLBCL population.

Table E7. Response after CAR introduction ⁺ cells By diagnosis FULL DLBCL, NOS tFL tCLL/MZL FL3B/PMBCL BOR, n ^a 88 57 19 10 2 ORR,% (95% CI) 74 (63.83) 74 (60, 85) 84 (60, 97) 50 (19, 81) 100 (16, 100) CR% (95% CI) 52 (41.63) 51 (37, 64) 63 (38, 84) 30 (7, 65) 100 (16, 100) Security, n ^b 91 59 19 10 3 Any CRS, % (95% CI) 35 (25, 46) 34 (22, 47) 42 (20, 67) 20 (3, 56) 67 (9, 99) sCRS (degrees 3-4),% (95% CI) 1 (0, 6) 2 (0.9) 0 0 0 Any NT,% (95% CI) 19 (11.28) 20 (11, 33) 21 (6, 46) 10 (0.45) 0 sNT (degrees 3-4),% (95% CI) 12 (6, 21) 14 (6, 25) 11 (1, 33) 10 (0.45) 0

^a Includes patients with a PD event, death, or 28-day follow-up. One patient had no repeat studies.

^b Includes all patients who received at least one dose of CAR ⁺ expressing cells in the 28 days prior to the data entry date or died.

[0869] As shown in Table E8 , a high rate of response and dose-dependent response was observed in the main patient population.

Table E8. Sustained response after CAR administration ⁺ cells Dose levels ^a DL1S DL2S BOR, n ^b 65 34 27 ORR (95% CI),% 80(68, 89) 77 (59.89) 82 (62, 94) CR (95% CI),% 55(43, 68) 47 (30, 65) 63 (42, 81) ≥ 3-mo f/u, n ^c 52 29 19 3-mo ORR (95% CI),% 65 (51.78) 59 (39, 77) 74 (49, 91) 3-mo CR (95% CI),% 54(40, 68) 41 (24.61) 68 (43, 87) ≥ 6-mo f/u, n ^d 38 20 14 6-mo ORR (95% CI),% 47(31, 64) 40 (19, 64) 50 (23, 77) 6-mo CR (95% CI),% 42 (26, 59) 30 (12, 54) 50 (23, 77)

^a Four patients (CORE) were treated for DL1D with similar results.

^b Includes patients with a PD event, death, or 28-day follow-up. One patient had no repeat studies.

^c The denominator is the number of patients who received CAR ⁺ cells ≥ 3 months ago, prior to the date of data collection, with an efficacy assessment at month 3 or before PD assessment or death.

^{The d} denominator is the number of patients who received CAR ⁺ cells ≥ 6 months ago, up to the date of the data snapshot, with efficacy estimated at month 6 or before PD assessment or death.

[0870] Three-month objective response rates (ORRs) among various patient subgroups in the high-risk DLBCL subgroup, which included all DLBCL patients treated at all dose levels in the core cohort, are shown in FIG. 22. The results showed a high long-term ORR in the high-risk DLBCL subgroup.

[0871] Results for duration of response (DOR) and overall survival (grouped by best overall response (non-responders, CR/PR, CR and/or PR)) are shown for the full cohort and core patient population in FIG. 23A-23D. The results also showed that 80% (16/20) of patients with CR at 3 months remained in CR at 6 months, and 92% (11/12) of patients with a response (CR or PR) at 6 months continued to show a long-term response. perspective.

[0872] FIG. 24 depicts the percentage of patients at this time point who were observed to experience laboratory abnormalities and treatment-related adverse events (TEAEs) (data for 5 patients with MCL treated with the respective product in DL1 from at least 28 days of follow-up, not included). In addition to the TEAEs shown in FIG. 24, the following terms of events were observed at Level 3-4 in ≥ 5% of patients: encephalopathy (8%), pancytopenia (5%), and febrile neutropenia (7%). Eight patients (9%) had infusion toxicity, defined as AE on the day of administration, consistent with CAR ⁺ cell administration, including facial flushing, headache, fever, pyrexia, chills, tremors, vomiting, rash, urticaria, pruritus, hypotension, wheezing, bronchospasm, dyspnea, nausea, vomiting, back pain, cough and infusion-related reaction. Events included chills (2), pyrexia (5), facial flushing (1), headache (1), hypotension (1), infusion-related reaction (1), rash (1), pruritus (1), and vomiting (1 ), with 6 grade 1 events, 1 grade 2 (chills), and 1 grade 3 (hypotension). TEAE in the main contingent did not differ significantly from that in the full contingent. The most common related TEAEs in patients treated in the outpatient group were CRS, hypotension, vomiting, anemia, and dyspnea.

[0873] Table E9 presents TEAE and neurotoxicity occurring in 25 percent or more of patients in the full or core cohort of patients who received DL1S and DL2S. No apparent dose-toxicity relationship was observed in the DLBCL population.

Table E9. TEAE ≥25% Full population, core population, and core population by dose level Symptom, n (%) FULL
(N=91) BASIC ^a
(n=67) BASIC DL1S
(n=34) MAIN DL2S
(n=29) Neutropenia ^b 85 (93) 63 (94) 33 (97) 26 (90) Anemia ^b 64 (70) 48 (72) 28 (82) 19 (66) Thrombocytopenia ^b 48 (53) 41 (61) 20 (59) 19 (66) Fatigue 34 (37) 25 (37) 11 (32) 12 (41) CRS 32 (35) 24 (36) 14 (41) 7 (24) Nausea 25 (27) 19 (28) 12 (35) 5 (17) Diarrhea 23 (25) 16 (24) 7 (21) 7 (24)

^a Includes 4 patients treated at dose level 1 on a two-dose regimen.

^b Laboratory abnormalities.

[0874] FIG. 25 depicts the number and percentage of patients observed to have CRS and/or NT at various time points after administration of CAR ⁺ cells. In this assessment, the median time to onset of a CRS or NT event was observed to be 5 (range 1-14) or 10 (range 3-23) days, respectively. During the first 72 hours after CAR ⁺ cell injection, 1 patient had NT (grade 1) and only 14% (13 of 91) had CRS (7 grade 1; 6 grade 2). The mean duration (Q1, Q3) of CRS or NT was 5 (4, 8) or 10.5 (7, 19) days, respectively. NT preceded CRS in 12 of 17 cases (71%). All measurable NT events resolved during analysis except one grade 1 tremor, and 2 patients died of progressive disease with ongoing NT (based on the event safety database, including additional patients analyzed after the analysis time point described in this Example) .

[0875] In the full cohort (n=91), selected patients with onset CRS or NT were administered anti-cytokine therapy with tocilizumab and/or dexamethasone as follows: Tocilizumab alone, 4% (n=4); Dexamethasone alone, 9% (n=8); Tocilizumab and dexamethasone, 8% (n=7). The median number of doses of dexamethasone was 6 (range, 2-99); and the mean number of doses of tocilizumab was 1 (range, 1-3).

[0876] Table E10 shows the results of toxicity in patients in the main population who received a single dose in DL1 or DL2. No deaths occurred from CRS or NT. The median time to onset of CRS was 5 days (range, 2-14) and for NT it was 11.5 days (range, 5-23). In the core population, 13% (n=9) received tocilizumab and 18% (n=12) received dexamethasone to alleviate toxicity. Eighteen percent of patients (12 of 67) showed terms of neurotoxicity consistent with encephalopathy, including encephalopathy (13%), 6% (4 of 67) had aphasia, and 3% (2 of 67) had seizures. In Table E10, the number of patients or % of total patients (parentheses) showing the indicated toxicity result is shown at all dose levels or specifically in patients treated with DL1 or DL2. Also shown in brackets are the upper and lower 95% confidence intervals.

Table E10. Toxicity in the general population receiving various dose levels All dose levels ^a
n=67 DL1S
n=34 DL2S
n=29 CRS, n (%) [95%CI] Any degree 24 (36) [24, 48] 14 (41) [25, 59] 7 (24) [10, 44] Degree 1/2 23 (34) [23, 47] 13 (38) [22.56] 7 (24) [10, 44] Grade 3/4 (sCRS) 1(1) [0, 8] 1 (3) [0, 15] 0 Neurotoxicity ^b .n (%) [95% CI] Any degree 14 (21) [12, 33] 8 (24) [11, 41] 5 (17) [6, 36] Degree 1/2 4 (6) [2, 15] 1 (3) [0, 15] 3 (10) [2, 27] Grade 3/4 (sNT) 10 (15) [7, 26] 7 (21) [9, 38] 2 (7) [1, 23] Any, n (%) [95%CI] CRS or NT, n (%) 28 (42) [30, 54] 15 (44) [27, 62] 10 (34) [18, 54] sCRS or sNT, n (%) 10 (15) [7, 26] 7 (21) [9, 38] 2 (7) [1, 23]

^a Four patients treated for DL1D with similar results.

^b Includes state of confusion, encephalopathy, aphasia, ataxia, cerebellar syndrome, delirium, decreased level of consciousness, dizziness, flattening of emotions, impaired hand-eye coordination, memory impairment, tremor, agitation, impaired attention, dysarthria, changes in mental status, muscle weakness , convulsions, drowsiness and urinary incontinence.

[0877] Of the twelve (12) patients receiving nonconforming products, 10 in DL1 and 2 in DL2, all were followed up for 28 days. CRS was observed in 33% of patients (4/12) and NT was not observed in any of the patients. Two patients received tocilizumab and 3 patients received dexamethasone. Toxicity rates were comparable to those observed in a larger cohort of patients administered the respective product. In patients receiving inappropriate products, pharmacokinetic (PK) expansion was higher in patients with CRS/NT, patients with high tumor burden or LDH levels.

C. Evaluation of outpatient administration

[0878] Data for a total of eight (8) patients were evaluated at this point in time when they were treated on an outpatient basis (mean age 58.5 and ECOG 0 or 1) at multiple clinical sites, including 3 patients whose data were available after the point in time at which they were analyzed for the purposes of this Example. The median duration of hospitalization was 15.6 days for patients treated in an inpatient setting (SD 9.6, n=86) and 9.3 days for patients treated on an outpatient basis (SD 11.9, n=8). A 40% reduction in hospital stay was observed in patients treated on an outpatient basis. The median number of days to hospitalization following outpatient administration of CAR ⁺ T cells was 5 days (range: 4-22). None required admission to the Intensive Care Unit (ICU) after outpatient administration.

[0879] Among those of 8 patients treated on an outpatient basis with more than 28 days of follow-up after administration, 1 remained on an outpatient basis for the duration of the dose-limiting period of toxicity. Seven (7) patients were admitted with fever (1 on study day 4, the rest on study day ≥ 5), 6 patients were admitted with CRS (4 grade 1, 2 grade 2) and 2 patients with grade 1 NT. None of the patients did not experience severe CRS or NT. One (1) patient was treated with tocilizumab without dexamethasone for CRS (Grade 2) and none of the patients were treated with dexamethasone for CRS or NT. One patient was admitted 3 days after CAR ⁺ T cell injection.

