RU2776823C2 - Compositions, products and methods related to dosing in cell therapy - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: options of a method for the determination of the risk of development of toxicity in an individual are proposed, including the analysis of a number of cells expressing a recombinant receptor in a blood sample of an individual, which was previously administered with a dose of cells expressing a recombinant receptor. The individual is at risk of development of toxicity, if the number of the specified cells in the blood sample is at least about 2 cells per microliter after no more than four days after the administration. The individual is also at risk of development of toxicity, if the number of the specified cells in the blood sample is at least about 5 or 10 cells per microliter after no more than five or six days after the administration. The individual is also at risk of development of toxicity, if the number of the specified cells in the blood sample is at least about 15 cells per microliter after no more than seven days after the administration.

EFFECT: invention provides the possibility of reduction in the risk of toxicity of T-cell therapy methods, while maintaining the efficiency of administered cells in an individual.

24 cl, 62 dwg, 18 tbl, 9 ex

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Applications: 62/464,371, filed Feb. 27, 2017, titled "COMPOSITIONS, ARTICLES OF MANUFACTURE AND METHODS RELATED TO DOSING IN CELL THERAPY"; 62/465817, filed March 1, 2017, entitled "COMPOSITIONS, ARTICLES OF MANUFACTURE AND METHODS RELATED TO DOSING IN CELL THERAPY"; 62/470180, filed March 10, 2017, entitled "COMPOSITIONS, ARTICLES OF MANUFACTURE AND METHODS RELATED TO DOSING IN CELL THERAPY"; 62/527002, filed June 29, 2017, entitled "COMPOSITIONS, ARTICLES OF MANUFACTURE AND METHODS RELATED TO DOSING IN CELL THERAPY"; 62/580416, filed November 1, 2017, entitled "COMPOSITIONS, ARTICLES OF MANUFACTURE AND METHODS RELATED TO DOSING IN CELL THERAPY"; 62/584740, filed November 10, 2017, entitled "COMPOSITIONS, ARTICLES OF MANUFACTURE AND METHODS RELATED TO DOSING IN CELL THERAPY"; and 62/596703, filed December 8, entitled "COMPOSITIONS, ARTICLES OF MANUFACTURE AND METHODS RELATED TO DOSING IN CELL THERAPY", the entire contents of which are incorporated herein by reference for all purposes.

Include a sequence listing as a link

[0002] This application is filed with a sequence listing in electronic format. The sequence list is in the form of a file named 735042009340SeqList.TXT, created on February 27, 2018, with a size of 37444 bytes. The complete information in electronic format from the sequence listing is provided by reference.

Field of invention

[0003] The present invention relates to cell therapy, comprising the administration of one or more doses of a T cell therapeutic composition, and to methods, compositions and articles for use therein. Cells from a T cell composition express recombinant receptors such as chimeric receptors, eg chimeric antigen receptors (CAR) or other transgenic receptors such as T cell receptors (TCR). The features of the embodiments of the present disclosure, including the number of cells or cell units administered and/or the activity of the cells administered, provide various benefits, such as a lower risk of toxicity in individuals treated with T cell compositions.

State of the art

[0004] Various methods of cell therapy are available for the treatment of diseases and conditions. Improved methods are needed, for example, to reduce the risk of toxicity from such methods. For example, improved methods are needed to reduce the risk of toxicity from cell therapy methods while maintaining the efficacy of the administered cells in the individual. The present invention relates to compositions, articles, methods and uses that meet such a need.

The essence of the invention

[0005] The present invention relates to embodiments relating to methods of cell therapy, such as methods of adoptive cell therapy, including methods that can be used to predict, reduce, prevent, alleviate one or more undesirable or adverse effects or toxicities, or their risks that may be associated with such therapies, such as toxicities, including severe toxicities, including severe or lethal neurotoxicity, including toxicities associated with cerebral edema or including cerebral edema. Among such present embodiments are articles of manufacture, compositions, such as compositions containing one or more unit doses of cells, and methods and uses, such as methods and uses related to or intended for methods of adoptive cellular immunotherapy, such as methods of therapy using engineered T cells, including CAR T cell therapy methods.

[0006] Among the present embodiments are articles such as articles comprising: (a) a container containing a unit dose of a T cell therapeutic composition comprising T cells containing a recombinant receptor, which is optionally a chimeric antigen receptor (CAR) , which specifically binds to an antigen, where a unit dose contains a target number of reference units (RU) within a given range, where RU in a given composition is determined by the formula RU=A x B, where: A is the number of cells, or a multiple of it, the fraction or a transformation of some phenotype present in a given composition, or is an average or weighted average of the number of cells, or a multiple thereof, a proportion or a transformation, of two or more phenotypes in a given composition; and B is a parameter value, or a fraction thereof, or a multiple, or a transformation that indicates the degree of or correlates with the degree of recombinant receptor dependent, optionally CAR dependent, activity in the composition; and (b) instructions for administering the composition, optionally one or more unit doses thereof, to an individual, optionally an individual having or suspected of having a disease or condition.

[0007] The present invention also provides an article of manufacture comprising: (a) a container containing a unit dose of a T cell therapeutic composition comprising T cells containing a recombinant receptor, optionally a chimeric antigen receptor (CAR), that specifically binds to an antigen, where: the unit dose contains or contains approximately (i) the target number of total recombinant receptor-expressing cells or the target number of total recombinant CD3+ receptor-expressing cells, or the target number of total recombinant CD8+ receptor-expressing cells, or (ii) the target number of reference units (RU ) within a given range, which is equal to or less than a threshold amount of RU, where a single dose does not contain more than a threshold value of RU, where the amount of RU in a given composition is determined by the formula: RU=A x B, where A is the number of cells, or a multiple of it , proportion or transformation, of some phenotype present in a given composition, or is the average or weighted average of the number of cells, or a multiple thereof, proportion or conversion, of two or more phenotypes in a given composition; and B is a parameter value, or a multiple thereof, or a transformation that indicates the degree of or correlates with the degree of recombinant receptor dependent, optionally CAR dependent, activity in a given T cell composition; and (b) instructions for administering the composition, optionally one or more unit doses thereof, to an individual, optionally an individual having or suspected to have a disease or condition.

[0008] The present invention also provides an article of manufacture comprising: (a) a container containing a unit dose of a T cell therapeutic composition comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds to an antigen, wherein the unit dose contains the target a dose of a T cell therapeutic composition, where: (i) if the value of a parameter that indicates the degree of or correlates with the degree of recombinant receptor-dependent activity of the composition is equal to or greater than a threshold value, the target dose is a first amount (or lies in a first range of amounts) cells of a given phenotype from the composition; (ii) if the parameter value is less than a threshold value, the target dose is a second number (or lies in a second range of numbers) of cells of a given phenotype from the composition, where the first number (or first range) is lower than the second number (or second range); and (b) instructions for administering the composition, optionally one or more unit doses thereof, to an individual, optionally an individual having or suspected of having a disease or condition.

[0009] The present invention also provides an article of manufacture comprising: (a) a container containing a unit dose of a T cell therapeutic composition comprising T cells containing a recombinant receptor, optionally a chimeric antigen receptor (CAR), that specifically binds to antigen, where: a single dose contains the target dose of the therapeutic composition of T-cells; and the T cell therapeutic composition is above the lower limit (LSL) and below the upper limit (USL) for B, where B is a parameter value that indicates the degree of or correlates with the degree of recombinant receptor dependent activity of the composition; and (b) instructions for administering the composition, optionally one or more unit doses thereof, to an individual, optionally an individual having or suspected of having a disease or condition.

[0010] The present invention relates to a method of treatment comprising administering to an individual suffering from a disease or condition, a single dose of a T cell composition containing cells containing a recombinant receptor, optionally a chimeric antigen receptor (CAR), that specifically binds to an antigen associated with a disease or condition, where the unit dose contains a target number of reference units (RU) within a given range, where the RU in a given composition is determined by the formula RU=A x B, where: A is the number of cells, or a multiple thereof, proportion or transformation, of some phenotype present in a given composition, or is the average or weighted average of the number of cells, or a multiple thereof, proportion or conversion, of two or more phenotypes in a given composition; and B is a parameter value, or a multiple thereof, or a transformation that indicates the degree of or correlates with the degree of recombinant receptor dependent, optionally CAR dependent, activity in the composition.

[0011] The present invention also relates to a method of treatment comprising administering to an individual suffering from a disease or condition, a single dose of a composition of T-cells containing cells containing a recombinant receptor, optionally, a chimeric antigen receptor (CAR), which specifically binds to an antigen associated with a disease or condition wherein the unit dose contains or contains approximately (i) a target number of total recombinant receptor-expressing cells or a target number of total recombinant receptor-expressing CD8+ cells, or (ii) a target number of reference units (RU) within a given range that is equal to or less than a threshold amount of RU, where a single dose does not contain more than a threshold value of RU, where the amount of RU in a given composition is determined by the formula: RU=A x B, where A is the number of cells, or its multiple, proportion or transformation, of some phenotype present in this song, and whether represents the average or weighted average of the number of cells, or a multiple thereof, proportion or transformation, of two or more phenotypes in a given composition; and B is a parameter value, or a multiple thereof, or a transformation that indicates the degree of or correlates with the degree of recombinant receptor dependent, optionally CAR dependent, activity in a given T cell composition.

[0012] In specific embodiments, A is total T cells, total CD3+ cells, total CD4+ or CD8+ cells, total CD3+CAR+ cells, total CD8+CAR+ cells, total CD4+ CAR+, or total the number of live or viable cells from any of the above, or its multiple or converted value. In some embodiments, A is total CD3+ cells, total CD8+, total CD3+CAR+ cells, total CD8+CAR+ cells, or total live or viable cells from any of the above, or a multiple or converted value thereof. In specific embodiments, A is the total number of apoptosis marker negative (–) cells that are CD3+ CAR+ cells, the total number of apoptosis marker negative (–) cells that are CD4+ CAR+, the total number of apoptosis marker negative (–) cells representing CD8+ CAR+ cells, or a multiple or converted value thereof, where the apoptosis marker is annexin V or active caspase 3. In specific embodiments, A is the total number of apoptosis marker-CD3+ CAR+ cells or the total number of apoptosis marker-CD8+ CAR+ cells, optionally, where the apoptosis marker is annexin V or active caspase 3.

[0013] The present invention also relates to a method of treatment comprising administering to an individual suffering from a disease or condition, a single dose of a T cell composition containing cells containing a recombinant receptor, optionally a chimeric antigen receptor (CAR), that specifically binds to an antigen associated with with a disease or condition where the unit dose contains the target dose of the T cell therapeutic composition: (i) if the value of a parameter that indicates the degree of or correlates with the degree of recombinant receptor dependent, optionally CAR dependent, activity of the composition is equal to or greater than a threshold value , the target dose is the first number (or lies in the first range of numbers) of cells of a given phenotype from the composition; (ii) if the parameter value is less than a threshold value, the target dose is the second number (or lies in the second range of numbers) of cells of a given phenotype from the composition, where the first number (or first range) is lower than the second number (or second range).

[0014] The present invention also relates to a method for assaying a therapeutic composition containing a single dose of a T cell composition, comprising: (a) analyzing a sample from a T cell composition containing T cells derived from an individual having a disease or condition and transduced a nucleic acid encoding a recombinant receptor, optionally a chimeric antigen receptor (CAR), that specifically binds to an antigen associated with a disease or condition, where the assay defines B for a cell composition, where B is a parameter value, or a multiple thereof, or a transformation that indicates the degree of or correlates with the degree of recombinant receptor dependent activity in the composition; and (b) evaluating the activity of the cell composition based on B, and/or evaluating whether the composition is above the lower specified limit (LSL) for B or below the upper specified limit (USL) for B.