[0880] Among 91 patients treated in inpatient and outpatient settings, 11 patients (12%) required ICU clearance to control toxicity; 8 patients (9%) required ICU clearance to control CRS or NT; 2 patients (2%) required ICU clearance to control acute respiratory events (one associated with CAR ⁺ T cell administration, one unrelated). Six (6) patients (6%) were intubated (based on the event safety database, including additional patients analyzed after the analysis time point described in this Example; n=94); 7 patients (7%) received vasopressors (based on the safety database of events defined as demonstrating hypotension in the first 28 days after CAR ⁺ T cell administration, as assessed by TEAE); and 2 patients (2%) underwent hemofiltration (based on the event safety database). The results showed that very few of the patients required ICU level and related procedures. The results support the feasibility of outpatient administration with safe ambulatory toxicity control, appropriate education and outpatient monitoring.

[0881] Evaluation of outpatient administration confirmed the feasibility of safe outpatient administration. 30% of patients were not re-admitted.

D. Pharmacokinetic evaluation

[0882] The number of CAR ⁺ T cells in peripheral blood and bone marrow at time points before administration (pretreatment or pre-antilymphocyte chemotherapy (LDC)) and subsequent treatment at various time points (with day of administration as day 1) in 87 patients in DLBCL contingent with estimated PK, flow cytometry using an antibody specific for the truncated receptor used as a surrogate marker, and quantitative polymerase chain reaction (qPCR) using primers specific for the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) present in the vector, encoding a chimeric antigen receptor (CAR). The area under the curve with plot numbers per microliter for the designated CAR ⁺ cell population between days 0 and 28 (AUC _0-28 ) and the maximum or peak concentration of CAR ⁺ cells in the blood (AUC _0-28 ; CAR ⁺ cells/μl of blood) was assessed. B-cell aplasia was assessed in peripheral blood by flow cytometry by CD19 staining. Cytokines were measured using the multiplex cytokine test. Data from all patients receiving various dose levels were pooled for safety analysis. For response analysis, data were stratified by dose level. Statistical analysis was two-sided without taking into account the multiplicity of comparisons.

[0883] FIG. 9A shows the detected numbers of CAR T cells per microliter of blood at various designated time points as assessed by flow cytometry or qPCR. FIG. 9B shows CAR ⁺ cells per microliter of blood versus microliter of bone marrow on day 11±3. As shown in FIG. 9A, levels of CAR-expressing cells in patient samples were monitored by both flow cytometry-based assays and qPCR assays. As shown in FIG. 9B, all patients (n=86 and 85 for flow cytometry and qPCR, respectively, excluding one patient with no flow cytometry and 2 patients with no qPCR) with assessed PK results showed detectable numbers of CARs. -expressing cells in blood and bone marrow. The results were consistent with the observation that CAR ⁺ T cells proliferated similarly in bone marrow and blood.

[0884] Levels over time of CD4 ⁺ and CD8 ⁺ CAR-expressing cells (as assessed by AUC _0-28 and AUC _0-28 ) were compared in different subgroups of patients receiving dose level 1 (DL1): diffuse large B-cell lymphoma de novo (DLBCL, NOS) or transformed from follicular lymphoma (tFL) (MAS; N=32), DLBCL transformed from marginal zone lymphoma or chronic lymphocytic leukemia (tMZL/tCLL; N=4), or mantle cell lymphoma (MCL; N= 5) that received CAR-expressing T cells in DL1. As shown in FIG. 10A and 10B, AUC _0-28 and AUC _0-28 varied among patients in different disease subgroups, with an increase in CD4 ⁺ and CD8 ⁺ CAR-expressing cells tending to be lower in the NON-MASTER subpopulations. PMBCL (n=2) and FL3B (n=1) are not indicated due to the limited number of patients. Growth in patients receiving DL2 was similar to that in patients receiving DL1.

E. Pharmacokinetic assessment of dose level

[0885] AUC _0-28 and AUC _0-28 for CD3 ⁺ , CD4 ⁺ and CD8 ⁺ CAR-expressing cells were also compared for patients receiving dose level 1 (DL1; n=32) and those receiving dose level 2 (DL2; n=27), in the main cohort (patients with DLBCL, NOS or high-grade B-cell lymphoma (double/triple hit); N=59). As shown in FIG. 11A and 11B and in Table E11, a higher mean AUC _0-28 was observed for CD3 ⁺ , CD4 ⁺ and CD8 ⁺ CAR-expressing cells was observed in patients who received DL2, compared with patients who received DL1. Similarly, a higher growth trend in patients who received DL2 was observed in the full DLBCL cohort. A longer duration of response (DOR) of 3 months was also observed among patients who received DL2 compared to those who receive DL1, without increasing toxicity. The mean time to AUC _0-28 (Tmax) for CD4 ⁺ and CD8 ⁺ CAR ⁺ cells was similar between patients who received DL1 and DL2.

[0886] Increased exposure of CAR ⁺ T cells was observed in DL2 compared to DL1, which corresponds to an increased duration of response without increased toxicity in DL2 patients.

Table E11. Pharmacokinetics in patients grouped by dose levels in the main population DL1S
(n=32) DL2S
(n=27) Total, DL1S and DL2S (n=59) CD3 ⁺ C _Max , median (cells/µl) 48.2 96.2 65.8 Q1, Q3 15.6, 151.3 30.2, 219.5 19.0, 204.2 Min, max 0.1, 7726.3 1.1, 1280.9 0.1, 7726.3 T _Max , median (days) 14.5 15.0 15.0 Q1, Q3 11, 15 11, 15 11, 15 Min, max 9, 24 8, 31 8, 31 AUC _0-28 , median (cells*day/µl) 477.7 823.1 542.4 Q1, Q3 165.9, 999.3 155.8, 3628.3 155.8, 3381.9 Min, max 1.8, 142816.7 16.5, 16087.8 1.8, 142816.7 CD4 ⁺ C _Max , median (cells/µl) 7.0 14.9 7.7 Q1, Q3 2.6, 46.0 2.0.46.8 2.5, 46.8 Min, max 0.1, 3039.9 0.2, 169.4 0.1, 3039.9 T _Max , median (days) 14.0 15.0 15.0 Q1, Q3 11, 15 11, 15 11, 15 Min, max 8, 24 8, 31 8, 31 AUC _0-28 , median (cells*day/µl) 71.1 166.1 91.5 Q1, Q3 26.4, 274.7 18.1.679.0 23.9.368.8 Min, max 1.2, 68990.3 2.9, 4266.8 1.2, 68990.3 CD8 ⁺ C _Max , median (cells/µl) 26.1 62.8 43.6 Q1, Q3 3.7, 111.2 26.2.171.7 9.1.151.6 Min, max 0.0, 5237.6 0.7.1261.8 0.0, 5237.6 T _Max , median (days) 15.0 15.0 15.0 Q1, Q3 11, 16 11, 17 11, 16 Min, max 4, 28 8, 31 4, 31 AUC _0-28 , median (cells*day/µl) 347.2 606.6 412.2 Q1, Q3 52.1,871.4 155.7.2463.4 72.1.1852.5 Min, max 0.3, 81865.9 4.7, 15570.0 0.3, 81865.9

F. Persistence

[0887] Persistence of CAR-expressing cells and CD19+ B cell aplasia (low numbers or absence of CD19+ B cells) were assessed at various time points in evaluable DLBCL patients treated with CAR ⁺ T cells based on detectable levels of CD3 ⁺ , CD4 ⁺ , or CD8 ⁺ CAR-expressing cells and levels of CD19+ B cells found in blood, respectively. The results are presented in Table E12. Among patients assessed in progression (progression time independent of BOR; n=37), mean 0.17 CD4 ⁺ CAR ⁺ cells/μl (range, 0-65.5 cells/μl) and mean 0.15 CD8 ⁺ CAR ⁺ cells/µl (range, 0-131.8 cells/µl) were observed in progression. Among patients assessed at relapse (during progression after reaching CR) (n=12), mean 0.17/μl (range, 0-35.1 cells/μl) CD4 ⁺ CAR-expressing cells and mean 0, 20 cells/μl (range, 0-131.8 cells/μl) CD8 ⁺ CAR-expressing cells were observed at relapse. Long-term persistence of CAR-expressing cells was observed in 75% of evaluable DLBCL patients at 12 months. Long-term persistence of B-cell aplasia was also observed in 75% of patients at 12 months, and in patients regardless of relapse status. The results are consistent with the conclusion that anti-CD19 CAR-expressing cells showed long-term persistence in the majority of patients and suggest the potential for long-term disease control even in relapsed patients.

[0888] Among patients who had a relapse, 91.7% (11/12) had detectable CAR-expressing cells in the blood at the time of relapse. This result is consistent with the conclusion that combination therapy or other intervention, in some embodiments, can be used to increase and/or enhance CAR-expressing cells, such as those that may be depleted.

Table E12. Long-term persistence of CAR ⁺ cells and CD19 applezia Month 3 Month 6 Month 9 Month 12 With progression With a relapse CAR T persistence in study patients, n 50 thirty 18 12 37 12 CD3 ⁺ ,% 100 80.0 77.8 75.0 91.9 91.7 CD4 ⁺ ,% 88.0 63.3 50.0 41.7 83.8 83.3 CD8 ⁺ ,% 90.0 70.0 55.6 50.0 83.8 75.0 CD19 ⁺ B cell aplasia
(< 1 cells/µl),% 96.0 93.3 77.8 75.0 97.3 100

G pharmacokinetic evaluation and toxicity

[0889] AUC _0-28 and AUC _0-28 of CD4 ⁺ and CD8 ⁺ CAR-expressing cells were also compared for patients in the general population with any grade (in this assessment, any of grades 1-4; no grade 5 CRS or NT not observed) cytokine release syndrome (CRS) or neurotoxicity (NT) with patients not assessed as showing degrees of CRS or NT. Median CD4 ⁺ CAR ⁺ AUC _0-28 (Q1, Q3) was 59 (18, 210) for no CRS (grade 0; n=43) and 267 (91, 1510) for any CRS (grade 1-4; n= 20) (p=0.001); median CD8 ⁺ CAR ⁺ AUC _0-28 (Q1, Q3) was 310 (36,900) for no CRS (grade 0; n=43) and 605 (174,5619) for any CRS (grade 1-4; n= 20) (p=0.021); median CD4 ⁺ CAR ⁺ AUC _0-28 (Q1, Q3) was 71 (23, 244) for no NT (grade 0; n=50) and 1269 (184, 3057) for any NT (grade 1-4; n= 13) (p=0.003); median CD8 ⁺ CAR ⁺ AUC _0-28 (Q1, Q3) was 304 (43, 799) for no NT (grade 0; n=50) and 2463 (607, 7691) for any NT (grade 1-4; n= 13) (p=0.004). As described above and shown in FIG. 12A-12D, higher levels of CD4 ⁺ and CD8 ⁺ CAR-expressing cells over time have been associated with CRS and NT.

H. Pharmacokinetic evaluation and response

[0890] The number of peak CD3 ⁺ CAR ⁺ cells/µl (CD3 ⁺ AUC _0-28 ) was assessed over time in patients who had the best overall response (BOR) CR, PR or PD. As shown in FIG. 13A and 13B, a trend towards better BOR was observed in taller patients with inter-patient variability.