[0015] The present invention also relates to a method for evaluating a T cell therapeutic composition, comprising evaluating a sample from a T cell composition containing T cells containing a recombinant receptor that specifically binds to an antigen associated with a disease or condition, by the activity of the cell composition based on B, and/or a judgment of whether the composition is above the Lower Set Limit (LSL) for B or below the Upper Set Limit (USL) for B.

[0016] The present invention also relates to a method for assessing the risk of toxicity of a T cell therapeutic composition, comprising: (a) evaluating a sample from a T cell composition administered to an individual by the number of reference units (RU) within a given range, where the composition T– cells contains T-cells containing a recombinant receptor that specifically binds to an antigen associated with a disease or condition, where RU in this composition is determined by the formula: RU=A x B, where: A is the number of cells, or a multiple of it, the proportion or transformation, cells of a certain phenotype present in the composition, or is the average or weighted average of the number of cells, or a multiple, proportion or transformation, of two or more phenotypes in the composition; and B is a parameter value, or a fraction thereof, or a multiple, or a transformation that indicates the degree of or correlates with the degree of recombinant receptor dependent, optionally CAR dependent, activity in the composition; and (b) comparing the amounts of RU to reference safe amounts of RU, wherein the comparison indicates whether or not the individual is at risk of experiencing an adverse event, optionally a serious adverse event, optionally severe neurotoxicity, grade equal to or greater than grade 4 or grade 5, or at least long-term neurotoxicity grade 3.

[0017] The present invention also relates to a method for preparing a therapeutic composition containing a single dose of a T cell composition, comprising: (a) analyzing a T cell composition containing T cells derived from an individual having a disease or condition and transduced with a nucleic acid , encoding a recombinant receptor, optionally a chimeric antigen receptor (CAR), that specifically binds to an antigen associated with a disease or condition, where the assay determines B for a cell composition, where B is a parameter value, or a multiple thereof, or a transformation that indicates the degree or correlates with the degree of recombinant receptor-dependent, optionally CAR-dependent, activity in the composition; and (b) filling the container with all or part of the composition and optionally another solution to obtain a unit dose of the T cell composition, where the unit dose contains the target number of reference units (RU) from the T cell composition, where the RU in this composition is determined by the formula: RU=A x B, where A is the number of cells, or a multiple, proportion or transformation, of some phenotype present in a given composition, or is the average or weighted average of the number of cells, or a multiple thereof, proportion or transformation, of two or more phenotypes in this composition.

[0018] The present invention also relates to a method for preparing a therapeutic composition containing a single dose of a T cell composition, comprising filling a container with all or part of the T cell composition containing T cells containing a recombinant receptor that specifically binds to an antigen associated with the disease or condition, and optionally, another solution, to obtain a unit dose of a T cell composition, where the unit dose contains the target number of reference units (RU) of the T cell composition, where RU in this composition is determined by the formula: RU=A x B, where A is the number of cells, or a multiple, proportion or transformation, of some phenotype present in a given composition, or is the average or weighted average of the number of cells, or a multiple, proportion or transformation, of two or more phenotypes in a given composition; and B is a parameter value, or a multiple thereof, or a transformation that indicates the degree of or correlates with the degree of recombinant receptor dependent, optionally CAR dependent, activity in the composition.

[0019] The present invention also relates to a method for preparing a T cell therapeutic composition for cell therapy, comprising filling a container with all or part of the composition containing T cells to a concentration of between about 10 million cells and about 70 million cells per ml, inclusive, and optionally, another solution.

[0020] The present invention relates to a unit dose of a therapeutic composition of T cells containing a number of cells containing a recombinant receptor, optionally a chimeric antigen receptor (CAR), specific for an antigen associated with a disease or condition, or expressed in a disease or condition, where the number of cells is between and between approximately 5.0×10 ⁶ and 2.25×10 ⁷ , 5.0×10 ⁶ and 2.0×10 ⁷ , 5.0×10 ⁶ and 1.5×10 ⁷ , 5.0×10 ⁶ and 1.0×10 ⁷ , 5.0×10 ⁶ and 7.5×10 ⁶ , 7.5×10 ⁶ and 2.25×10 ⁷ , 7.5×10 ⁶ and 2 .0×10 ⁷ , 7.5×10 ⁶ and 1.5×10 ⁷ , 7.5×10 ⁶ and 1.0×10 ⁷ , 1.0×10 ⁷ and 2.25×10 ⁷ , 1, 0×10 ⁷ and 2.0×10 ⁷ , 1.0×10 ⁷ and 1.5×10 ⁷ , 1.5×10 ⁷ and 2.25×10 ⁷ , 1.5×10 ⁷ and 2.0 x10 ⁷ , 2.0 x 10 ⁷ , and 2.25 x 10 ⁷ recombinant receptor-expressing cells, each including optionally recombinant receptor-expressing cells that are CD8+ or that are negative (–) for an apoptosis marker and CD8+, optional , where the apoptosis marker is p is annexin V or active caspase 3.

[0021] The present invention relates to a method for determining whether an individual is at risk of toxicity, comprising assaying the number of recombinant receptor-expressing cells in the blood of an individual, where said individual has previously been dosed with recombinant receptor-expressing cells, wherein the individual is at risk of developing toxicity if: (i ) no more than four days after the start of administration, the number of cells expressing the recombinant receptor in the blood of the individual is at least or about 2 expressing the recombinant receptor cells per microliter; (ii) no more than five or six days after the start of administration, the number of recombinant receptor-expressing cells in the individual's blood is at least or about 5 recombinant receptor-expressing cells per microliter, or at least or about 10 recombinant receptor-expressing cells per microliter ; or (iii) no more than seven days after the start of administration, the number of recombinant receptor-expressing cells in the individual's blood is at least or about 15 recombinant receptor-expressing cells per microliter.

[0022] The present invention also relates to a method for determining whether an individual is at risk of toxicity, comprising: (a) administering to an individual suffering from a disease or condition, a dose of recombinant receptor-expressing cells; and (b) after administration of the cells, analysis of the number of cells expressing the recombinant receptor in the blood of the individual, where the individual is at risk of developing toxicity if: (i) no more than four days after the start of administration, the number of cells expressing the recombinant receptor in the blood of the individual is at least or about 2 recombinant receptor expressing cells per microliter; (ii) no more than five or six days after the start of administration, the number of recombinant receptor-expressing cells in the individual's blood is at least or about 5 recombinant receptor-expressing cells per microliter, or at least or about 10 recombinant receptor-expressing cells per microliter. microliter; or (iii) no more than seven days after the start of administration, the number of recombinant receptor-expressing cells in the individual's blood is at least or about 15 recombinant receptor-expressing cells per microliter.

[0023] The present invention also relates to a method for evaluating a stimulatory reagent for use in preparing a T cell therapeutic composition, comprising: (i) evaluating a T cell composition obtained using a stimulatory reagent for recombinant receptor dependent activity or phenotype; and (ii) comparing the recombinant receptor-dependent activity or phenotype with the same activity or phenotype obtained for the control composition, or comparing with a standard unit of recombinant receptor-dependent activity or phenotype, where the stimulant is determined to be available for release for use in the method obtaining a therapeutic T cell composition, if the recombinant receptor-dependent activity or phenotype of the cell composition obtained using the stimulatory reagent differs by no more than 40%, or no more than 30%, or no more than 20%, or no more than by 10%, or not more than 5% of the same activity obtained for the control composition, or from the standard unit.

[0024] In some of any such embodiments, the degree or level of recombinant receptor-dependent activity or phenotype of the T cell therapeutic composition, in particular aspects, represents features or properties specific to the therapeutic composition and/or its products associated with the risk of adverse side effect following administration of the T cell therapeutic composition to an individual. In some aspects, such risks are higher or occur at a higher frequency in specific individuals who have one or more risk factors, e.g., (i) individuals who have been subjected to fewer cycles of prior therapy, optionally less than two cycles of prior therapy, up to initiation of administration of the T cell therapeutic composition, (ii) individuals who are young, optionally less than 30 years of age, (iii) individuals whose CD4:CD8 ratio in the post-apheresis sample from the individual is below a certain threshold, optionally below 1:1, or less than 1.5:1 or less than 0.5:1 or less, for example, when the dose administered is based on the total number of T cells or the total number of T cells expressing an engineered or recombinant receptor such as CAR, for example, without specifying the number or the ratio of CD4+ or CD8+ T cells per dose; (iv) individuals having a weight greater than the average weight among the group of individuals being treated; (v) individuals with a platelet count of less than or about less than 120,000; (vi) individuals having B-cell leukemia, optionally acute lymphocytic leukemia (ALL); (vii) individuals having a high disease burden prior to, e.g. immediately prior to or within one month prior to initiation of administration of the T cell therapeutic composition, optionally as determined based on a percentage of bone marrow blast cells greater than or equal to 5%, the sum of the products of the diameters (SPD), or lactate dehydrogenase levels; or (viii) individuals lacking the molecular subtype of acute lymphoblastic leukemia (ALL) with the Philadelphia chromosome (Ph+) and/or the similar subtype with the Ph-chromosome (Ph-like); (ix) individuals undergoing transitional chemotherapy prior to initiation of administration of the T cell therapeutic composition; (x) individuals pre-treated with anti-lymphocyte therapy, optionally including administration of fludarabine and/or cyclophosphamide, prior to administration of the T cell therapeutic composition; and/or (xi) individuals whose level, amount, or concentration of interleukin-15 (IL-15) in a blood sample prior to administration of the T cell therapeutic composition is above a threshold value. In some embodiments, the present invention relates to embodiments, for example, relates to methods, articles, compositions, doses, and dosing methods responsible for such features or properties, for example, in connection with dosing in an individual, and thus, for preventing, ameliorating or reducing the risk or likelihood of an individual experiencing an adverse event or toxicity, such as severe or Grade 3 neurotoxicity or greater, or Grade 3, Grade 4, or Grade 5 long-term neurotoxicity following administration of the T cell therapeutic composition.

[0025] In some of any such embodiments, exemplary features include the number or frequency (or number/kg) of viable cells, and/or cells of various phenotypes, at individual doses administered to individuals. Illustrative such phenotypes include the expression of one or more surface markers, as assessed by flow cytometry. For example, in some aspects, the phenotypes include CD3+, CD8+, CD4+ and/or CAR+. Also present among the phenotypes are viability and phenotypes associated with, or indicative of, or believed to be indicative of functional, healthy, or biologically active cells. In specific aspects, such phenotypes include the absence or slight presence or expression of markers indicative of apoptosis, apoptotic cells, or various, e.g., early or late stages of one or more of death or entry into the apoptotic pathway (e.g., annexin V, caspase 3) . In specific aspects, such markers include the ability of cells to produce cytokines or other factors in a manner that is not specific to the CAR antigen, for example, by staining intracellular cytokines (ICS) in response to PMA/ionomycin and/or FOXP3. In some aspects, markers and phenotypes include markers and phenotypes associated with one or more of T cell activation, depletion, stem cell-like properties, naive T cells, longevity, T cell subgroups, memory phenotype(s), and phenotypes. one or more subgroups of T-memory cells, such as one or more of T _CM , T _EM and T _SCM .