I. Pharmacokinetic assessment by blood analytes and patient parameters

[0891] Plasma levels of cytokines before treatment with CAR ⁺ T cells (pre-anti-lymphocyte chemotherapy), including interleukin 7 (IL-7), IL-15, macrophage inflammatory protein (MIP-1α), were assessed in patients showing CAR ⁺ CD3 ⁺ blood C _max > 500 CAR ⁺ T cells/µl (N=55) compared with patients who showed CAR ⁺ CD3 ⁺ blood C _max < 500 CAR ⁺ T cells/µl (N=7). As shown in FIG. 14A, increased plasma cytokine levels before CAR ⁺ T cell treatment were observed to be associated with CAR ⁺ CD3 ⁺ C _max > 500 CAR ⁺ T cells/µl (Wilcoxon P-values <.05 (no variance regulation); except for IL -7 p=0.07).

[0892] Peak plasma levels of various cytokines (IL-6, IL-10, IL-16, interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), MIP-1α, MIP-1β, monocyte chemoattractant protein- 1 (MCP-1) and CXC chemokine motif 10 (CXCL10)) were also evaluated in patients showing CAR ⁺ CD3 ⁺ blood C _max > 500 CAR ⁺ T cells/µl (N=68) compared with patients showing CAR ⁺ CD3 ⁺ blood C _max < 500 CAR ⁺ T cells/µl; N=9). As shown in FIG. 14B, higher peak cytokine levels were observed to be associated with CAR ⁺ CD3 ⁺ C _max >500 CAR ⁺ T cells/µl (Wilcoxon P values <0.05; no regulation diversity).

[0893] The ratio between the volumetric amount of tumor size measurement of the product before treatment with CAR ⁺ T cells (pre-antilymphocyte chemotherapy (LDC)) (SPD), as an indicator of tumor mass, and AUC _0-28 CD3 ⁺ CAR ⁺ T cells, representing CAR ⁺ T exposure over time. As shown in FIG. 15, a positive correlation was observed between baseline SPD and CD3 ⁺ AUC _0-28 , with a Spearman correlation of 0.32 and p=0.019.

J. Pretreatment Patient Parameters and Results of Response and Toxicity

[0894] Analyte levels, including cytokines and inflammatory markers such as Ferritin, C-reactive protein (CRP), D-dimer (fibrin breakdown product), IL-6, IL-10, IL-15, IL-16 TNF- α, MIP-1α, and MIP-1β, before treatment with CAR ⁺ T cells (pre-LDC) were compared for patients with any grade (here grades 1-4) of cytokine release syndrome (CRS) or neurotoxicity (NT) with patients without any CRS or NT (grade 0). In this cohort of grade 1-4 CRS patients, all but CRS events were defined as grade 1 or 2. As shown in FIG. 16A (CRS) and FIG. 16B (NT), higher peak plasma cytokine levels and inflammatory marker levels were observed to be associated with CRS and NT based on univariate analysis (Wilcoxon P-value <0.05 for all analytes except ferritin for CRS (p=0, 14) and CRP for CRS (p=0.09)). For CRS, after sizing tumor mass in multivariate analysis, MIP-1β, IL-10, and TNF had p<0.05; for NT IL-15, IL-6, MIP-1α, and TNF had a p<0.05.

[0895] Pre-LDC patient parameters such as lactate dehydrogenase (LDH) levels and tumor volume measurement such as product size sum (SPD) as an indicator of tumor mass were compared between patients who were observed not to developed CRS or neurotoxicity compared to patients who were observed to develop CRS or NT. As shown in FIG. 17, patients with CRS or NT showed higher levels of pre-treatment patient parameters such as SPD (cm ² ) and LDH (U/L) levels; such levels were observed to correlate with CRS or NT with one-way statistical analysis. Other patient parameters that have been observed to be associated with CRS and NT include a shorter time to diagnosis (p=0.05 and p=0.09, for CRS and NT, respectively). Patient parameters that were not observed to be associated with CRS or NT included age (p=0.19 and p=0.54, respectively) and prior number of treatments (p=0.67 and p=0.59, respectively), disease stage 0-2 versus 3-4 (p=0.79, p=0.51), and patient body weight (p=0.35 and p=0.44, respectively).

[0896] FIG. 18A shows pre-treatment SPD and LDH levels among individual patients (dots; where shading of individual dots indicates whether or not individual patients showed degrees of neurotoxicity (left group) or degrees of CRS (right group). In FIG. 18A, dotted lines on the y and X delineate ≥50 cm SPD2 and ^LDH ≥500 U/L, respectively As shown in FIG. NT and CRS The estimated ratio of inequality for the development of CRS or NT in patients above or below the SPD and LDH levels indicated by the dotted lines in Fig. 18A, with 95% confidence intervals (CI) is shown in Fig. 18B and 18C. greater than 1 indicates an increased likelihood or likelihood of developing CRS or NT As shown, SPD of 50 ^cm2 or greater and LDH of 500 U/L or greater have been observed to be associated with an increased risk of developing CRS or NT.SPD of 50 ^cm2 or greater and an LDH of 500 U/L or higher have been observed to be associated with an approximately 8-fold increased risk of developing any grade of CRS and NT, and an SPD of less than 50 ^cm2 and an LDH of less than 500 U/L have shown a reduced risk of any grade CRS and NT. The results were consistent with the association of baseline patient parameters, including high tumor burden and inflammatory biomarkers, with CAR ⁺ T cell growth and increased CRS and neurotoxicity.

[0897] Various patient parameters before treatment (pre-LDC), including markers associated with tumor load (SPD), inflammatory cytokines, and other blood analytes, including LDH, ferritin, CRP, D-dimer, SAA-1, IL-6 , IL-10, IL-15, IL-16, TNF-α, IFN-γ, MIP-1α, and CXCL10 were compared for patients with and without a sustained response of 3 months with one-way statistical analysis. As shown in FIG. 19, some tumor mass markers, markers of inflammation or inflammatory cytokines, were observed to be lower in patients who showed long-term response (p value <0.05 for all parameters except SPD (p=0.1274)). Similar results were observed in patients receiving DL2 when analyzed individually. There was an inverse association of baseline patient parameters, including high tumor burden and inflammatory biomarkers, with long-term response. In some aspects, this inverse association may be due to higher growth and depletion of CAR ⁺ T cells.

[0898] Relationships between patient factors, clinical correlates, and blood analytes with the development of CRS and NT grades were assessed using statistical analysis based on one-way non-parametric analyses. Table E13 lists the results of the univariate analysis. In this assessment, age <40 years and no previous HSCT correlated with CRS or NT levels. Patients <40 years of age were not observed to have statistically different rates of higher tumor mass from older patients. Patients with an ECOG score of 2 did not have a statistically different higher tumor burden compared to patients with an ECOG score of 0-1. Patients without prior HSCT or double/triple hit or double expressor were not associated with CRS or NT.

Table E13. Univariate analysis of key subgroups CRS NT Variable, n (%) Any degree Degree 1/2 Degree 3/4 Any degree Degree 1/2 Degree 3/4 COMPLETE population (N=91) Age < 40 years (n=8) 5 (63) 4 (50) 1 (13) 3 (38) 0 3 (38) 40-64 years old (n=49) 19 (39) 19 (39) 0 9 (18) 5 (10) 4 (8) ≥ 65 years (n=34) 8 (24) 8 (24) 0 5 (15) 13) 4 (12) Pre-LD ECOG PS 0-1 (n=81) 28 (35) 27 (33) eleven) 15 (19) 4 (5) 11 (14) 2 (n=10) 4 (40) 4 (40) 0 2 (20) 2 (20) 0 Double/triple hit or double expressor Yes (n=30) 12 (40) 12 (40) 0 6 (20) 3 (10) 3 (10) No (n=22) 6 (27) 6 (27) 0 4 (18) 2 (9) 2 (9) Previous HSCTs Yes (n=39) 10 (26) 10 (26) 0 5 (13) 4 (10) 13) No (n=52) 22 (42) 21 (40) 12) 12 (23) 2(4) 10 (19)

K. Peak blood analytes, response and toxicity

[0899] Peak plasma levels of blood analytes after treatment, including cytokines and inflammatory markers such as CRP, Serum Amyloid A1 (SAA-1), IL-2, IL-6, IL-10, IL-15, TNF-α, MIP-1α, MIP-1β, MCP-1, CXCL10, and Chemokine Motif Ligand C-C 13 (CCL13) were compared for patients with cytokine release syndrome (CRS) or neurotoxicity (NT) grades 1-4 with patients who were observed to did not have CRS or NT. As shown in FIG. 20A (CRS; CRS grades 0, n=51; CRS grades 1-4, n=28) and FIG. 20B (NT; NT grade 0, n=63; NT grade 1-4, n=16), higher peak plasma cytokine levels and levels of inflammatory markers have been observed to be associated with CRS and NT (Wilcoxon P values <0.001 for absence of CRS versus any CRS and for absence of NT versus any NT other than IL-15 (P=0.05 and 0.006, respectively)).

[0900] Peak plasma levels of blood analytes, including cytokines and inflammatory markers such as CRP, SAA-1, IL-5, IL-6, IL-7, IL-8, IL-15, Lymphotoxin alpha (LT-α) , TNF-α, IFN-γ, MIP-1α, MIP-1β, MCP-1, CXCL10, and transforming growth factor beta (TGF-β), were evaluated for patients with best complete response (BOR), complete response (CR), or partial response (PR) (N=57) compared with levels in patients with stable disease (SD) or progressive disease (PD) (N=17); or for patients with 3-month SD or PD (SD/PD) (N=31) compared with patients who showed CR/PR at 3 months (N=35). As shown in FIG. 21A (Best Overall Response (BOR)) and FIG. 21B (month 3 response), lower peak plasma levels of cytokines and levels of inflammatory markers were observed to be associated with better BOR and response at month 3 (Wilcoxon P-value <0.05 without diversity regulation).

[0901] In this study, administration of an anti-CD19 CAR ⁺ cell composition was administered to patients with relapsed/refractory aggressive non-Hodgkin's lymphoma (NHL) having high-risk disease features. Observed responses including sustained responses including 81% RRR, 63% CR at DL2, with 80% of patients with CR at 3 months remaining at CR at 6 months at all dose levels, mean DOR of patients treated at all dose levels of 9. 2 months, with the average CR duration not reached at this point in the analysis in this example. The results were also consistent with manageable levels of toxicity and a favorable safety profile, which in some embodiments may be consistent with outpatient administration. Low rates of severe CRS (1%) and severe neurotoxicity (12%) were observed with few events after the first 72 hours. The results were consistent with the feasibility of outpatient administration.

[0902] Pharmacokinetic evaluations showed that higher growth of CAR ⁺ T cells was generally associated with an increased frequency of CRS and NT. Patients receiving DL2 showed a higher exposure to CAR T compared to patients receiving DL2, which, in general, corresponded to an increased duration of response without an increased level of toxicity. In some aspects, pre-treatment patient factors such as pre-LDC, including homeostatic and inflammatory cytokines and tumor burden, have been observed to be associated with and/or stimulate very high growth and toxicity. Injected CAR ⁺ T cells were shown to grow in the blood and bone marrow of all patients, with variability among patients and between types of disease. Injected CAR ⁺ T cells also showed long-term persistence, with 75% (9/12) of evaluable patients having detectable CAR T cells at 12 months. CAR T cells and B cell aplasia were observed to still be present at the time of relapse (11/12 and 12/12 patients, respectively), suggesting that tumors may evade CAR T cells and that combined strategies may be effective to prevent recurrence or increase, stimulate or enhance depleted CAR T cells. Overall, there was a trend towards a higher response with higher growth with variability among patients, suggesting that other patient factors and/or disease characteristics, such as tumor burden, may contribute to determining response.

Example 4: Characteristics of the T cell therapeutic composition for administration and method for preparing the composition

[0903] Examples of therapeutic compositions of T cells containing autologous T cells expressing a chimeric antigen receptor (CAR) specific for CD19, used for administration in Examples 1 and 2 above, were evaluated for more than a hundred phenotypic, functional and health-related trait cells using flow cytometry and in vitro tests. Therapeutic cell compositions generated for patients enrolled in a clinical trial evaluating anti-CD19 CAR-T cell therapy for the treatment of relapsed/refractory B-cell non-Hodgkin's lymphoma (N=63; main cohort) were investigated. Cells were evaluated before and after development for various traits. Examples of characteristics to be assessed are presented in Table E14. The memory and cellular health phenotypes of CAR T cells were examined using flow cytometry. The functionality of T cells was assessed using in vitro antigen-specific bioassays. Characterization and release testing was performed on therapeutic compositions of T cells that underwent a representative number of freeze-thaw cycles.

Table E14: Representative Traits Characteristics Measured in Therapeutic Cell Compositions Containing Anti-CD19 CAR T Cells Cell composition characteristic class representative trait cell health Viability
Active intracellular caspase 3
Annexin V memory phenotype CCR7 (C-C chemokine type 7 receptor) cell function Inflammatory cytokines such as TNF-α
(tumor necrosis factor α)

[0904] To obtain cell compositions for administration, autogenous cells were isolated from patients via leukapheresis. Leukapheresis samples were subjected to a process to generate CAR-expressing cells. The process included cell washing with automated washing and immunoaffinity selection to purify CD4 ⁺ and CD8 ⁺ T cells, resulting in two compositions enriched in CD8 ⁺ (in which an average of 99%, Inter Quartile Range (IQR) 98-100% , cells were CD8 ⁺ ) and CD4 ⁺ (in which an average of 99%, IQR 99-100%, cells were CD4 ⁺ ) cells, respectively.

[0905] Cells enriched in CD4 ⁺ and CD8 ⁺ compositions were separately subjected to lentiviral transduction with a vector encoding an anti-CD19 CAR with a 41BB costimulatory region. The transduced populations were then separately incubated in the presence of cell growth stimulating reagents. Grown CD8 ⁺ and CD4 ⁺ cells were composed and kept by freezing separately and stored until administration. To reduce variation between batches and/or compositions of cells derived from different patients, such as those having different patient characteristics, in parameters representative of cell health, cells were run at constant volumes from batch to batch. The cell products showed a close range of viable cell concentrations (based on evaluation of cell compositions for one patient cohort, CD8 ⁺ : mean 31x10 ⁶ cells/ml, IQR 28-40x10 ⁶ cells/ml, N=38; CD4 ⁺ : mean 35x10 ⁶ cells/ml ml, IQR 31-40x10 ⁶ , N=36).

[0906] As shown in FIG. 26 and presented in Table E15, automated T cell purification resulted in pure populations of CD8 ⁺ and CD4 ⁺ T cells. This strategy reduced the chance of transducing non-T cells and resulted in high purity of T cells in the therapeutic cell composition, regardless of growth duration.

Table E15: Purity of T cells (% of total leukocytes) by method steps Leukapheresis post cleaning Mid process The composition of the medicinal product CD4 frequency ⁺ T cells Median 17.6 95.3 98.9 99.2 IQR 11.7-24.7 92.1-98.5 98.1-99.2 98.7-99.6 CD8 frequency ⁺ T cells Median 19.2 96.0 98.9 99.3 IQR 13.2-30.9 93.4-98.6 98.0-99.2 98.4-99.7

[0907] At the injection site of the composition, the cells were thawed and administered separately, according to the target volume of each composition corresponding to the number of CD8 ⁺ CAR ⁺ and CD4 ⁺ CAR ⁺ cells at an appropriate dose (such as for DL1 containing 5x10 ⁷ total CAR-expressing T cells (2.5x10 ⁷ each of CAR-expressing CD4 ⁺ and CAR-expressing CD8 ⁺ cells), or DL2 containing 1x10 ⁸ total CAR-expressing T cells (5x10 ⁷ each of CAR-expressing CD4 ⁺ and CAR-expressing CD8 ⁺ cells)).

[0908] During the clinical trial, there was a process change from high volume formulations to low volume formulations. After the change, the composition of therapeutic cells was in a constant low volume with a tightly controlled range of viable cell concentrations. In some cases, a low volume formulation has been used instead of a high volume formulation. Parameters representative of the health of CAR-expressing T cells in administration formulations, such as by measurement, post-thaw, viability, cell surface Annexin V expression, and levels of active intracellular Caspase 3, were assessed in high-volume and low-volume cell formulations. .

[0909] Changing the process from high volume formulations to low volume formulations resulted in increased process reliability and reduced variability in cell health traits. The values for the concentration, percentage of viable cells, and percentage of active caspase 3 negative cells among CD4 ⁺ and CD8 ⁺ T cells of individual cell compositions are shown in FIG. 27A-27C and in Table E16. The mean percentage of cells expressing Annexin V was 11% (IQR 9-18%; N=33) of CD8 ⁺ CAR ⁺ T cells and 10% (IQR 8-17%; N=31) of CD4 ⁺ CAR ⁺ T cells. . Caspase 3 expression was observed to be similar to that of Annexin V. The change to low volume formulations resulted in increased process reliability and reduced variability in cell health traits.

Table E16: Signs of cell health CD4 ⁺ CD8 ⁺ High composition volume Low composition volume High composition volume Low composition volume Cell concentration
x10 ⁶ cells/ml Median 17.1 37.2 12.1 30.8 IQR 15.9-19.3 31.7-40.4 10.9-15.3 28.5-38.0 Cell viability% Median 82.8 82.5 72.0 80.3 IQR 79.5-84.7 80.4-84.3 69.3-76.6 76.4-83.3 % Caspase-3 negative cells Median 82.8 82.5 IQR 79.5-84.7 80.4-84.3

[0910] The numbers of CAR ⁺ CD4 ⁺ and CAR ⁺ CD8 ⁺ T cells in the compositions for administration were precisely controlled. The number of cells actually administered to the exemplary group of patients was observed to be 8% or less than the target cell number for the administered dose:

2.4-2.7x10 ⁷ (target ±8%) CD4 ⁺ CAR ⁺ T cells and 2.4-2.7x10 ⁷ (target ± 8%) CD8 ⁺ CAR ⁺ T cells for patients injected with cells in DL1 (n=48)

4.6-5.1x10 ⁷ (target ±8%) CD4 ⁺ CAR ⁺ T cells or 4.6-5.1x10 ⁷ (target ± 8%) CD8 ⁺ CAR ⁺ T cells for patients injected with cells in DL2 (n=20).

[0911] The dose range administered was found to have low variability among different exemplary patient groups:

48-52 ×10 ⁶ CD3 ⁺ CAR ⁺ T cells in DL1 (n = 34)

96-101×10 ⁶ CD3 ⁺ CAR ⁺ T cells in DL2 (n = 29)

24-27 x 10 ⁶ CD4 ⁺ CAR ⁺ or CD8 ⁺ CAR ⁺ T cells in DL1 (n = 34)

· 46-51 x 10 ⁶ CD4 ⁺ CAR ⁺ or CD8 ⁺ CAR ⁺ T cells in DL2 (n = 29).

[0912] As shown in FIG. 28A, CAR-expressing T cell formulations administered to patients were observed to show high T cell purity and low batch to batch variation. In terms of, for example, process and product controls, therapeutic cell compositions containing CAR T cells have been observed to have low batch-to-lot variability in cell-specific T cell function.

[0913] In vitro accumulation of antigen-specific cytokines and intracellular cytokine staining (ICS) showed similar low batch-to-batch differences in cytokine production for a variety of cytokines (IL-2, TNF-α, and IFN-γ). In an exemplary ICS experiment, composition cells were stimulated with CD19, stained for cytokines, including TNF-α and surface proteins, including CC chemokine receptor type 7 (CCR7) as a memory phenotype marker, and analyzed by flow cytometry. The number of cells in the administration composition that were positive for cytokines or surface proteins was determined. FIG. 28B shows the number of CD4 ⁺ CAR ⁺ and CD8 ⁺ CAR ⁺ cells, CD4 ⁺ CAR ⁺ TNF-α+ and CD8 ⁺ CAR ⁺ TNF-α+ cells, CD4 ⁺ CAR ⁺ CCR7+ and CD8 ⁺ CAR ⁺ CCR7+ and CAR T compositions present -cells for introduction into DL1 and DL2. These results are presented in Table E17. The results show low variability in the number of CD4 ⁺ CAR ⁺ and CD8 ⁺ CAR ⁺ cells, CD4 ⁺ CAR ⁺ TNF-α+ and CD8 ⁺ CAR ⁺ TNF-α+ cells, CD4 ⁺ CAR ⁺ CCR7+ and CD8 ⁺ CAR ⁺ CCR7+ cells. For example, a dense range was observed for the number of cells positive for TNF-α production (n=61).

Table E17: Controlled dose, T cell phenotypes and cell-specific function (x 10 ⁶ cells) CD4 ⁺ CAR ⁺ CD8 ⁺ CAR ⁺ CD4 ⁺ CAR ⁺
TNF- α ⁺ CD8 ⁺ CAR ⁺
TNF- α ⁺ CD4 ⁺ CAR ⁺
CCR7 ⁺ CD8 ⁺ CAR ⁺
CCR7 ⁺ DL1 DL2 DL1 DL2 DL1 DL2 DL1 DL2 DL1 DL2 DL1 DL2 Median 25.1 50.1 25.0 50.0 23.0 47.4 19.9 41.7 11.1 15.8 8.9 15.0 IQR 24.7-25.3 49.6-50.0 24.8-25.2 49.6-50.4 21.3-23.5 46.6-48.5 18.3-21.8 39.9-46.0 5.5-14.1 11.0-26.0 3.0-14.8 8.6-25.3

[0914] A parameter indicative of the production of CAR ⁺ tumor necrosis factor alpha (TNFα) cells after CD19 stimulation showed a narrow range among different batches with a relative standard deviation (RSD) of 37% for CD4 ⁺ CAR ⁺ T cells (N=59 ) and 51% for CD8 ⁺ CAR ⁺ T cells (N=61).