[0026] In some of any such embodiments, the features related to the therapeutic cell composition include the total number of CD8+CAR+ T cells per dose administered, the total number or frequency (e.g., among CD8+CAR+ T cells) of CD8+CAR+ T– cells at the dose administered are observed to be negative for a factor indicative of apoptosis, such as annexin V surface staining (annexin V–) or caspase 3 cleavage (indicative of non-apoptotic cells). Specific examples of cell composition-related features include total number of CD3+CAR+ T cells per dose administered, total number or frequency (e.g., among CD3+CAR+ T cells) of CD3+CAR+ T cells per dose observed to be negative by a factor indicative of apoptosis, such as annexin V surface staining (annexin V–) or caspase 3 cleavage (indicative of non-apoptotic cells).

[0027] In some of any such embodiments, exemplary parameters are properties specific to therapeutic compositions and/or their preparation, including viable cell concentration (VCC), cell multiplicity between inoculation and collection of drug product; and number of copies of the vector (VCN) in the administered dose. Among the exemplary parameters are also parameters generally associated with or indicative of function or activity or other features or capabilities of the cells in the compositions. Among these are various features associated with cell stimulation outcomes, including various measures of proliferation and activation or activity, including those induced in a manner induced or dependent on TCR and/or CAR. Illustrative of such parameters related to function or activity are measures or levels (such as their amount or concentration, or level per cell) of production of one or more factors (such as various cytokines) by cells in the composition, in response to antigen-specific CAR or other stimulation.

[0028] In some of any such embodiments, parameters indicative of CAR-targeted antigen-specific activities and functions (such as CAR antigen-dependent cytokine secretion and cytotoxicity) of the compositions are assessed in co-culture assays. Cells from the therapeutic composition to be evaluated are incubated in the presence of CD19 expressing target cells. For cytokine assays, cumulative amounts (pg/ml) of cytokine(s) (e.g., IL-2, IFN-gamma, TNF-alpha, IL-6, sCD137, MIP1b, MIP1a, IL-10, IL-4, IL- 13, IL-5 or GM-CSF) can be assessed after incubation of target cells and the number of different compositions, including the same number of transduced or CAR+ cells, in the same volume, for example, over a period of approximately 22-24 hours. Such assays provide an indication of antigen-specific cytokine secretion per CAR+ cell per dose. In other assays, cytolytic activity against CD19-expressing target cells was assessed after incubation with T cells.

[0029] In some of any such embodiments, measure of activity or functional effect in response to a non-specific CAR antigen stimulus, such as production of a cytokine or other factor following stimulation with an anti-CD3/anti-CD28 antibody, such as in the form of a coating magnetic beads. In such assays, cultures containing fewer CAR+ cells among the T cells in culture may have a higher relative score.

[0030] In some of any such embodiments, relationships in relation to the number of copies of the vector (per cell or per genome) are considered surrogate indicators of cell endurance and/or a set of variables associated with the relative abundance of modified or unmodified cells in the composition and/or overall density cells in the composition. For example, in some embodiments, cells from blood samples that are generally less healthy at the start of the process have a lower likelihood of surviving during the transduction phase, and/or cell death during transduction could increase transduction efficiency. Thus, in specific instances, a higher viral vector copy number per composition, such as introduced via transduction with a lentiviral or gamma-retroviral vector, may indicate a composition in which a greater percentage of cells were unhealthy at the start of the process and, in particular aspects, may have properties that correlate, for example, inversely, with neurotoxicity. In some embodiments, a higher transduction frequency may result in a composition having a higher frequency of modified (eg, CAR+) cells during cryopreservation, storage, and thawing. In specific aspects where the dose is based on numbers of modified cells (or subtypes thereof, e.g., CAR+CD3+), such compositions with a higher frequency of modified cells may have a lower overall cell density, which, in specific embodiments, correlates with a decrease in the level or the frequency of biologically active or non-apoptotic cells.

[0031] In some of any such embodiments, various measures of the number(s) or normalized numbers of cells negative for surface staining of apoptosis markers (including annexin V) at doses administered to an individual (or the frequency of such apoptosis marker negative cells among cells product, eg among CAR+CD3+ or CAR+CD8+) correlate or are associated with an adverse event such as toxicity, eg neurotoxicity. In some aspects, annexin V and caspase-3 are observed to be broadly similar. While such parameters can also be considered to be related to the function or activity of the CAR-T cell composition, and thus can be expected to be related to efficacy, these variables are generally not significantly correlated with treatment response outcome, e.g. whether or not the individual will achieve a complete response or no response.

[0032] In some of any such embodiments, a combination of features or parameters of a CAR-T cell therapeutic composition (e.g., antigen-specific activity and/or viability), features and/or characteristics of an individual (e.g., age, weight, and/or disease) , and other treatments (eg, number of previous treatment cycles) is associated with a risk of lethal neurotoxicity, eg after treatment with a CAR-T cell composition.

[0033] In some of any such embodiments, cerebral edema correlates and/or is associated with early and rapid proliferation of CAR-T cells and/or with increased levels of IL-15, an inflammatory biomarker and CAR-T growth factor. In some aspects, the dose and function of CD8+ CAR+ cells, together with the characteristics and/or traits of the individual, control, correlate with, and/or are associated with early and/or rapid proliferation of CAR-T cells.

[0034] In some of any such embodiments, the characteristics and/or traits of the individual are or include young age (e.g., less than 30 years of age), prior treatment (e.g., with two or less prior regimens), intensive transitional chemotherapy, and/or high-intensity use of fludarabine and/or cyclophosphamide, eg for anti-lymphocyte therapy.

[0035] In some of any such embodiments, cerebral edema, eg, cerebral edema associated with and/or following CAR-T cell therapy, is associated with endothelial injury and complete disruption of the blood-brain barrier (BBB). In specific aspects, cerebral edema is not associated with brain CAR-T cell infiltration, central nervous system (CNS) leukemia, or previous cycles of therapy for CNS leukemia.

[0036] In some of any such embodiments, the identification of high-risk patients before or before treatment, such as using CAR-T cell therapy, is key to potential management and risk mitigation, such as the risk or likelihood of toxicity, such as neurotoxicity or cerebral edema. In specific aspects, the risk of lethal neurotoxicity can be mitigated, reduced, and/or reduced by minimizing variability in CAR-T cell compositions, such as minimizing variability among cell compositions in terms of viability, CD4+/CD8+ ratio, and activity. In specific aspects, the use of a specific formulation product, eg, a formulation of CAR-T cells with specific parameters, may reduce dose and function variability.

[0037] In some of any such embodiments, the present methods, articles, compositions, doses, and dosing methods provide advantages in that they take into account, and, as appropriate, regulate or correct, potential sources of variability, including variability arising from due to changing reagents, and/or patient-to-patient variability. For example, in particular embodiments, it may be advantageous to generate modified T cells by a method involving the use of a T cell stimulation/expansion reagent (or a plurality of reagents), with confirmation by release assay of being below or within the acceptable range of a variable, compared to threshold level of a parameter, e.g., specific activity relative to that parameter of a therapeutic composition (such as an indication of the amount or relative amount of a reagent required to induce a particular degree of antigen-specific activity in a final T cell composition obtained by a T cell modification method, e.g. with a control reagent or standard unit, as appropriate for that assay). In specific embodiments, the embodiments provided include doses of cells in which the cells are obtained by a method that includes a release assay to confirm that any variability in such a specific activity parameter (e.g., antigen-specific level of a pro-inflammatory cytokine) is within an acceptable range and/or below the upper the set limit. In some embodiments, the methods provided include such a release assay. In specific embodiments, the preparation of cell compositions is carried out using reagents and/or methods for which the variability of such parameters is within an acceptable range. In specific embodiments, the present methods, compositions, and articles of manufacture provide advantages in that they use dosing and/or cell preparation methods that mitigate the risk associated with the potential variability of such specific activity parameters, for example, by minimizing the variability of a second parameter that , together with a specific activity parameter, correlates with the risk of toxicity. For example, by minimizing the variability in the frequency of biologically active or healthy cells (eg, non-apoptotic cells) obtained by the method, the impact of changes in specific activity-related parameters on safety can be minimized. In some embodiments, a dose or dosing method comprising the feature(s) associated with the number or frequency of biologically active or non-apoptotic cells, such as biologically active or non-apoptotic modified cells, or modified CD8+ cells, reduces the risk associated with variability in antigen-specific activity parameters. . In specific aspects, this is achieved by dosing, including an upper limit for such cells and/or determining the dose based on reference units, for example, based on a formula that takes into account the number of biologically active cells.

[0038] In specific embodiments, using a method with reduced variability in the frequency of such biologically active cells among modified cells reduces the risk that particular patients (e.g., patients having cells that are less prone to apoptosis or healthier) may inadvertently administer a higher dose of biologically active cells than intended, when dosing based on the number of modified T cells in general. In addition, it has been observed that methods such as this method with greater control of phenotype and function reduce the degree of variability for the ability of cells obtained by this method to produce pro-inflammatory cytokines in an antigen-specific manner. The results of the above study are consistent with the interpretation that, in particular in combination with the amounts of biologically active modified cells, a dosing method taking into account such cell-specific parameters of activity (for example, such that a specific target range of such activity - or not more than a threshold values are given at a given dose) can be used to provide a dose capable of providing the desired clinical or therapeutic outcome while still remaining within the safety margin or reducing the risk of unwanted toxicity, such as neurotoxicity.

[0039] In some embodiments, the use of a method that consistently produces higher frequencies of biologically active modified cells allows the use of cell doses that are much lower (in terms of numbers of modified, e.g., CAR+ cells) than other dosing methods. in which, more frequently, the modified cells are positive for apoptotic markers or are otherwise less healthy. For example, based on the observations herein, and given the observation that available dosing methods generally do not take into account the frequency of apoptotic cells, in specific embodiments, the numbers of modified (e.g., CAR+) T cells (e.g., modified CD8+ and/or CD4+ cells) are as low as 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 million cells.

[0040] In some of any such embodiments, the present embodiment includes evaluating or determining the number of cells, or a multiple or fraction thereof, or transformation, of a given phenotype present in a given composition. In some embodiments, A is the average or weighted average of the number of cells, or a multiple thereof, proportion or conversion, of two or more phenotypes in a given composition. In specific embodiments, the target number of units is less than a threshold number of units, optionally a safe number of reference units, where the safe number of reference units is, in relation to a group of individuals analyzed after treatment with a T cell therapeutic composition containing T -cells expressing the recombinant receptor, optionally CAR, the smallest number of drug reference units administered to an individual among those individuals in the group who continued to develop an adverse event. In specific embodiments, the adverse event is a serious adverse event, optionally severe neurotoxicity, optionally a grade equal to or greater than grade 4 or grade 5, or at least long-term grade 3 neurotoxicity.

[0041] In some embodiments, the target number of reference units is less than the safe number of reference units by an amount corresponding to a safety factor and/or by an amount in the range of 1.5- to 3-fold, optionally, approximately 2 - a multiple, or a multiple of the standard deviation in a group of individuals who did not experience an adverse event, optionally, neurotoxicity of degree 0-2, optionally, where the multiple is in the range from 1.5- to 3-fold. In specific embodiments, the target number of reference units is equal to or greater than the reference effective number of reference units, where the reference effective amount is, in relation to the group of individuals analyzed after treatment using a therapeutic composition of T cells containing the recombinant receptor, optionally, CAR, the number of units a drug administered to one or more individuals within a group for which the desired therapeutic outcome is indicated, optionally a partial response or a complete response (CR).