[0915] The results were consistent with the observation that a composition containing an accurate and constant dose of CD4 ⁺ and CD8 ⁺ CAR T cells, conditions for controlling and optimizing the cultivation of CD4 ⁺ and CD8 ⁺ T cells, low variability in cytokine production and / or a constant composition and the volume of the composition to be administered can result in stable cell health in the composition. In embodiments of the invention, aspects of such a manufacturing and control process contribute to the low variability of features of such genetically engineered cell compositions using cells from, and generated for administration, many different patients. Such aspects in some aspects include the use of an accurate, constant flat dose of administered CD4 ⁺ and CD8 ⁺ cells among patients; conditions for controlling and optimizing CD4 ⁺ and CD8 ⁺ T cell culture, such as those that result in low batch variability between pharmaceutical product phenotypes (eg, CCR7) and in vitro function (eg, IL-2, TNF-α, and IFN production -γ after antigen stimulation), for example, among different patients; and the use of a constant composition and volume of the pharmaceutical product, which can result in or promote consistency among method-generated therapeutic cell compositions in terms of traits representative of cell health.

Example 5 Biomarker Evaluation Before and After Administration of Tumor Biopsies from Patients with Relapsed and Refractory Non-Hodgkin's Lymphoma (NHL) for Administration of Anti-CD19 CAR Expressing Cells

[0916] Expression of several biomarkers was assessed in tumor biopsies collected from patients before and/or after administration of CAR-expressing cells.

A. Tumor biopsy specimens

[0917] Tumor biopsies were collected from selected patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) (R/R) or mantle cell lymphoma (MCL) who were treated with therapeutic CAR ⁺ T cell compositions containing autologous T- cells expressing the CD19-specific chimeric antigen receptor (CAR) described in Examples 1 and 2 above, based on the time point evaluation in Example 1.A.2. Tumor biopsies were taken before CAR ⁺ T cell injection (before treatment) and 7-20 days after administration (after treatment). The results are described in this example for evaluation through time point in Example 1.A.2 in the ongoing study. The results of 43 biopsies (26 before treatment; 17 after blowing and 15 matched pairs) from 28 total patients (25 DLBCL and 3 µl) were examined.

B. Evaluation of Biomarkers, Response Results and Safety

[0918] Infiltration of CAR ⁺ T cells in tumor biopsies was quantified using in situ hybridization (ISH) probes specific for mRNA encoding anti-CD19 CAR. CAR ⁺ T cells, non-CAR T cells, and B cells were enumerated using multiplex immunofluorescence (IF) assays detecting a surrogate cell surface marker for CAR-expressing cells, CD4, CD8, CD19, CD20, CD73, FOXP3, CD163, IDO and PD-L1. Tumor biopsy sections were stained with hematoxylin and eosin (H-E) and evaluated for tissue quality and tumor identification. Immunofluorescence images were analyzed using image analysis software. Potential correlations with response outcomes were assessed using statistical analysis based on univariate t-analyses, and p-values were 2-tailed without taking into account the multiplicity of comparisons.

[0919] Patients were assessed for response and safety outcomes, including assessment of tumor burden at various time points after CAR ⁺ T cell administration, including at 3 months post-administration, and determining whether the patient had progressive disease (PD), stable disease (SD), partial response (PR), or complete response (CR). Safety outcomes assessed included neurotoxicity (neurological complications including symptoms of confusion, aphasia, encephalopathy, myoclonic seizures, convulsions, lethargy, and/or mental status changes) classified on a scale of 1-5 according to the National Cancer Institute-Common Toxicity Criteria (CTCAE) , version 4.03 (NCI-CTCAE v4.03).

C. Results

[0920] The observed objective response rate (ORR; including CR and PR) was 71% (20/28) in patients for whom biopsies were evaluated. Grades 1, 2 CRS was observed in 36% (10/28; grades 1, 2) of patients for whom biopsies were evaluated, and Grades 2-4 NT were observed in 18% (5/28) of patients for whom biopsies were evaluated.

[0921] Pre-treatment tumor biopsies were observed to contain variable cell compositions: tumor cells (median: 77%; range 5-96%), CD4 ⁺ cells (0.90%; 0.02-15%) and CD8 ⁺ cells (1.5%; 0-23%). The results showed that patients with CR or PR at 3 months after CAR ⁺ T cell administration had a higher percentage of endogenous CD4 ⁺ cells in tumors before treatment compared with PD (CR, median PR: 7.9%; median PD: 0 .38%, p<0.0001). The percentages of CD8 ⁺ cells in pre-treatment tumors did not differ between 3-month response groups (CR, median PR: 1.9%; median PD: 0.47%; p=0.6496).

[0922] In post-treatment biopsies, CAR ⁺ T cells were observed to infiltrate the tumor and accounted for up to 22% of the cells in the biopsy sample. The rate of tumor infiltration in post-treatment specimens (7-20 days after injection) was observed to be higher in patients who continued to achieve CR (median: 3.9%) or PR (median: 1.1%) compared with patients who continued to achieve best complete response (BOR) SD or PD (median: 0.51%). Although both CD4 ⁺ and CD8 ⁺ CAR T cells were observed to infiltrate the tumor area at time point post-treatment (7-20 days post-treatment), patients who continued to achieve CR were observed to have more high ratio of CD8 ⁺ CAR ⁺ T cells to CD4 ⁺ CAR ⁺ T cells at this post-treatment time point compared with patients who continued to achieve BOR SD or PD (median CR: 0.83; SD, median PD: 0.14 ; p=0.0097).

[0923] Comparison of relevant pre- and post-treatment biopsies from individual patients showed that patients ultimately achieving BOR CR or PR tended to have greater increases in post-treatment CD8 ⁺ cells (CAR ⁺ T and non-CAR T) in tumors along compared with patients eventually achieving BOR SD or PD (CR, median change in PR: +5.3%; SD, median change in PD: +0.06%; p=0.1225).

[0924] Expression of immunosuppressive factors including CD73, FOXP3, CD163, IDO, and PD-L1 varied among patients before treatment (CD73 (median: 1.5%; range 0-42%), FOXP3 (0.10%; 0 -1.5%), IDO (0.06%; 0-11%), CD163 (1.2%; 0-24%) and PD-L1 (0.16%; 0-56%)) and after treatment (CD73 (1.6%; 0-53%), FOXP3 (0.09%; 0-4.3%), IDO (0.28%; 0-15%), CD163 (3.6%; 0-22%) and PD-L1 (3.3%; 0-65%)). The post-treatment increase in CD8 ⁺ cells in matched biopsies has been observed to be associated with post-treatment increases in IDO (R ² =0.64) and PD-L1 (R ² =0.61) expression. This result is consistent with the conclusion that infiltration of CD8 ⁺ CAR ⁺ cells at the time of assessment may indicate a potential likelihood of reaching a response rate or duration of response, and that the presence and/or activity of such cells may lead to upregulation of TME factors.

D. Conclusion

[0925] A sustained response at month 3 following CAR ⁺ T cell administration has been observed to be associated with higher levels of CD4 ⁺ cells in pre-treatment tumors. In tumor cells following CAR ⁺ treatment, both CD4 ⁺ and CD8 ⁺ T cells have been observed to infiltrate the tumor and adjacent tissue. ORR has been associated with an increase in CAR ⁺ T cells in tumor biopsies. The increase in CD8 ⁺ levels in the tumor biopsy after treatment compared with CD8 ⁺ levels in the tumor biopsy before treatment was associated with increased expression of IDO and PD-L1. In some embodiments, therapies targeting these pathways, such as administered during or after administration of CAR-T cells, may enhance one or more therapeutic outcomes or their duration following administration of CAR ⁺ T cells.

Example 6: Further evaluation of response and safety outcomes in patients with relapsed and refractory non-Hodgkin's lymphoma (NHL) after administration of anti-CD19 CAR-expressing cells

[0926] The results of response and safety were evaluated in patients at a subsequent time point in the clinical study described in Examples 1 and 3 above.

A. Patients and Treatment

[0927] The analysis at this point in time presented in this example is based on an assessment of a total of 102 patients in the entire cohort (73 in the main cohort) who were injected with anti-CD19CAR-expressing cells. The full cohort included patients who had DLBCL (DLBCL, de novo NOS and transformed from follicular lymphoma; high-grade B-cell lymphoma (double/triple hit); DLBCL transformed from CLL or MZL; PMBCL; and FL3B, ECOG 0 -2, after 2 lines of therapy; the main cohort for analysis included patients with DLBCL, NOS and transformed from follicular lymphoma (tFL) or high-grade B-cell lymphoma (double/triple hit) and with Eastern Cooperative Oncology Group Performance Status ( ECOG PS) 0 or 1. Approximately 90% of treated patients in the general and core cohort had at least one high-risk disease predictive of a short mean overall survival (OS) of 3-6 months (see Crump et al., Blood (2017) 130:1800-1808 and Van de Neste et al., Bone Marrow Transplant (2016) 51(1):51-7), such as double/triple hit expressors, primary refractory disease refractory to 2 or more lines of therapy, never achieved CR, never received autologous stem cell transplantation (ASCT) or PS ECOG 2.

[0928] At this point in time, a total of 134 patients were subjected to leukapheresis, of which 2 had compositions not available. The product was available to 99% of apheresis patients (132/134) in the DLBCL cohort. Of the 18 patients for whom products were available, 5 withdrew from the study and 13 developed progressive disease or died. A total of 114 patients were injected with anti-CD19 CAR-expressing cells, of which 12 received inappropriate anti-CD19 CAR-expressing cells (compositions did not necessarily meet some specifications, but were considered safe to administer). Patients received DL1 (n=45), a double dose of DL1 (n=6), or DL2 (n=51). Seven (7) patients with mantle cell lymphoma (MCL) were injected with CAR ⁺ cells in DL1. At this point in time, eight (8) patients were treated on an outpatient basis.

[0929] Demographic and baseline characteristics of patients of the full and main contingent at the time point are presented in Table E18.

Table E18. Patient characteristics: DLBCL population Characteristic FULL (n=102) BASIC (n=73) Average age (range), years 61 (20-82) 60 (20-82) ≥0 (20-82) (range) 37 (36) 24 (33) Subtype B-NHL, n (%) DLBCL, NOS de novo 63 (62) 53 (73) Transformed from FL (tFL) 23 (23) 20 (27) Transformed from MZL (tMZL) /CLL (tCLL) 6(6)/6(6) 0 Follicular Grade 3B/PMBCL 1(1)/3(3) 0 Molecular Subtype, n (%) Double/triple hit ^a 19 (19) 16 (22) Patient characteristics, n (%)L ECOG PS 0-1 93 (91) 73 (100) IPI 3-5 43 (42) 26 (36) CNS involvement 2(2) eleven) Chemorefractive ^b 71 (70) 49 (67) Previous lines of therapy, median (range) 3 (1-8) 3 (2-8) Didn't reach CR 49 (48) 36 (49) Any HSCT 41 (40) 28 (38) Previous autologous 38 (37) 28 (38) Previous allogeneic 5 (5) 0 HSCT, hematopoietic stem cell transplant. IPI, International Predictive Index; SD, stable disease; WHO, World Health Organization.
^a At trial initiation, included in DLBCL, NOS histology; based on new WHO criteria (Swerdlow et al., (2016) Blood 127(20):2375-2390), now considered to be well-differentiated B-cell lymphoma, with myc and bcl2 and/or bcl6 permutations with DLBCL histology (double/triple hit ).
^b SD or PD for duration of chemotherapy regimen or relapse < 12 months after autologous SCT.