[0042] In specific embodiments, A is the average or weighted average of the number of cells, or a multiple thereof, proportion or conversion, of two or more phenotypes in a given composition. In some embodiments: the target number is the target number of recombinant receptor-expressing CD3+ cells that are negative (-) for an apoptosis marker and CD3+, optionally, where the apoptosis marker is annexin V or active caspase 3; or the target amount is the target amount of recombinant receptor-expressing CD8+ cells that are negative (-) for an apoptosis marker and CD8+, optionally, where the apoptosis marker is annexin V or active caspase 3.

[0043] In specific embodiments, the target number of cells in (i) is between and between about 5.0x10 ⁶ and 2.25x10 ⁷ 5.0x10 ⁶ and 2.0x10 ⁷ .5 .0×10 ⁶ and 1.5×10 ⁷ , 5.0×10 ⁶ and 1.0×10 ⁷ , 5.0×10 ⁶ and 7.5×10 ⁶ , 7.5×10 ⁶ and 2, 25×10 ⁷ , 7.5×10 ⁶ and 2.0×10 ⁷ , 7.5×10 ⁶ and 1.5×10 ⁷ , 7.5×10 ⁶ and 1.0×10 ⁷ , 1.0 ×10 ⁷ and 2.25×10 ⁷ , 1.0×10 ⁷ and 2.0×10 ⁷ , 1.0×10 ⁷ and 1.5×10 ⁷ , 1.5×10 ⁷ and 2.25× 10 ⁷ , 1.5×10 ⁷ and 2.0×10 ⁷ , 2.0×10 ⁷ and 2.25×10 ⁷ recombinant receptor-expressing cells, including each optional recombinant receptor-expressing CD3+ or CD8+ cells or which are negative (-) for an apoptosis marker and CD8+, optionally, where the apoptosis marker is annexin V or active caspase 3.

[0044] In specific embodiments, the target number of cells in (i) is: between at least or at least about 5×10 ⁶ , 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9×10 ⁶ , 10×10 ⁶ , and approximately 15×10 ⁶ recombinant receptor-expressing cells, including each, optionally, recombinant receptor-expressing cells that are CD8+ or that are negative (–) for an apoptosis marker and CD8+, optionally, where the apoptosis marker is annexin V or active caspase 3; between at least or at least about 5.55×10 ⁶ , 6.66×10 ⁶ , 7.77×10 ⁶ , 8.99×10 ⁶ , 1.0×10 ⁷ , 1.1×10 ⁷ and approximately 1.67×10 ⁷ expressing the recombinant receptor, including each optionally expressing the recombinant receptor cells that are CD8+, or which are negative (-) for the apoptosis marker and CD8+, optionally, where the apoptosis marker is annexin V or active caspase 3; between at least or at least about 6.25×10 ⁶ , 7.5×10 ⁶ , 8.75×10 ⁶ , 1.0×10 ⁷ , 1.13×10 ⁷ , 1.25×10 ⁷ and approximately 1.9 x 10 ⁷ expressing the recombinant receptor, including each optionally expressing the recombinant receptor cells that are CD8+ or that are negative (-) for the apoptosis marker and CD8+, optionally, where the apoptosis marker is annexin V or active caspase 3; between at least or at least about 7.14×10 ⁶ , 8.5×10 ⁶ , 1.0×10 ⁷ , 1.14×10 ⁷ , 1.29×10 ⁷ , 1.42×10 ⁷ and about 2.14 x 10 ⁷ expressing the recombinant receptor, including each optionally expressing the recombinant receptor cells that are CD8+ or that are negative (-) for the apoptosis marker and CD8+, optionally, where the apoptosis marker is annexin V or active caspase 3.

[0045] In some embodiments, the target number of cells in (i) is between at least or at least about 5×10 ⁶ , 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9×10 ⁶ , 10×10 ⁶ and approximately 15×10 ⁶ recombinant receptor-expressing cells that are negative (-) for an apoptosis marker and CD8+, including each, optionally, where the apoptosis marker is annexin V or active caspase 3. In specific embodiments, target the number of cells in (i) is between at least or at least about 6.25×10 ⁶ , 7.5×10 ⁶ , 8.75×10 ⁶ , 1.0×10 ⁷ , 1.13×10 ⁷ , 1.25×10 ⁷ and approximately 1.9×10 ⁷ expressing the recombinant receptor of CD8+ cells, including each.

[0046] In specific embodiments, the target reference amount of RU is less than a threshold number of units, or is less than the reference safe amount of RU, where the reference safe amount of RU is, in relation to the group of individuals analyzed after treatment using the therapeutic composition T– cell containing T-cells expressing a recombinant receptor, optionally CAR, the smallest number of reference units of the drug administered to an individual among those individuals in the group who continued to develop an adverse event.

[0047] In some embodiments, A is the number of cells of some phenotype present in a given composition, and B is a parameter value that indicates the degree of or correlates with the degree of recombinant receptor-dependent activity in the composition. In specific embodiments, A and/or B is a quantity or value transform, respectively, where the transform includes a log transform, a power transform, or a logit transform. In specific embodiments, A is the number of cells of some phenotype present in a given composition, and B is a multiple or transformation of a parameter value indicative of the degree of or correlated with the degree of CAR-dependent activity in a given T cell composition, optionally, where B represents the logarithmic transformation of the value.

[0048] In some embodiments, the logarithmic transformation is decimal log (log ₁₀ (x)), natural log (ln(x)) or binary log (log ₂ (x)). In specific embodiments, is the number of viable cells in the composition and/or is the number of cells that are not apoptotic, do not have a factor indicative of early apoptosis or apoptosis, are not in the early stages of apoptosis, or are not in the late stages of apoptosis , and/or represents the number of cells in a certain state of differentiation, and/or represents the number of cells with memory cells/stem cells, or represents its multiple or transformation.

[0049] In specific embodiments, the phenotype includes positive expression of a surface marker that is one or more of CD3, CD4, or CD8, and/or includes positive expression of a recombinant receptor, optionally CAR, or a surrogate recombinant receptor expression marker. In some embodiments, the phenotype is CD3+ CAR, CD4+/CAR+, CD8+/CAR+. In specific embodiments, the phenotype includes the absence of a factor indicative of apoptosis or one or more stages of the apoptotic cascade or pathway, optionally, expression of an apoptosis marker. In specific embodiments, the phenotype includes negative expression of an apoptosis marker, optionally an early apoptosis or late apoptosis marker. In some embodiments, the apoptosis marker is surface phosphatidylserine and/or detected using annexin V, or is an active caspase or a caspase proform, optionally an active caspase 3 or a caspase 3 proform. In specific embodiments, the phenotype includes annexin-.

[0050] In specific embodiments, the phenotype includes an indicator of the production of one or a combination of cytokines, optionally not specific for the antigen or recombinant receptor and/or polyclonal production, where one or more cytokines is IL-2, IL-13, IL-17 , IFN-gamma or TNF-alpha. In some embodiments, the production index is measured in an assay, optionally an intracellular cytokine staining assay, comprising incubating a sample from a T cell composition with a polyclonal agent, an antigen specific agent, or a recombinant receptor binding agent, optionally CAR. In specific embodiments, the agent is or contains PMA and ionomycin, or is or contains a T cell receptor or a T cell receptor complex agonist. In specific embodiments, the phenotype comprises negative expression of an activation marker, where the activation marker is selected from CD25, CD127, LAG3, Ki67, and combinations thereof.

[051] In some embodiments, the phenotype includes negative expression of a depletion marker, where the depletion marker is a PD1 or FOXP3 gene product, or a combination thereof. In specific embodiments, the phenotype includes a naïve or memory cell phenotype, optionally wherein the memory cell phenotype includes an effector memory T cell phenotype, a central memory T cell phenotype, or a CD45RA-expressing effector memory T cell phenotype (Temra).

[0052] In some embodiments, the threshold for recombinant receptor-dependent activity is less than the reference safety level, where the reference safety level is, as applied to a group of individuals analyzed after treatment with a T cell therapeutic composition containing T cells expressing recombinant receptor, optionally CAR, the lowest level of CAR-dependent activity of the therapeutic composition administered to an individual among those individuals in the group who continued to develop an adverse event. In specific embodiments, the indicator is determined in an assay comprising culturing or incubating for a fixed time, optionally 24 hours, a given composition or a sample thereof in the presence of an antigen, antigen-expressing cells, and/or an agent that specifically binds to a recombinant receptor, optionally , CAR. In specific embodiments, the assay is an ELISA.

[0053] In some embodiments, a factor index is: (i) a concentration, relative concentration, amount, or relative amount of a factor; or (ii) the amount or relative amount of factor per unit of cells administered from a given composition, or (iii) the amount or relative amount of factor per unit of cells administered from a given composition per unit of time, optionally one hour; or (iv) a level indicative of any of (i)-(iii). In specific embodiments, one or more of the factors is one or a combination of soluble factors, optionally one or a combination of cytokines, chemokines, or soluble receptors, optionally soluble costimulatory receptors. In specific embodiments, one or more of the factors is one or a combination of pro-inflammatory cytokines, Th2 cytokines, and Th17 cytokines. In some embodiments, one or more of the factors is one or a combination of IL-2, IFN-gamma, TNF-alpha, IL4, IL-5, IL-10, IL-13, GM-CSF, sCD137, MIP1a, and M1Pb . In specific embodiments, one or more of the factors is one or a combination of IL-2, IFN-gamma, TNF-alpha, and IL-10. In specific embodiments, one or more of the factors is a combination of any two or more of the above soluble factors, and the parameter is the arithmetic or geometric mean of the scores for the two or more factors.

[0054] In some embodiments, the parameter is the arithmetic or geometric mean of, optionally, amount or concentration, for at least two of TNF-alpha, IFN-gamma, and IL-2, or of TNF-alpha, IFN-gamma and IL-2. In particular embodiments, the parameter is a normalized score value, where the normalization is compared to a reference factor score. In specific embodiments, the reference score is the average of scores among a plurality of, optionally, at least 10, at least 15, at least 20 reference chimeric antigen receptor (CAR) T cell therapeutic compositions in which: (i) for each of the reference T cell therapeutic compositions, an acceptable safety profile was observed or determined as a result after administration to an individual, optionally where the individual has a disease or condition with antigen expression or association with an antigen; and/or (ii) for each of the reference T cell therapeutic compositions, the desired efficacy is observed or determined as a result after administration to an individual, optionally where the individual has a disease or condition with antigen expression or association with an antigen. In some embodiments, the acceptable safety profile is no observable grade 2 or higher neurotoxicity, or no grade 3 or higher neurotoxicity. In specific embodiments, the acceptable safety profile is no observable grade 3 or higher neurotoxicity.

[0055] In specific embodiments, the effectiveness is a partial response or a complete response (CR). In some embodiments, the benchmark is the score, in the same assay, of a factor in a reference T cell composition that is prepared in the same manner as the T cell therapeutic composition but does not express the recombinant receptor, optionally CAR, does not specifically recognize antigen and/or does not express any recombinant receptor, optionally any CAR. In specific embodiments, this parameter is normalized to control for patient-specific variability in the score of one or more factors.

[0056] In specific embodiments, the parameter is the normalized value of a factor score, compared to the same score in the same assay, for a control factor, where the control factor level in the T cell therapeutic composition is known to be absent or has been observed to lack a feature or correlation , or a significant correlation with the outcome of an adverse event or toxicity, or their likelihood or risk, where the outcome of an adverse event or toxicity, optionally, is a severe neurotoxicity. In some embodiments, the control factor is a factor that is not statistically correlated and/or correlated with the occurrence of an adverse event among a plurality of individuals who continued to develop an adverse event after administration of the T cell composition, optionally, the control factor is or comprises one or a combination of IL-5, IL-13, GM-CSF and IL-6, optionally wherein the control factor score is the arithmetic or geometric mean of two or more of the above.

[0057] In specific embodiments, the implementation, the parameter does not include cytolytic activity or its indicator. In particular embodiments, the parameter does not include a recombinant receptor dependent or antigen specific cytolytic activity or an index thereof. In some embodiments, the phenotype is CD8+ CAR+ cells or an apoptosis marker - CD8+ CAR+ cells, optionally wherein the apoptosis marker is annexin V or active caspase 3; and the parameter is a score for a pro-inflammatory cytokine, which is optionally one or a combination of TNF-alpha, IL-2, and IFN-gamma, or a normalized value thereof.

[0058] In specific embodiments, the adverse event is a grade 4 or 5 neurotoxicity, and the threshold number of units: is or is approximately 1.75×10 ⁷ if A is negative (-) for the apoptosis marker and CD8+CAR+, and B is TNF-alpha or its normalized value; is or is about 2.19×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha or a normalized value thereof; is or is approximately 1.25×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma or its normalized value; is or is approximately 1.56×10 ⁷ if A is CD8+CAR+ and B is IFN-gamma or its normalized value; is or is approximately 1.5×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IL-2 or a normalized value thereof; is or is approximately 1.88×10 ⁷ if A is CD8+CAR+ and B is IL-2 or its normalized value; is or is approximately 1.5×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IFN-gamma or a normalized value thereof; is or is approximately 1.88×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof; is or is approximately 2.0×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IL-2 or a normalized value thereof; is or is about 2.5×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha and IL-2 or its normalized value; is or is approximately 1.25×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma and IL-2 or a normalized value thereof; is or is about 1.56×10 ⁷ if A is CD8+CAR+ and B is IFN-gamma and IL-2 or its normalized value; is or is approximately 1.75×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha, IFN-gamma and IL-2 or a normalized value thereof; is or is approximately 2.19×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma and IL-2 or its normalized value, optionally, where the apoptosis marker is annexin V or active caspase 3.

[0059] In specific embodiments, the adverse event is a grade 4 or 5 neurotoxicity, and the range of target reference units: is between or about between 2.0×10 ⁵ and 1.75×10 ⁷ , inclusive, if A is negative (–) for apoptosis marker and CD8+CAR+, and B is TNF-alpha or its normalized value; is between or approximately between 2.5×10 ⁵ and 2.19×10 ⁷ inclusive if A is CD8+CAR+ and B is TNF-alpha or a normalized value thereof; is between or approximately between 4×10 ⁵ and 1.25×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma or a normalized value thereof; is between or approximately between 5x10 ⁶ and 1.56x10 ⁷ inclusive if A is CD8+CAR+ and B is IFN-gamma or a normalized value thereof; is between or approximately between 2.0×10 ⁵ and 1.5×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IL-2 or a normalized value thereof; is between or approximately between 2.5x10 ⁵ and 1.88x10 ⁷ inclusive if A is CD8+CAR+ and B is IL-2 or a normalized value thereof; is between or approximately between 3.0×10 ⁵ and 1.5×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IFN-gamma or its normalized value; is between or approximately between 3.75×10 ⁵ and 1.88×10 ⁷ inclusive if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof; is between or approximately between 3.0×10 ⁵ and 2.0×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IL-2 or its normalized value; is between or approximately between 3.75×10 ⁵ and 2.5×10 ⁷ inclusive if A is CD8+CAR+ and B is TNF-alpha and IL-2 or a normalized value thereof; is between or approximately between 4.0×10 ⁵ and 1.25×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma and IL-2 or its normalized value; is between or approximately between 5.0×10 ⁵ and 1.56×10 ⁷ inclusive if A is CD8+CAR+ and if B is IFN-gamma and IL-2 or its normalized value; is between or approximately between 4.0×10 ⁵ and 1.75×10 ⁷ inclusive if A is negative (–) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha, IFN-gamma and IL –2 or its normalized value; is between or approximately between 5.0×10 ⁵ and 2.19×10 ⁷ inclusive if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma and IL-2 or a normalized value thereof, optional where the apoptosis marker is annexin V or active caspase 3.

[0060] In some embodiments, the adverse event is at least a long-term grade 3 neurotoxicity and the cut-off number of units: is or is about 1.0×10 ⁶ if A is negative (-) for apoptosis marker and CD8+ CAR+, and B is TNF-alpha or its normalized value; is or is approximately 1.25×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha or a normalized value thereof; is or is approximately 2.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma or its normalized value; is or is approximately 2.5×10 ⁶ if A is CD8+CAR+ and B is IFN-gamma or its normalized value; is or is approximately 3.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IL-2 or a normalized value thereof; is or is approximately 3.75×10 ⁶ if A is CD8+CAR+ and B is IL-2 or its normalized value; is or is approximately 1.5×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IFN-gamma or a normalized value thereof; is or is about 1.88×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof; is or is approximately 2.5×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IL-2 or a normalized value thereof; is or is approximately 3.12×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha and IL-2; or its normalized value is or is approximately 3.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma and IL-2 or its normalized value; is or is approximately 3.75×10 ⁶ if A is CD8+CAR+ and B is IFN-gamma and IL-2 or its normalized value; is or is approximately 2.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha, IFN-gamma and IL-2 or a normalized value thereof; is or is approximately 2.5×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma and IL-2 or its normalized value, optionally, where the apoptosis marker is annexin V or active caspase 3.

[0061] In specific embodiments, the adverse event is at least Grade 3 long-term neurotoxicity, and the target reference unit range is: between or approximately between 3.0 x 10⁵ and 1.0×10⁶, inclusive, if A is negative (-) for apoptosis marker and CD8+CAR+, and B is TNF-alpha or its normalized value; is between or approximately between 3.75×10⁵ and 1.25×10⁶, inclusive, if A is CD8+CAR+ and B is TNF-alpha or its normalized value; is between or approximately between 4×10⁵ and 2.0×10⁶, inclusive, if A is negative (-) for the apoptosis marker CD8+CAR+, and if B is IFN-gamma or its normalized value; is between or approximately between 5×10⁶ and 2.5×10⁶, inclusive, if A is CD8+CAR+ and B is IFN-gamma or its normalized value; is between or approximately between 2.0×10⁵ and 3.0×10⁶, inclusive, if A is negative (-) for the apoptosis marker CD8+CAR+, and if B is IL-2 or its normalized value; is between or approximately between 2.5×10⁵ and 3.75×10⁶, inclusive, if A is CD8+CAR+ and B is IL-2 or its normalized value; is between or approximately between 3.0×10⁵ and 1.5×10⁶, inclusive, if A is negative (-) for the apoptosis marker CD8+CAR+, and if B is TNF-alpha and IFN-gamma or its normalized value; is between or approximately between 3.75×10⁵ and 1.88×10⁶, inclusive, if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof; is between or approximately between 3.0×10⁵and 2.5×10⁶, inclusive, if A is negative (-) for the apoptosis marker CD8+CAR+, and if B is TNF-alpha and IL-2 or its normalized value; is between or approximately between 3.75×10⁵ and 3.12×10⁶, inclusive, if A is CD8+CAR+ and B is TNF-alpha and IL-2 or its normalized value; is between or approximately between 4.0×10⁵, inclusive, and 3.0×10⁶if A is negative (-) for the apoptosis marker CD8+CAR+, and if B is IFN-gamma and IL-2 or its normalized value; is between or approximately between 5.0×10⁵ and 3.75×10⁶, inclusive, if A is CD8+CAR+ and B is IFN-gamma and IL-2 or its normalized value; is between or approximately between 4.0×10⁵and 2.0×10⁶, inclusive, if A is negative (-) for the apoptosis marker CD8+CAR+, and if B is TNF-alpha, IFN-gamma, and IL-2 or its normalized value; is between or approximately between 5.0×10⁵ and 2.5×10⁶, inclusive, if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma and IL-2 or its normalized value, optionally, where the apoptosis marker is annexin V or active caspase 3.

[0062] In specific embodiments, the T cell therapeutic composition contains between about 10 million cells per ml and about 70 million cells per ml, or between about 10 million viable cells per ml and about 70 million viable cells per ml, each including. In some embodiments, the T cell therapeutic composition contains between about 15 million cells or viable cells per ml and about 60 million cells or viable cells per ml, including each. In specific embodiments, the T cell composition contains more than 10 million cells or viable cells per ml. In specific embodiments, the T cell therapeutic composition contains more than 15 million cells, or more than 15 million cells per ml. In some embodiments, the composition further comprises a cryoprotectant and/or the article of manufacture further comprises instructions for thawing the composition prior to administration to an individual.

[0063] In specific embodiments, the disease or condition is cancer, optionally myeloma, lymphoma, or leukemia. In specific embodiments, the disease or condition is a B-cell malignancy, optionally a B-cell malignancy, selected from the group consisting of 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 antigen is an 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, cancer-testicular antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, chemokine ligand 1 with C-C motif (CCL-1), CD19, CD20 , CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD138, CD171, epidermal growth factor protein (EGFR), truncated epidermal growth factor protein (tEGFR), mutated 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 receptor-like protein 5 (FCRL5; also known as a homologue 5 Fc receptor or FCRH5), fetal acetylcholine receptor (fetal AchR), folate binding protein (FBP), folate receptor alpha , ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), G protein-coupled receptor 5D (GPCR5D), Her2/neu (erb-B2 receptor tyrosine kinase), Her3 (erb-B3), Her4 (erb–B4), erbB dimers, melanoma-associated human high molecular weight antigen (HMW–MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA–A1), human leukocyte antigen A2 (HLA–A2), IL– receptor 22 alpha (IL-22Ra), IL-13 receptor alpha 2 (IL-13Ra2), kinase insert domain containing receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope from L1-CAM , member A of family 8 containing leucine-rich repeats (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, mesothelin, c-Met, mouse cytomegalovirus (CMV), mucin 1 ( MUC1), MUC16, natural killer group 2 D member ligands (NKG2D), melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, preferably e. melanoma receptor antigen (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblastic cell glycoprotein (TPBG, also known as 5T4), tumor-associated glycoprotein 72 (TAG72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor antigen 1 (WT-1), pathogen-specific or universal label-associated antigen , and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV or other pathogens. The antigens targeted by the receptors in some embodiments include antigens associated with B-cell cancer, such as any of a number of known B-cell markers. In some embodiments, the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig-kappa, Ig-lambda, CD79a, CD79b, or CD30.