B. Safety and response outcomes after treatment

[0930] Table E19 shows the security result of the main and full contingent. As shown, no deaths from CRS or NT were observed. In the full cohort, the median time to onset of CRS was 5 days (range, 2-12 days) and time to NT was 10 days (range, 3-23 days). In the full cohort, 17% (n=17) received tocilizumab and 21% (n=21) received corticosteroids as an intervention to treat toxicity. In the core population, no increase in CRS or NT was observed in DL2 compared to DL1.

Table E19. Safety Outcomes After CAR Introduction ⁺ cells FULL BASIC All dose levels
n=102 All dose levels ^a n=73 DL1S
n=33 DL2S
n=37 CRS, n (%) Any degree 38 (37) 27 (37) 14 (42) 11 (30) Degree 1/2 37 (36) 26 (36) 13 (39) 11 (30) Grade 3/4 (sCRS) eleven) eleven) 13) 0 Neurotoxicity, n (%) Any degree 23 (23) 18 (25) 8 (24) 9 (24) Degree 1/2 10 (10) 7(10) 13) 6 (16) Grade 3/4 (sNT) 13 (13) 11 (15) 7 (21) 3 (8) Any, n (%) CRS or NT 44 (43) 32 (44) 15 (45) 15 (41) sCRS or sNT 13 (13) 11 (15) 7 (21) 3 (8)

^a Three patients treated with DL1D (dose level 1, two-dose regimen) with similar results.

[0931] FIG. 29 depicts the percentage of patients in the full cohort at this time point (n=102) who were observed to experience laboratory abnormalities and treatment-related adverse events (TEAEs) (data for 6 patients with MCL treated with the respective product in DL1 from at least 28 days of follow-up are not included; TEAEs and laboratory abnormalities present in 20% or more of patients are indicated).

[0932] As shown in Table E20, high response rates were observed in patients with relapsed or refractory (R/R) DLBCL. The results were consistent with the dose-response effect on treatment outcome in the main population. Patients with a tumor burden above the threshold (as indicated by a sum of product size (SPD) tumor volume measurement of greater than 50 cm ² ) were similarly divided between patients receiving DL1 and DL2 (approximately 1/3 of the patients in each group).

Table E20. Response after CAR introduction ⁺ cells FULL BASIC All dose levels (n=102) All dose levels ^a (n=73) DL1S (n=33) DL2S (n=37) ORR (95% CI), % 75(65-83) 80 (68-88) 79(61-91) 78(62-90) CR (95% CI), % 55 (45-65) 59 (47-70) 55 (36-72) 62(45-78) 3-mo ORR (95% CI), % 51 (41-61) 59 (47-70) 52(34-69) 65 (48-80) 3-mo CR (95% CI), % 38 (29-48) 45 (34-57) 36 (20-55) 51 (34-68) 6-mo ORR (95% CI), % 40 (31-50) 47 (35-59) 42 (26-61) 49 (32-66) 6-mo CR (95% CI), % 34 (25-44) 41 (30-53) 33 (18-52) 46 (30-63)

^a Three patients treated with DL1D (dose level 1, two-dose regimen) with similar results.

[0933] The six-month objective response rate (ORR) among various patient subgroups in the high-risk DLBCL subgroup, which included all DLBCL patients treated at all dose levels in the core cohort, is shown in FIG. 30. The results showed a high long-term RRR in the high-risk subgroup of DLBCL to anti-CD19 CAR ⁺ T cell administration.

[0934] Results for response time (DOR, with a median follow-up of 8 months) and overall survival (grouped by best overall response (non-responders, CR/PR, CR and/or PR) with a median follow-up of 12 months) are shown for the entire cohort and the main contingent of patients in FIG. 31A-31D. The results showed that in the core cohort, 88% of patients with CR at 3 months continued to show CR at 6 months, and 93% of patients who showed CR at 6 months continued to show prospect of a long-term response.

[0935] The results were consistent with the observation that administration of an anti-CD19 CAR+ cell composition that contains a precise and stable dose of CD4 ⁺ and CD8 ⁺ CAR+ T cells results in a sustained response in patients with aggressive R/R NHL with poor prognosis and/ or heavy pretreatment. The results showed a favorable sustained response rate in the core cohort with levels of 49% ORR and 46% CR at 6 months, and 93% of patients (at all dose levels) in CR at 6 months remained responsive at this time point. The results were also consistent with controlled toxicity and a favorable safety profile, including low rates of severe CRS (1%) and severe neurotoxicity (13%), which, in some aspects, allows for outpatient administration.

[0936] The present invention is not intended to be limited in scope to the particular disclosed embodiments given, for example, to illustrate various aspects of the invention. Various modifications to the described compositions and methods will be apparent from the foregoing description and disclosure. Such modifications may be practiced without departing from the true scope and spirit of the disclosure and are intended to be included within the scope of the present disclosure.

SEQUENCES

SEQID NO. SUBSEQUENCE DESCRIPTION 1 ESKYGPPCPPCP spacer (IgG4 hinge) (aa)
Homo sapiens 2 GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCT spacer (IgG4 hinge) (nt)
homo sapiens 3 ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Hinge-CH3 spacer
Homo sapiens 4 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Hinge-CH2-CH3 spacer
Homo sapiens 5 RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLC EVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH IgD-hinge-Fc
Homo sapiens 6 LEGGGEGRGSLTCGDVEENPGPR T2A
artificial 7 MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGC WGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM tEGFR
artificial 8 FWVLVVVGGVLACYSLLVTVAFIFWV CD28 (amino acids 153-179 Accession No. P10747)
Homo sapiens 9 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV CD28 (amino acids 114-179 Accession No. P10747)
Homo sapiens 10 RSKRSRLLHSDYMNMTPRRPPGPTRKHYQPYAPPRDFAAYRS CD28 (amino acids 180-220 P10747)
Homo sapiens eleven RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 (LL-GG)
Homo sapiens 12 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 4-1BB (amino acids 214-255 Q07011.1)
Homo sapiens 13 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3 zeta
Homo sapiens 14 RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3 zeta
Homo sapiens 15 RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3 zeta
Homo sapiens 16 RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDK CNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM tEGFR
artificial 17 EGRGSLLTCGDVEENPGP T2A artificial 18 GSGATNFSLLKQAGDVEENPGP P2A 19 ATNFSLLKQAGDVEENPGP P2A 20 QCTNYALLKLAGDVESNPGP E2A 21 VKQTLNFDLLKLAGDVESNPGP F2A 22 PGGG-(SGGGG)5-P- where P is proline, G is glycine, and S is serine linker 23 GSADDAKKDAAKKDGKS Linker 24 GSTSGSGKPGSGEGSTKG Linker 25 gacatccagatgacccagaccacctccagcctgagcgccagcctgggcgaccgggtgaccatcagctgccgggccagccaggacatcagcaagtacctgaactggtatcagcagaagcccgacggcaccgtcaagctgctgatctaccacaccagccggctgcacagcggcgtgcccagccggtttagcggcagcggctccggcaccgactac agcctgaccatctccaacctggaacaggaagatatcgccacctacttttgccagcagggcaacacactgccctacacctttggcggcggaacaaagctggaaatcaccggcagcacctccggcagcggcaagcctggcagcggcgagggcagcaccaagggcgaggtgaagctgcaggaaagcggccctggcctggtggcccccagccagagcct gagcgtgacctgcaccgtgagcggcgtgagcctgcccgactacggcgtgagctggatccggcagccccccaggaagggcctggaatggctgggcgtgatctggggcagcgaccacctactacaacagcgccctgaagagccggctgaccatcatcaaggacaacagcaagagccaggtgttcctgaagatgaacagcctgcagaccg acgacccgccatctactactactgcgccaagcactactactacggcggcagctacgccatggactactggggccagggcaccagcgtgaccgtgagcagc scFv encoding sequence 26 _{X1PPX2P _}
X ₁ is glycine, cysteine or arginine
X ₂ is cysteine or threonine Hinge 27 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Hinge 28 Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Hinge 29 ELKTPLGDTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP Hinge thirty Glu Ser Lys Tyr Gly Pro Cys Pro Ser Cys Pro Hinge 31 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Hinge 32 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Hinge 33 Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Hinge 34 Glu Val Val Val Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Hinge 35 RASQDISKYLN FMC63 CDR L1 36 SRLHSGV FMC63 CDR L2 37 GNTLPYTFG FMC63 CDR L3 38 DYGVS FMC63 CDR H1 39 VIWGSETTYYNSALKS FMC63 CDR H2 40 YAMDYWG FMC63 CDR H3 41 EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS FMC63 VH 42 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT FMC63 VL 43 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTI IKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS FMC63 scFv 44 KASQNVGTNVA SJ25C1 CDR L1 45 SATYRNS SJ25C1 CDR L2 46 QQYNRYPYT SJ25C1 CDR L3 47 SYWMN SJ25C1 CDR H1 48 QIYPGDGDTNYNGKFKG SJ25C1 CDR H2 49 KTISSVVDFYFDY SJ25C1 CDR H3 50 EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTVTVSS SJ25C1 VH 51 DIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKR SJ25C1 VL 52 GGGGSGGGGSGGGGS Linker 53 EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQ SPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKR SJ25C1 scFv 54 HYYYGGSYAMDY FMC63HC-CDR3 55 HTSRLHS FMC63 LC-CDR2 56 QQGNTLPYT FMC63 LC-CDR3 57 ACACGGCCTCGTGTATTACTGT IGH primer 58 ACCTGAGGAGACGGTGACC IGH primer

--->

SEQUENCE LIST

<110> JUNO THERAPEUTICS, INC.