[0064] In specific embodiments, the product further comprises information indicating that the container contains the target number of units. In some embodiments, the container is the first container, and the product further comprises additional containers, where each of the additional containers contains a unit dose containing the target number of T cell composition units. In specific embodiments, the additional containers contain between about 10 million cells or viable cells per ml and about 70 million cells or viable cells per ml, between about 15 million cells or viable cells and about 60 million cells or viable cells per ml, including each , more than 10 million cells or viable cells per ml, more than 15 million cells or viable cells per ml, or a combination thereof. In specific embodiments, the unit dose contains no more than 15×10 ⁶ CD8+CAR+ cells that are negative by detection using annexin V or active caspase 3 or caspase 3 proform. In some embodiments, the unit dose further comprises a number of CAR positive CD4+ cells, where the number is in a ratio or approximately in a 1:1 ratio with CD8+CAR+ cells.

[0065] In specific embodiments, a composition of T cells is obtained by a method in which: frequency, (1) among CAR+ cells in the composition, (2) among CAR+CD3+ cells in the composition, and/or (3) among CAR+CD8+ cells in the composition, cells of a phenotype indicating signs of biologically active cells and/or the absence of apoptosis, or early or late stages of apoptosis, differ by no more than 40% or no more than 30% or no more than 20%, or not more than 10%, or not more than 5%, from the average of the specified frequency in the set of compositions of T-cells obtained by this method, and/or differs from such an average by no more than one standard deviation; or the frequency, (1) among CAR+ cells in the composition, (2) among CAR+CD3+ cells in the composition, and/or (3) among CAR+CD8+ cells in the composition, of cells of a phenotype indicative of the absence of apoptosis, or of early or late stages apoptosis differs by no more than 40%, or no more than 20%, or no more than 10%, among the plurality of T cell compositions obtained by this method.

[0066] In specific embodiments, the method includes: (a) incubating a population of cells containing T cells with an agent containing a nucleic acid molecule encoding a recombinant receptor under conditions to introduce a nucleic acid encoding a recombinant receptor into cells in the population ; and (b) stimulating the cells, before, during and/or after said incubation, wherein the stimulation comprises incubating the cells in the presence of a stimulating condition that induces a primary signal, signaling, stimulating, activating and/or proliferating the cells. In some embodiments, the method further comprises, before (a), isolating a population of cells from a biological sample. In specific embodiments, isolation comprises selecting cells based on surface expression of CD3 or based on surface expression of one or both of CD4 and CD8, optionally by positive or negative selection.

[0067] In some embodiments, the biological sample is or contains a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or a leukapheresis product.

[0068] In specific embodiments, the stimulatory condition includes incubation with a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and/or one or more intracellular signaling domains of one or more costimulatory molecules.

[0069] In specific embodiments, the stimulatory reagent contains a primary agent that specifically binds to a member of the TCR complex and a secondary agent that specifically binds to a costimulatory T cell molecule. In some embodiments, the primary agent specifically binds to CD3 and/or a costimulatory molecule selected from the group consisting of CD28, CD137 (4-1-BB), OX40, or ICOS. In specific embodiments, the primary and secondary agents contain antibodies and/or are present on the surface of a solid support, optionally a bead.

[0070] In specific embodiments: the stimulatory reagent is a reagent for which it is determined that the recombinant receptor dependent activity or antigen specific activity, optionally recombinant receptor dependent or antigen specific dependent production or accumulation of a pro-inflammatory cytokine, differs by no more than 40%, or no more than 30%, or no more than 20%, or no more than 10%, or no more than 5%, of the average pro-inflammatory cytokine production or accumulation among the multiple T cell compositions produced this way using a stimulating reagent, and / or differs from such an average by no more than one standard deviation; and/or a stimulatory reagent is a reagent for which the recombinant receptor dependent activity or antigen specific activity, optionally recombinant receptor dependent or antigen specific dependent production or accumulation of a pro-inflammatory cytokine is determined to differ by no more than 40% or no more than 30%, or not more than 20%, or not more than 10%, or not more than 5%, among the many compositions of T-cells obtained by this method; and/or the stimulatory reagent is a reagent for which it is determined that the recombinant receptor dependent activity or antigen specific activity, optionally recombinant receptor dependent or antigen specificity dependent production or accumulation of a pro-inflammatory cytokine, for a cell composition obtained using a stimulating reagent, differs by no more than 40%, no more than 30%, no more than 20%, or no more than 10%, or no more than 5% from the control composition, where the control composition and cell composition are obtained using the same method, including derived from the same population of cells, except that the control composition is implemented in the presence of a control stimulatory reagent, or a standard number of units of recombinant receptor-dependent activity. In some embodiments, the control stimulatory reagent, when used in the method, is known to result in a T cell composition in which the recombinant receptor dependent activity or antigen specific activity is within an acceptable range of variability.

[0071] In specific embodiments, the container is filled with all or part of the T cell composition, and optionally with another solution, to a concentration between about 10 million cells and about 70 million cells per ml, inclusive. In specific embodiments, the container is filled with another solution and the solution contains a cryoprotectant, optionally DMSO. In some embodiments, the concentration is between about 15 and about 60 million cells per ml, inclusive. In specific embodiments, the concentration is greater than 10 million cells per ml. In specific embodiments, the concentration is greater than 15 million cells per ml. In some embodiments, the DMSO concentration is or is about or does not exceed 7.5%. In specific embodiments, the concentration is greater than 60 million cells per ml. In specific embodiments, the DMSO concentration is greater than 7.5%, optionally between or about between 7.5% and 9.0%, inclusive.

[0072] In some embodiments, the filling is carried out in an automatic manner, optionally in a closed system. In specific embodiments, the adjusted unit dose is less than, optionally, less than 1.5 times, less than 2 times, less than 3 times, less than 4 times the average unit dose for the group of individuals treated using a T cell therapeutic composition comprising T cells expressing a recombinant receptor, optionally a CAR. In specific embodiments, a sample from the T cell composition, optionally a cryopreserved sample, is evaluated after administration of the T cell composition to an individual. In some embodiments, if B is greater than USL, the individual is determined to be at risk of toxicity. In specific embodiments, if B is greater than USL, the individual to whom the composition was administered is monitored and/or treated with an agent to alleviate or reduce the likelihood of a toxicity outcome or cytokine release syndrome following administration of the cell composition, and optionally, until a sign or symptom of a toxicity outcome occurs. .

Brief description of the drawings

[0073] FIG. 1 is a graph showing the concentrations of CAR+ cells/µl of blood at different time points after administration of T cell compositions containing anti-CD19 CAR expressing cells. Data are shown for individuals who experienced grade 3 or less neurotoxicity (stars), grade 4 (pentagons), or grade 5 (circles).

[0074] FIG. 2 is a graph showing the concentrations of CAR+ cells/µl of blood at different time points after administration of T cell compositions containing anti-CD19 CAR expressing cells. Data are shown for individuals who experienced grade 2 or less neurotoxicity (squares), grade 3 neurotoxicity (stars), long-term grade 3 neurotoxicity (diamond), grade 4 neurotoxicity (pentagon), or grade 5 neurotoxicity (circles with arrows).

[0075] FIG. 3A-3V are block diagrams quantifying various parameters of cell compositions containing cells expressing anti-CD19 CAR. The box plots on the left compare measurements for cell compositions derived from individuals who experienced grade 4 or less neurotoxicity with measurements for cell compositions derived from individuals who experienced grade 5 neurotoxicity. The box plots on the right compare measurements for cell compositions derived from individuals who experienced a complete response (CR), with measurements for cell compositions derived from individuals who did not experience a response (NR). Measurements for cell compositions derived from individuals whose response is not available are also shown in this diagram (NA).

[0076] The block diagrams of FIG. 3A shows the total number of CD8+CAR+ apoptosis marker negative cells administered to individuals.

[0077] The block diagrams of FIG. 3B shows the percentage of CD8+CAR+ cells positive for IL-13 after PMA/ionomycin by cytokine internal staining (ICS) at administered doses.

[0078] The block diagrams of FIG. 3C shows the number/kg of injected CD8+CAR+ negative for apoptosis marker (annexin V–) cells.

[0079] The block diagrams of FIG. 3D shows the percentage of CD4+CAR+ cells positive for CD127+.

[0080] The block diagrams of FIG. 3E show total infused cells/kg.

[0081] The block diagrams of FIG. 3F shows the percentage of CD4+CAR+ cells positive for IL-13 after PMA/ionomycin by ICS.

[0082] The block diagrams of FIG. 3G show the frequency of apoptosis marker (annexin V) positive cells among injected CD8+CAR+ cells.

[083] The block diagrams of FIG. 3H show FOXP3 levels by qPCR.

[084] The block diagrams of FIG. 3I show the total number of injected CD3+CAR+ negative apoptosis marker (annexin V) cells.

[0085] The block diagrams of FIG. 3J show the number/kg of injected CD3+CAR+ negative for the apoptosis marker (annexin V–) cells.

[0086] The block diagrams of FIG. 3K shows the viable cell concentration for the cryopreserved drug product (CDP).

[0087] The block diagrams of FIG. 3L show the total number of infused cells.

[0088] The block diagrams of FIG. 3M shows IL2 ICS in CD4+CAR+ cells.

[0089] The block diagrams of FIG. 3N show the frequency of apoptosis marker (annexin V) positive cells among injected CD3+CAR+ cells.

[0090] The block diagrams of FIG. 3O shows the frequency of FOXP3+ cells among CD4+ cells.

[0091] The block diagrams of FIG. 3P show the frequency of CD3+ cells.

[0092] The block diagrams of FIG. 3Q shows the number/kg of CD8+CAR+ cells injected.

[0093] The block diagrams of FIG. 3R shows non-antigen/CAR specific IL-10 production per total cells (CD3/28 stimulated).

[0094] The block diagrams of FIG. 3S show ICS TNF-alpha for CD4+CAR+ cells.

[0095] The block diagrams of FIG. 3T shows ICS for TNF-alpha CD8+CAR+ cells.

[0096] The block diagrams of FIG. 3U shows non-antigen/CAR specific IL-6 production per total cells (CD3/28 stimulated).

[0097] The block diagrams of FIG. 3V show the number of injected CD3+CAR+ cells/kg.

[0098] FIG. 4 shows block diagrams quantifying TNF-alpha production following CD19 stimulation with cell compositions containing cells expressing anti-CD19 CAR. The box plot on the left compares measurements for cell compositions derived from individuals who developed grade 4 or less neurotoxicity with measurements for cell compositions derived from individuals who experienced grade 5 neurotoxicity. The box plots on the right compare measurements for cell compositions derived from individuals who experienced CR, with measurements for cell compositions derived from individuals who experienced NR. Measurements for cell compositions derived from individuals whose response is not available are also shown in this diagram (NA).

[0099] FIG. 5A-B are block diagrams quantifying cytokine production following CD19 stimulation with cell compositions containing cells expressing anti-CD19 CAR. The box plot on the left compares measurements for cell compositions derived from individuals who experienced grade 4 or less neurotoxicity with measurements for cell compositions derived from individuals who experienced grade 5 neurotoxicity. The box plots on the right compare measurements for cell compositions derived from individuals who experienced CR, with measurements for cell compositions derived from individuals who experienced NR. Measurements for cell compositions derived from individuals whose response is not available are also shown in this diagram (NA).

[0100] The block diagrams of FIG. 5A shows antigen-stimulated IL-2 production.

[0101] The block diagrams of FIG. 5B shows antigen-stimulated IFN-gamma production.