ALBERTSON, Tina

<120> ARTICLES AND METHODS FOR THE TREATMENT USING ADOPTIONAL CELL THERAPY

<130> 735042012140

<140> Not yet assigned

<141> At the same time

<150> 62/514.774

<151> 2017-06-02

<150> 62/515,530

<151> 2017-06-05

<150> 62/521.366

<151> 2017-06-16

<150> 62/527,000

<151> 2017-06-29

<150> 62/549.938

<151> 2017-08-24

<150> 62/580.425

<151> 2017-11-01

<150> 62/593.871

<151> 2017-12-01

<150> 62/596.764

<151> 2017-12-08

<150> 62/614.957

<151> 2018-01-08

<160> 58

<170> FastSEQ for Windows Version 4.0

<210> 1

<211> 12

<212> protein

<213> Homo sapiens

<220>

<223> Spacer (IgG4 hinge)

<400> 1

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

1 5 10

<210> 2

<211> 36

<212> DNA

<213> Homo sapiens

<220>

<223> Spacer (IgG4 hinge)

<400> 2

gaatctaagt acggaccgcc ctgcccccct tgccct 36

<210> 3

<211> 119

<212> protein

<213> Homo sapiens

<220>

<223> Hinge-CH3 Spacer

<400> 3

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg

1 5 10 15

Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys

20 25 30

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

35 40 45

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

50 55 60

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

65 70 75 80

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

85 90 95

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

100 105 110

Leu Ser Leu Ser Leu Gly Lys

115

<210> 4

<211> 229

<212> protein

<213> Homo sapiens

<220>

<223> Hinge-CH2-CH3 Spacer

<400> 4

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys

225

<210> 5

<211> 282

<212> protein

<213> Homo sapiens

<220>

<223> IgD-hinge-Fc

<400> 5

Arg Trp Pro Glu Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala

1 5 10 15

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

20 25 30

Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys

35 40 45

Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro

50 55 60

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

65 70 75 80

Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val Val Gly

85 90 95

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

100 105 110

Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser Asn Gly

115 120 125

Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu Trp Asn

130 135 140

Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu Pro Pro

145 150 155 160

Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln Ala Pro Val Lys

165 170 175

Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu Ala Ala Ser

180 185 190

Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn Ile Leu Leu

195 200 205

Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly Phe Ala Pro

210 215 220

Ala Arg Pro Pro Pro Gln Pro Gly Ser Thr Thr Phe Trp Ala Trp Ser

225 230 235 240

Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr Tyr Thr

245 250 255

Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala Ser Arg

260 265 270

Ser Leu Glu Val Ser Tyr Val Thr Asp His

275 280

<210> 6

<211> 24

<212> protein

<213> Artificial sequence

<220>

<223> T2A

<400> 6

Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp

1 5 10 15

Val Glu Glu Asn Pro Gly Pro Arg

20

<210> 7

<211> 357

<212> protein

<213> Artificial sequence

<220>

<223>tEGFR

<400> 7

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

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

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

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

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu

195 200 205

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

210 215 220

Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu

225 230 235 240

Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met

245 250 255

Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala

260 265 270

His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val

275 280 285

Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His

290 295 300

Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro

305 310 315 320

Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala

325 330 335

Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly

340 345 350

Ile Gly Leu Phe Met

355

<210> 8

<211> 27

<212> protein

<213> Homo sapiens

<220>

<223>CD28

<300>

<308> UniProt P10747

<309> 1989-07-01

<400> 8

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 9

<211> 66

<212> protein

<213> Homo sapiens

<220>

<223>CD28

<300>

<308> UniProt P10747

<309> 1989-07-01

<400> 9

Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn

1 5 10 15

Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu

20 25 30

Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly

35 40 45

Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe

50 55 60

Trp Val

65

<210> 10

<211> 41

<212> protein

<213> Homo sapiens

<220>

<223>CD28

<300>

<308> UniProt P10747

<309> 1989-07-01

<400> 10

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 11

<211> 41

<212> protein

<213> Homo sapiens

<220>

<223>CD28

<400> 11

Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 12

<211> 42

<212> protein

<213> Homo sapiens

<220>

<223> 4-1BB

<300>

<308> UniProt Q07011.1

<309> 1995-02-01

<400> 12

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 13

<211> 112

<212> protein

<213> Homo sapiens

<220>

<223> CD3 zeta

<400> 13

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

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

50 55 60

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

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

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

100 105 110

<210> 14

<211> 112

<212> protein

<213> Homo sapiens

<220>

<223> CD3 zeta

<400> 14

Arg Val Lys Phe Ser Arg Ser Ala Glu Pro Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

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

50 55 60

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

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

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

100 105 110

<210> 15

<211> 112

<212> protein

<213> Homo sapiens

<220>

<223> CD3 zeta

<400> 15

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

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

50 55 60

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

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

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

100 105 110

<210> 16

<211> 335

<212> protein

<213> Artificial sequence

<220>

<223>tEGFR

<400> 16

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

1 5 10 15

Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

<210> 17

<211> 18

<212> protein

<213> Artificial sequence

<220>

<223> T2A

<400> 17

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

1 5 10 15

GlyPro

<210> 18

<211> 22

<212> protein

<213> Artificial sequence

<220>

<223> P2A

<400> 18

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

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210> 19

<211> 19

<212> protein

<213> Artificial sequence

<220>

<223> P2A

<400> 19

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

1 5 10 15

Pro Gly Pro

<210> 20

<211> 20

<212> protein

<213> Artificial sequence

<220>

<223>E2A

<400> 20

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

1 5 10 15

Asn Pro Gly Pro

20

<210> 21

<211> 22

<212> protein

<213> Artificial sequence

<220>

<223> F2A

<400> 21

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

1 5 10 15

Glu Ser Asn Pro Gly Pro

20

<210> 22

<211> 9

<212> protein

<213> Artificial sequence

<220>

<223> Linker

<220>

<221> REPEAT

<222> (5)...(9)

<223> SGGGG is REPEAT 5 times

<400> 22

Pro Gly Gly Gly Ser Gly Gly Gly Gly

15

<210> 23

<211> 17

<212> protein

<213> Artificial sequence

<220>

<223> Linker

<400> 23

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

1 5 10 15

Ser

<210> 24

<211> 18

<212> protein

<213> Artificial sequence

<220>

<223> Linker

<400> 24

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr

1 5 10 15

Lys Gly

<210> 25

<211> 735

<212> DNA

<213> Artificial sequence

<220>

<223>scFv

<400> 25

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> 26

<211> 5

<212> protein

<213> Artificial sequence

<220>

<223> hinge

<220>

<221> OPTION

<222> (1)...(1)

<223> Xaa1 = glycine, cysteine or arginine

<220>

<221> OPTION

<222> (4)...(4)

<223> Xaa4 = cysteine or threonine

<400> 26

Xaa Pro Pro Xaa Pro

15

<210> 27

<211> 15

<212> protein

<213> Artificial sequence

<220>

<223> hinge

<400> 27

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

1 5 10 15

<210> 28

<211> 12

<212> protein

<213> Artificial sequence

<220>

<223> hinge

<400> 28

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

1 5 10

<210> 29

<211> 61

<212> protein

<213> Artificial sequence

<220>

<223> hinge

<400> 29

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

20 25 30

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

35 40 45

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

50 55 60

<210> 30

<211> 12

<212> protein

<213> Artificial sequence

<220>

<223> hinge

<400> 30

Glu Ser Lys Tyr Gly Pro Cys Pro Ser Cys Pro

1 5 10

<210> 31

<211> 12

<212> protein

<213> Artificial sequence

<220>

<223> hinge

<400> 31

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

1 5 10

<210> 32

<211> 9

<212> protein

<213> Artificial sequence

<220>

<223> hinge

<400> 32

Tyr Gly Pro Pro Cys Pro Pro Cys Pro

15

<210> 33

<211> 10

<212> protein

<213> Artificial sequence

<220>

<223> hinge

<400> 33

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

1 5 10

<210> 34

<211> 14

<212> protein

<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> protein

<213> Artificial sequence

<220>

<223> FMC63 CDR L1

<400> 35

Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn

1 5 10

<210> 36

<211> 7

<212> protein

<213> Artificial sequence

<220>

<223> FMC63 CDR L2

<400> 36

Ser Arg Leu His Ser Gly Val

15

<210> 37

<211> 9

<212> protein

<213> Artificial sequence

<220>

<223> FMC63 CDR L3

<400> 37

Gly Asn Thr Leu Pro Tyr Thr Phe Gly

15

<210> 38

<211> 5

<212> protein

<213> Artificial sequence

<220>

<223> FMC63 CDR H1

<400> 38

Asp Tyr Gly Val Ser

15

<210> 39

<211> 16

<212> protein

<213> Artificial sequence

<220>

<223> FMC63 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> protein

<213> Artificial sequence

<220>

<223> FMC63 CDR H3

<400> 40

Tyr Ala Met Asp Tyr Trp Gly

15

<210> 41

<211> 120

<212> protein

<213> Artificial sequence

<220>

<223> FMC63 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> protein

<213> Artificial sequence

<220>

<223> FMC63 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> protein

<213> Artificial sequence

<220>

<223> FMC63 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> protein

<213> Artificial sequence

<220>

<223> SJ25C1 CDR L1

<400> 44

Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala

1 5 10

<210> 45

<211> 7

<212> protein

<213> Artificial sequence

<220>

<223> SJ25C1 CDR L2

<400> 45

Ser Ala Thr Tyr Arg Asn Ser

15

<210> 46

<211> 9

<212> protein

<213> Artificial sequence

<220>

<223> SJ25C1 CDR L3

<400> 46

Gln Gln Tyr Asn Arg Tyr Pro Tyr Thr

15

<210> 47

<211> 5

<212> protein

<213> Artificial sequence

<220>

<223> SJ25C1 CDR H1

<400> 47

Ser Tyr Trp Met Asn

15

<210> 48

<211> 17

<212> protein

<213> Artificial sequence

<220>

<223> SJ25C1 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> protein

<213> Artificial sequence

<220>

<223> SJ25C1 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> protein

<213> Artificial sequence

<220>

<223> SJ25C1 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> protein

<213> Artificial sequence

<220>

<223> SJ25C1 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> protein

<213> Artificial sequence

<220>

<223> Linker

<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> protein

<213> Artificial sequence

<220>

<223> SJ25C1 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 Glyn 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> protein

<213> Artificial sequence

<220>

<223> FMC63 CDR H3

<400> 54

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

1 5 10

<210> 55

<211> 7

<212> protein

<213> Artificial sequence

<220>

<223> FMC63 CDR L2

<400> 55

His Thr Ser Arg Leu His Ser

15

<210> 56

<211> 9

<212> protein

<213> Artificial sequence

<220>

<223> FMC63 CDR L3

<400> 56

Gln Gln Gly Asn Thr Leu Pro Tyr Thr

15

<210> 57

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> IGH primer

<400> 57

acacggcctc gtgtattact gt 22

<210> 58

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> IGH primer

<400> 58

acctgaggag acggtgacc 19

<---

Claims (87)