[0102] FIG. 6A and 6B show two-dimensional scatterplots of the number of apoptosis marker- (annexin V-) CD8+CAR+ cells at the administered dose and antigen-stimulated production of TNF-alpha. Data points for cell compositions derived from individuals who experienced neurotoxicity of grade 4 or less (circles) or grade 5 (triangles) are shown in FIG. 6A. Data points for cell compositions derived from individuals who experienced grade 0-2, 3 neurotoxicity, grade 3 long-term neurotoxicity (3p), grade 4 and 5 neurotoxicity are shown in FIG. 6b. FIG. 6B shows data points associated with individuals who developed grade 5 neurotoxicity and were treated with anti-lymphocyte chemotherapy using cyclophosphamide (CY) or fludarabine and cyclophosphamide (Flu/CY). The oval indicates the range of these parameters for most of the 11 exemplary cell compositions evaluated in a clinical study in which none of the individuals showed grade 5 neurotoxicity or cerebral edema.

[0103] FIG. 7 shows a two-dimensional scatterplot of the number of apoptosis marker-(Annexin V-) CD8+CAR+ cells at the administered dose and antigen-stimulated production of TNF-alpha. Data points are shown for compositions of cells derived from individuals who experienced neurotoxicity of grade 4 or less (squares and circles), or grade 5 (triangles). Cell compositions derived from individuals from the additional clinical study are shown (squares).

[0104] FIG. 8 is a graph showing individual data points for the number of administered apoptosis marker (annexin V)-CD8+CAR+ cells x TNF-alpha production for individuals who experienced varying degrees of neurotoxicity.

[0105] FIG. 9 is a graph showing individual data points for the number of administered apoptosis marker- (annexin V-) CD8+CAR+ cells x TNF-alpha production for individuals with different responses.

[0106] FIG. 10 is a graph showing individual data points for the number of administered apoptosis marker- (annexin V-) CD8+CAR+ cells x IFN-gamma production for individuals who experienced varying degrees of neurotoxicity.

[0107] FIG. 11 is a graph showing individual data points for the number of administered apoptosis marker- (annexin V-) CD8+CAR+ cells x IFN-gamma production for individuals with different clinical responses.

[0108] FIG. 12 is a graph showing individual data points for the number of administered apoptosis marker- (annexin V-) CD8+CAR+ cells x IL-2 production for individuals who experienced varying degrees of neurotoxicity.

[0109] FIG. 13 is a graph showing individual data points for the number of administered apoptosis marker- (annexin V-) CD8+CAR+ cells x IL-2 production for individuals with different clinical responses.

[0110] FIG. 14A and 14B are graphs showing individual data points for the number of administered apoptosis marker- (annexin V-) CD8+CAR+ cells (A) x normalized TNF-alpha production (B) for individuals who experienced varying degrees of neurotoxicity (FIG. 14A ), or for individuals with different responses (FIG. 14B).

[0111] In FIG. 15A and 15B are graphs showing individual data points for the number of administered apoptosis marker- (annexin V-) CD8+CAR+ cells (A) x normalized IFN-gamma production (B) for individuals who experienced varying degrees of neurotoxicity (FIG. 15A ), or for individuals with different responses (FIG. 15B).

[0112] FIG. 16A and 16B are graphs showing individual data points for the number of administered apoptosis marker- (annexin V-) CD8+CAR+ cells (A) x normalized IL-2 production (B) for individuals who experienced varying degrees of neurotoxicity (FIG. 16A ), or for individuals with different responses (FIG. 16B).

[0113] FIG. 17A and 17B are graphs showing individual data points for the number of injected apoptosis marker-(annexin V)-CD8+CAR+ cells (A) x normalized production of TNF-alpha and IFN-gamma (B) for individuals who experienced varying degrees of neurotoxicity. (FIG. 17A), or for individuals with different responses (FIG. 17B).

[0114] FIG. 18A and 18B are graphs showing individual data points for the number of administered apoptosis marker- (annexin V-) CD8+CAR+ cells (A) x normalized TNF-alpha and IL-2 production (B) for individuals who experienced varying degrees of neurotoxicity. (FIG. 18A), or for individuals with different responses (FIG. 18B).

[0115] FIG. 19A and 19B are graphs showing individual data points for the number of administered apoptosis marker- (annexin-) CD8+CAR+ cells (A) x normalized IFN-gamma and IL-2 production (B) for individuals who experienced varying degrees of neurotoxicity ( FIG. 19A), or for individuals with different responses (FIG. 19B).

[0116] FIG. 20A and 20B are graphs showing individual data points for the number of administered apoptosis marker- (annexin-) CD8+CAR+ cells (A) x normalized TNF-alpha, IFN-gamma, and IL-2 production (B) for individuals in which experienced neurotoxicity of varying degrees (FIG. 20A), or for individuals with different responses (FIG. 20B).

[0117] FIG. 21A and 21B are graphs showing cell phenotypes from compositions containing anti-CD19+ CAR cells prepared by the method described in Example 1 (A) and the alternative method described in Example 2 (B). FIG. 21A is a block diagram showing the percentage of apoptosis marker- (annexin V-) cells among CD8+CAR+ cells. FIG. 21B is a graph showing individual cell composition values for dosed annexin-negative CD8+CAR+ cells and cytokine production for cell compositions prepared by Illustrative Method A and Illustrative Method B. The shaded boxes show the observed distributions.

[0118] FIG. 22 shows individual data points for the number/kg of CD3+CAR+ cells (x10 ⁶ cells/kg) administered to individuals who developed grade 0-2 or grade 3-5 neurotoxicity.

[0119] FIG. 23A-C are block diagrams comparing T cell compositions containing cells expressing anti-CD19 CAR. FIG. 23A and 23B are block diagrams comparing T cell compositions containing cells treated with the same reagent from different batches during production. FIG. 23A quantifies TNF-alpha production by the cell after stimulation with CD19. FIG. 23B shows the frequency of CD27+/CD28+ cells among CD8+CAR+ cells. Data points are shown for T cell compositions associated with grade 0-2 neurotoxicity (squares), 3 (stars with arrows), long-term grade 3 neurotoxicity (3p; diamonds), grade 4 neurotoxicity (pentagons with arrows), and 5 (circles). . FIG. 23C shows TNF-alpha production after stimulation with CD19-expressing cells in T-cell compositions containing anti-CD19 CAR-expressing cells prepared using the same media stored under different conditions.

[0120] FIG. 24 is a box plot showing the frequency of T cells in CD4+ and CD8+ enriched T cell compositions at various steps in the process for preparing modified cell compositions containing CAR T cells described in Example 8. Frequency (% of total) is shown. leukocyte counts) of CD4+ and CD8+ cells in the compositions.

[0121] FIG. 25 is a graph showing CD3+ T cell levels at individual doses at dose level 1 (DL1) and dose level 2 (DL2).

[0122] FIG. 26 is a block diagram showing the concentration, viability, and frequency of caspase-3 negative CD4+ and CD8+ T cells in therapeutic cell formulations containing CAR-T cells at high or low formulation volume.

[0123] FIG. 27 shows graphs showing controlled dose, T cell phenotypes, and cell specific function of an individual cell composition containing CAR T cells that were infused. Data points are shown for the amounts of CD4+CAR+, CD8+CAR+, CD4+CAR+TNF-α, CD8+CAR+TNF-α, CD4+CAR+CCR7+, CD8+CAR+CCR7+ infused into individual individuals.

[0124] FIG. 28 is a graph showing (i) for each of a number of individuals, the individual unboxed dots show the maximum PK score (number of CAR+ T cells/μl of blood (PK score) at peak (individually, time point at which measured the highest number of CAR+ T cells, among the time points at which the number of CAR+ T cells was assessed after an individual was treated with compositions expressing CAR against CD19 T cells) (where the points also indicate the highest degree of neurotoxicity observed for each individual: degree neurotoxicity 0–2 (squares), grade 3 (stars), long-term grade 3 neurotoxicity (diamonds), grade 4 (arrowed pentagons) or grade 5 (circles) neurotoxicity, and (ii) median levels, for those of these patients who appeared to have an early or late CAR+ T-cell peak (shown as upper and lower dot-boxed lines with range bars, respectively) IL-1 5 (picograms/ml) in serum at various indicated time points before and after administration of such T cell compositions. The oval shows individuals who were observed to have early, rapid proliferation of CAR-T cells compared to other individuals.

Detailed description

[0125] The entire contents of all publications, including patent documents, scientific articles, and databases, referenced in this application are incorporated by reference for all purposes to the same extent as if the contents of each individual publication were individually referenced. . If the definition given in the present description conflicts with or otherwise does not correspond to the definition given in patents, applications, published applications and other publications given in the present description by reference, the definition given in this description takes precedence over the definition given in the present description as a reference.

[0126] The section headings used in this specification are for organizational purposes only and should not be construed as limiting the subject matter described.

I. DOSES AND DOSING OF T-CELL THERAPEUTIC COMPOSITIONS TO REDUCE THE RISK OF TOXICITY

[0127] The present invention relates to methods, compositions and articles of manufacture for use in relation to cellular therapy, such as therapy using modified T-cells, for the treatment of diseases and conditions, including various tumors. The present embodiments relate to T cell therapeutic compositions containing modified T cells, such as cells modified to express recombinant proteins, such as cells expressing recombinant receptors engineered to recognize and/or specifically bind molecules associated with a disease or condition, and leading to a response, such as an immune response against such molecules, upon binding to such molecules. Receptors may include chimeric receptors, such as chimeric antigen receptors (CARs), and other transgenic antigen receptors, including transgenic T cell receptors (TCRs).

[0128] The present embodiments, in some aspects, relate to aspects in which certain parameters associated with the function or activity of a T cell therapeutic composition, for example, antigen-specific or recombinant receptor-dependent activity, can predict the risk of an adverse event or toxicity occurring. , such as severe neurotoxicity, in an individual subjected to the administration of the T cell composition and/or may provide information about the activity of the therapeutic T cell composition. Aspects of the present embodiments also relate to administering or providing a unit dose containing a number of units, such as a target number of units that is a function of the number of cells of a particular phenotype, such as a phenotype indicative of biologically active cells or cell populations, and cell function or activity. present in the T cell therapeutic composition, eg, antigen specific or recombinant receptor dependent activity, including the amount to be used in relation to dosing of the T cell therapeutic composition. In some aspects, the present methods can alleviate or reduce the risk of toxicity in an individual subjected to a dose of a T cell therapeutic composition while still maintaining the activity of the T cell composition. The present invention also relates to cell-containing products designed to be administered following such dosing regimens.

[0129] In some embodiments, the methods, doses, unit doses, compositions, and articles of manufacture presented are based on observations, which may provide advantages in taking certain parameters and their combinations into account when determining an appropriate dose for cell therapy and/or releasing cell compositions for therapy. . In particular methods available, doses are based on numbers of particular cell types, such as cells modified to have a particular activity, such as cells positive for the modified receptor. For example, in specific available methods and doses, the dose is based on an observed or suspected relationship between the number (or number per patient weight) of viable modified T cells, or a subset thereof, such as viable, cytotoxic (e.g., CD8+) modified T cells. In various contexts, such amounts may be related to efficacy and/or safety outcomes such as response and/or risk of toxicities such as neurotoxicity (NTx), cerebral edema and CRS. The present invention relates to embodiments based on the observation that despite this, such indicators, in some contexts, do not provide a consistent adequate prediction of the risk of types of toxicity, for example, severe neurotoxicity or cerebral edema, in particular, without taking into account other variables . Accordingly, methods for determining dose and evaluating product for release based on such indicators alone may not be entirely satisfactory. For example, such methods may, in some contexts, fail to define an appropriate safety margin and/or therapeutic window for safe and effective cell therapy. The present invention relates to methods (including methods of treatment, dosing, dose determination and release analysis) and doses, and therapeutic compositions aimed at overcoming such shortcomings.