1. A method of treating a patient with or suspected of having a CD19-expressing B-cell malignant tumor, comprising administering to the patient a dose of CD4 ⁺ and CD8 ⁺ T cells, each of the CD4 ⁺ and CD8 ⁺ T cells, individually, includes a chimeric antigenic receptor (CAR), which binds to CD19, and: (a) CAR contained in CD4+ T cells and in CD8+ T cells is the same; (b) administration comprises administering a plurality of individual compositions, wherein the plurality of individual compositions comprises a first composition comprising one of CD4 ⁺ T cells and CD8 ⁺ T cells and a second composition comprising the other of CD4 ⁺ T cells and CD8 ⁺ T- cells; And (c) the introduction of the first composition and the second composition is carried out with an interval of from about 0 to about 12 hours, with an interval of from about 0 to about 6 hours, or with an interval of from about 0 to about 2 hours. 2. The method of claim. 1, in which the introduction of the first composition and the introduction of the second composition is carried out with an interval of from about 1 minute to about 1 hour. 3. The method of claim 1 or 2, wherein the first composition and the second composition are administered no more than 2 hours apart. 4. The method according to any one of paragraphs. 1-3, in which the first composition includes CD4 ⁺ T cells. 5. The method according to any one of paragraphs. 1-3, in which the first composition includes CD8 ⁺ T cells. 6. The method according to any one of paragraphs. 1-5, in which the introduction of the first composition is carried out before the introduction of the second composition. 7. The method according to any one of paragraphs. 1-6, in which the dose of CD4 ⁺ and CD8 ⁺ T cells includes a specific ratio of CD4 ⁺ cells expressing CAR to CD8 ⁺ cells expressing CAR, which is from about 1:3 to about 3:1. 8. The method of claim 7 wherein the specified ratio is or approximately 1:1. 9. The method according to any one of paragraphs. 1-8, in which the dose of CD4 ⁺ and CD8 ⁺ T cells is from about 1x10 ⁵ total CAR-expressing cells to about 5x10 ⁸ total CAR-expressing cells. 10. The method according to any one of paragraphs. 1-9, in which the dose of CD4 ⁺ and CD8 ⁺ T cells includes from or from about 1x10 ⁷ to or up to about 2x10 ⁸ total recombinant receptor-expressing T cells, inclusive. 11. The method according to any one of paragraphs. 1-10, in which the dose of CD4 ⁺ and CD8 ⁺ T cells is from about 1x10 ⁷ total CAR-expressing cells to about 1.5x10 ⁸ total CAR-expressing cells. 12. The method according to any one of paragraphs. 1-11, wherein the B cell cancer is myeloma, leukemia, or lymphoma. 13. The method according to any one of paragraphs. 1-12, wherein the B cell cancer is acute lymphocytic leukemia (ALL), adult ALL, chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), or diffuse large B cell lymphoma (DLBCL). 14. The method according to any one of paragraphs. 1-13, wherein B cell cancer is ALL. 15. The method according to any one of paragraphs. 1-13, wherein B cell cancer is ALL adults. 16. The method according to any one of paragraphs. 1-13, wherein the B cell cancer is CLL. 17. The method according to any one of paragraphs. 1-13, wherein the B cell cancer is NHL. 18. The method according to any one of paragraphs. 1-13, wherein the B cell cancer is DLBCL. 19. The method according to any one of paragraphs. 1-18, wherein the CAR includes an extracellular antigen-recognition domain specifically binding to an antigen and an intracellular signaling domain including a CD3-zeta (CD3ζ) chain and a co-stimulatory signaling region that is a CD28 or 4-1BB signaling domain. 20. The method according to any one of paragraphs. 1-19, wherein the T cells are primary T cells obtained from a patient. 21. The method according to any one of paragraphs. 1-20, wherein the T cells are autologous to the patient. 22. The method according to any one of paragraphs. 1-21 in which the patient is or has been identified as having an ECOG status of 0 or 1. 23. The method according to any one of paragraphs. 1-22, in which: at least 35%, at least 40%, or at least 50% of patients treated according to the method achieve a complete response (CR); at least 60%, 70%, 80%, 90%, or 95% of patients who achieve CR show a CR that lasts for 3 months or more; at least 60%, 70%, 80%, 90%, or 95% of patients who achieve CR at one month and/or three months remain in response, remain in CR, and/or survive or remain alive without progression for 3 months or more after reaching CR; at least 50%, at least 60%, or at least 70% of patients treated according to the method achieve an objective response (OR); at least 60%, 70%, 80%, 90%, or 95% of patients who achieve an OR show an OR that lasts for 3 months or more; at least 35%, at least 40%, or at least 50% of patients who achieve OR remain in response or survive for 3 months or more after achieving OR; or at least 40%, at least 50%, at least 60%, at least 70% of patients who, at or before cell dosing, had or were identified as having double/triple hit lymphoma or relapse after autologous transplant stem cells (ASCT) have achieved an OR or an OR that lasts for 3 months or more. 24. The method according to any one of paragraphs. 1-23, in which more than or more than about 50%, about 60%, about 70%, or about 80% of patients treated according to the method do not show cytokine release syndrome (CRS) grade 3 or greater, do not show grade 3 neurotoxicity 3 or more or more than 40% or 50% or 55% show no neurotoxicity or CRS. 25. The method according to any one of paragraphs. 1-24, wherein at least 40%, at least 50%, at least 60%, at least 70% of patients who, at or prior to cell dosing, had or were identified as having double/triple hit lymphoma or relapse after autologous stem cell transplant (ASCT) administration, achieved an OR, or an OR that lasts for 3 months or more. 26. The method according to claim 25, in which: CR or OR lasts for more than 3 months; at least 20%, at least 25%, at least 35%, at least 40%, or at least 50% of patients treated according to the method achieve a CR that lasts for more than 3 months; at least 60%, 70%, 80%, 90%, or 95% of patients treated according to the method and who achieve CR remain in CR, or remain in response, or survive for 3 months or more ; at least 60%, 70%, 80%, 90%, or 95% of patients treated according to the method who achieve a CR in one month or three months remain in response, remain in CR, survive, or remain alive without progression for 3 months or more; at least 50%, at least 60%, or at least 70% of patients treated according to the method achieve an objective response (OR); at least 60%, 70%, 80%, 90%, or 95% of patients achieve an OR that lasts for 3 months or more; or at least at least 35%, at least 40%, or at least 50% of patients treated according to the method and achieving an OR remain in response or survive for 3 months or more. 27. The method according to any one of paragraphs. 1-26, in which: at or prior to administration of the cell dose, the patient is or has been identified as having lymphoma associated with or involving central nervous system (CNS) involvement; or at least 70%, at least 80%, at least 90%, or at least 95% of patients treated according to the method, who at or before dosing of cells showed or were identified as having CNS-involved lymphoma, achieved elimination of CNS disease. 28. The method according to any one of paragraphs. 1-27, wherein at or before the cell dose is administered, the patient has a brain lesion, optionally a temporal lobe lesion. 29. The method according to any one of paragraphs. 1-28, in which: at least 35%, at least 40%, or at least 50% of patients treated according to the method achieve a complete response (CR) or remission of CNS disease; at least 60%, 70%, 80%, 90%, or 95% of patients who achieve CR remain in CR for 3 months or more; at least 60%, 70%, 80%, 90%, or 95% of patients who achieve CR or CNS disease remission at one or three months remain in response, remain in CR, survive, or remain alive without progression for 3 months or more; at least 50%, at least 60%, or at least 70% of patients treated according to the method achieve an objective response (OR) or remission of CNS disease; at least 60%, 70%, 80%, 90%, or 95% of patients achieve OR within 3 months or more, or within 6 months or more; at least 60%, 70%, 80%, 90%, or 95% of patients who achieve OR or remission of CNS disease remain in response or survive for 3 months or more and/or for 6 months or more; the brain lesion is reduced in size or volume by more than or more than about 25%, 50%, 75%, or more; or reduction, or remission, or clearance of CNS disease is achieved in at least 35%, at least 40%, or at least 50% of patients treated according to the method. 30. The method according to any one of paragraphs. 1-29, in which: more than or more than about 30%, 35%, 40% or 50% of patients treated according to the method do not show cytokine release syndrome (CRS) or neurotoxicity of any degree; at least or about 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of patients treated according to the method do not show onset of CRS earlier than 3 days after administration or do not show the onset of neurotoxicity earlier than 5 days after administration; or the mean onset of neurotoxicity among patients treated according to the method occurs at or after the mean peak, or the mean time to resolve CRS in patients treated according to the method and/or the mean onset of neurotoxicity among patients treated according to the method is greater than or approximately 8, 9, 10 or 11 days. 31. The method according to any one of paragraphs. 1-30, wherein the patient has not been administered a treatment agent or agent capable of treating, preventing, delaying, attenuating, or reducing the development or risk of developing toxicity after the cell dose has been administered to the patient prior to administration of the cell dose. 32. The method of claim 28, wherein the agent is or includes an anti-IL-6 antibody, an anti-IL-6R antibody, or a steroid. 33. The method of claim 28 or 29, wherein the agent is or includes a steroid. 34. The method according to any one of paragraphs. 31-33, wherein the agent is or includes tocilizumab, siltuximab, or dexamethasone. 35. The method according to any one of paragraphs. 1-34, in which: administration and any monitoring is carried out on an outpatient basis or does not require admission or overnight stay in the hospital; And if the patient shows a prolonged fever or a fever that is or has not been reduced by or has not been reduced by more than 1ºC after treatment with an antipyretic, the patient is hospitalized or overnight in the hospital, or a treatment agent or agent is administered to treat or prevent or reduce or attenuation of neurotoxicity or cytokine release syndrome or the risk thereof. 36. The method according to any one of paragraphs. 13, 17 and 19-35 wherein the NHL is selected from the group consisting of aggressive NHL, diffuse large B cell lymphoma (DLBCL), NOS (de novo and transformed from indolent), primary mediastinal large B cell lymphoma (PMBCL) , mantle cell lymphoma (MCL) and/or follicular lymphoma (FL), optionally stage 3B follicular lymphoma (FL3B). 37. The method according to any one of paragraphs. 13, 17 and 19-36, wherein NHL includes diffuse large B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), NOS (de novo or transformed from indolent lymphoma), or stage 3B follicular lymphoma (FL3B ). 38. The method according to any one of paragraphs. 13, 17 and 19-37, in which the NHL includes the DLBCL. 39. The method of claim 38, wherein the DLBCL does not include DLBCL transformed from MZL and CLL (Richter), or the patient dosed with cells has DLBCL characterized as de novo or transformed from indolent and/or does not have DLBCL , transformed from MZL and CLL. 40. The method according to any one of paragraphs. 13, 17 and 19-37, in which the NHL includes PMBCL. 41. The method according to any one of paragraphs. 13, 17 and 19-37, in which the NHL includes NOS. 42. The method according to any one of paragraphs. 13, 17 and 19-37, in which the NHL includes the FL. 43. The method according to any one of paragraphs. 1-21 and 23-42, wherein the patient is or has been identified as having an Eastern Cooperative Oncology Group Performance Status (ECOG) status of 0, 1, or 2. 44. The method according to any one of paragraphs. 13, 17 and 19-43, in which at or just before the cell dose, the patient relapsed after treatment with or became refractory to one or more prior NHL therapies other than a different dose of CAR expressing cells. 45. The method according to any one of paragraphs. 13, 17, and 19-44, in which at or just prior to cell dose administration, the patient relapsed from remission after treatment with or became refractory to two or more prior NHL therapies other than another dose of CAR expressing cells. 46. The method according to any one of paragraphs. 1-45, wherein at or before the dose of cells: the patient is or has been identified as having double/triple hit lymphoma; the patient is or has been identified as having a chemo-refractory lymphoma, optionally a chemo-refractory DLBCL; the patient has not achieved complete remission (CR) in response to prior therapy; or the patient relapsed within 1 year or less than 1 year after receiving an autologous stem cell transplant (ASCT). 47. The method according to any one of paragraphs. 1-46, wherein CAR comprises, in that order, a CD19-specific scFv transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule. 48. The method according to any one of paragraphs. 1-47, wherein CAR comprises, in that order, a CD19-specific scFv transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule that is or contains a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from a primary a signaling molecule containing ITAM, which is the signaling domain of CD3-zeta. 49. The method according to any one of paragraphs. 1-48 wherein, prior to administration, the patient is preconditioned with an antilymphocyte therapy comprising administration of fludarabine, cyclophosphamide, or fludarabine and cyclophosphamide. 50. The method according to any one of paragraphs. 1-49, in which the administration of the dose of cells is carried out through outpatient delivery. 51. The method according to any one of paragraphs. 1-50, in which the dose of cells is administered parenterally, optionally intravenously.

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