[0130] In some aspects, the present work has found that certain product-specific features and clinical or patient-specific factors may affect the risk, relative risk, or likelihood of toxicity following cell therapy. In some instances, variability in such product-specific and/or patient-specific features may result in variability in features of cell therapeutic composition doses administered to different individuals within a group of individuals and/or outcomes observed in such different individuals following such administration. In some aspects, such variability is observed despite certain similarities in doses administered to different individuals, for example, when the administered dose or composition contains cells modified to express the same modified receptor, such as a chimeric antigen receptor, containing the same vector, or transduced or transfected using the same vector; in some aspects, different doses or compositions administered to different individuals include or contain or consist of the same number (or number per weight or other characteristic of the individual) of cells (or cells of a particular phenotype). In some aspects, controlling for variability in one or more features of cell compositions can minimize such risks and/or minimize such variability in dose or outcome features. In some aspects, minimizing or controlling variability, such as the degree of variability (or increase in uniformity), of one or more product or composition features among individuals subjected to a particular therapy may minimize the impact of variability on outcome or dose features among patients with patient-specific variability. factors or clinical factors that may otherwise influence such outcome measures or doses. For example, in some aspects, reducing the variability of a product or composition feature(s) may reduce the variability in risk or likelihood, or may reduce the risk or likelihood of an individual or population of individuals developing treatment-related toxicity, such as neurotoxicity or cerebral edema, or their degree, for example, without reducing or significantly reducing the likelihood of achieving a particular response or clinical outcome. In some cases, reducing the variability attributed to clinical or patient-specific factors may minimize the risk of toxicity, such as neurotoxicity or cerebral edema, following cell therapy.

[0131] In some aspects, variability among unit doses of the cell composition may be due to one or more aspects of the manufacturing processes used in obtaining or manufacturing a therapeutic agent based on modified cells. In some cases, changes to the raw material or its handling or storage may influence the variables observed in this work that influence the risk of toxicity and/or outcomes. In some aspects, variability from batch to batch or during storage/handling of raw materials, and/or the use of different types of raw materials among the methods carried out for a number of individuals, can be influenced, for example, by increasing variability, or increasing or decreasing certain aspects of the obtained compositions of cells such as aspects that, if changed, may lead to variability in the risk of toxicity or clinical outcomes among individuals undergoing cell therapy methods, in particular among those individuals who differ in certain patient-specific features. In some embodiments, the present invention relates to methods including evaluating, testing, and/or monitoring the potential impact or variability for one or more such product features or risks, or likelihood, due to (e.g., one or all of) raw materials (multiple raw materials), batch(es), reagent(s) or their storage or handling or modification. In some embodiments, such methods include assays, such as assays conducted prior to the use of such raw material, batch, storage, or manipulation, upon preparation of the cell composition to be administered to an individual, or prior to such administration. In some aspects, assays evaluate the effect of a raw material, batch, change, or method of manipulation or storage, on one or more traits of cell composition, such as traits that were observed in this work to affect the risk of toxicity or outcome, or aggravate the effect of variability. from patient to patient of such outcomes. In some aspects, the assays evaluate whether there is an acceptably low degree of variability compared to another material, batch, or method of storage or manipulation, or an acceptably low effect on such one or more features. In some aspects, a raw material, batch, or method of storage or manipulation is released for the manufacture of a cell-based therapeutic agent to be administered to a patient - or the patient is subjected to such cell therapy - if, for example, only if, or only after the analysis confirms, that such variability or influence is within an acceptable range or value or limit. In some of any such embodiments, such estimated parameters may include the number or percentage of biologically active cells, non-apoptotic cells, healthy cells, cells with a certain activity (or the number or percentage of biologically active, non-apoptotic or healthy, or active cells of a given phenotype), and /or an indicator indicative of activity, such as activity per cell, of the composition. In some embodiments, determining that the variability of one or more of these traits is below a certain level using a new raw material or batch, or storage method or manipulation, may reduce the risk of toxicity or decrease in response after the introduction of such new raw material or batch, or storage method, or manipulations. In some embodiments, among such methods to which the present invention pertains are methods that analyze the composition prior to product release and/or adjust the dosing strategy based on such parameters.

[0132] In some embodiments, one or more risks or outcomes, such as the risk of toxicity, or the appropriate dose may depend on or vary with one or more treatment- or patient-specific variables. For example, among illustrative clinical signs and patient signs that may correlate, in some embodiments, with the risk of certain toxicities, including neurotoxicity or cerebral edema, or their degrees, included the general health or age of the patient (for example, whether the patient's age is greater than or less than or equal to 30 years) prior to treatment history, including the number of previous treatment cycles and/or whether the patient was subjected to antilymphocyte and/or maintenance chemotherapy prior to treatment, such as treatment including fludarabine. In particular, among those patient or clinical signs that may increase the risk, relative risk, and/or likelihood of toxicity following a dose of cells in cell therapy (e.g., CAR-T-cell therapy), for example, fewer cycles of previous therapy, for example, two or less cycles of previous therapy, young age, for example, age younger than 30 years, the ratio of CD4+ and CD8+ T cells in a sample from a patient after apheresis, or the number of cells injected into an individual, due to cell dosing based on mass, for example, where more cells are administered to individuals with greater mass. Such features may influence the resulting features of the modified cell compositions obtained from the individual and/or affect the patient's infusion features, which may contribute to the risk of toxicity following cell therapy (eg, using CAR-T cells). The present invention relates to methods that reduce the variability in outcomes and/or the risk of toxicity among patients with variability in one or more of these patient-specific or treatment-specific features. The present invention also relates to methods taking into account such patient- or treatment-specific features when determining or formulating or administering a dose of a cell-based therapeutic agent to a patient or patients.

[0133] In some embodiments, the methods presented, such as routes of administration, and dosages include (or include administering) fewer cells, or fewer biologically active or active cells (or modified cells) per dose administered to an individual who is healthier, is younger, e.g., less than 30 years of age, fewer cycles of prior therapy, e.g. two or fewer cycles of prior therapy, or had certain prior therapy such as anti-lymphocyte therapy or chemotherapy such as including fludarabine, and/or transplant. In other embodiments, the dosing process or method features contribute to the variability of such patient-specific factors, such as such that such patient-specific factors do not lead to variability or do not lead to significant variability in toxicity or other risk. In some embodiments, the methods provided include using a fixed dose, e.g., total CAR+ cells, total CAR+CD8+ T cells, and/or CAR+/CD4+ T cells, so as to administer a precise or fixed dose of that type(s). ) cells in each of the groups of individuals being treated, including individuals of variable weight. In some aspects, patient-specific variables are important for dosing if the therapy is autologous, such that the properties of the patient's cells may be important for the health status or function or endurance of the cells in the composition.

[0134] In some aspects, certain patient characteristics or clinical factors may increase the proliferation of cells introduced by cell therapy in the individual who was subjected to therapy, which, in some cases, may contribute, such as an increase, or may be associated with risk or associated with an increased risk, relative risk, or likelihood of toxicity, such as neurotoxicity, following cell therapy. In some embodiments, the patient's characteristics or clinical factors associated with the risk of toxicity, such as neurotoxicity or cerebral edema, include disease type (eg, ALL); disease burden (eg, as determined by volume measurement or the presence of an inflammatory marker); whether the individual has been subjected to prior transitional chemotherapy or antilymphocyte therapy such as those involving fludarabine (Flu) or cyclophosphamide and fludarabine (CY/Flu); peak early proliferation of recombinant receptor (eg, CAR) T cells in the blood, eg, within 2, 3, 4, 5, 6, or 7 days after initiation of cell therapy; and/or the presence of peak levels of IL-15, eg, greater than or equal to 30 pg/ml, in blood or serum, eg, within 2, 3, 4, 5, 6, or 7 days after initiation of cell therapy. In some embodiments, the present methods, such as the present methods and/or dosing methods, such as methods for controlling dose and/or variability, may minimize or reduce such risks, and/or minimize the effect of patient symptoms or clinical factors on such risks or outcome. . Among these methods are methods in which such factors are taken into account when adjusting or determining the dose for such individuals, and methods in which process or dosing features are used that minimize the effect of such factors on the risk of toxicity or outcome.

[0135] In some embodiments, the present compositions, dosages, and methods provide the advantage that they can limit variability in the dose of cells administered to individuals, factors that have been observed to correlate with adverse events, e.g., types of toxicities, such as types of neurotoxicity. including severe neurotoxicity and/or cerebral edema. Such variability is limited or reduced, in some aspects, by a dose or dose determination method that takes into account or corrects for such variability, such as dosing based on cell number or cell phenotype, which has a property known to be correlated with an adverse event. In some aspects, variability is limited by using a release analysis that takes into account such variability. In some embodiments, in addition to the numbers of modified T cells, the dose and/or release of the product is further based on one or more of a combination of important variables. Such indicators, in some aspects, include parameters indicative of the degree to which viable cells are biologically active or healthy (e.g., not programmed to die), which, in some aspects, is determined based on the absence of a detectable apoptotic marker and/or on based on relatively low levels of intracellular production of specific factors compared to dying or apoptotic cells. In some aspects, the indicators include the frequency of modified CD8+ cells, as opposed to taking into account only the number of modified T cells in general and/or the ratio of CD4:CD8 cells among modified cells. In some aspects, indicators include parameters indicative of the degree of antigen or cell-specific activity present in the composition, such as indicators of the degree to which cells in the composition are capable of exhibiting particular activities or functions, such as the secretion of particular factors (such as pro-inflammatory cytokines). ) in response to antigen- or cell-specific stimulation.

[0136] In some embodiments, the dose is based on the number of biologically active modified T cells; in some aspects, biologically active refers to a property of cells not programmed to undergo cell death, such as a property of non-apoptotic cells or cells that do not show signs of entering the apoptotic pathway. In some aspects, the dose is based on the number of biologically active modified CD8+ T cells, and/or when the dose is based on the total number of biologically active modified T cells, an upper limit or threshold number of biologically active modified CD8+ T cells is also indicated.

[0137] In some embodiments, ensuring that a unit dose contains a relatively constant amount of biologically active modified cells is achieved in part by a method for producing cells exhibiting variability in such a biologically active population that is low, or below acceptable or threshold variability, among compositions obtained by this method, including compositions obtained from samples from a number of different individuals, for example, individuals having different characteristics, such as individuals of different ages, with different number of cycles and / or types of previous therapy, and indications and their subtypes or severity, or degree. In some aspects, the frequency of such a biologically active population is obtained in this way, which differs by no more than 20%, or no more than 10%, or no more than 5%, from the average of the specified frequency in the set of compositions of T cells obtained by this method, and/or differs from such mean by no more than one standard deviation, or differs by no more than 20%, or no more than 10%, or no more than 5%, among the plurality of T cell compositions obtained in this way, among such diverse samples and patients. In some such aspects, the dose is based on the number (or number per patient weight or height or blood volume) of modified T cells or modified CD8+ T cells, taking into account the expected frequency of biologically active or non-apoptotic cells among such cells, and/or variability in such frequency among compositions of cells modified from representative populations of individuals. In some aspects, the population size of the cells in the dose is chosen to provide an adequate margin of safety, eg, below a threshold, taking into account such variability and frequency.

[0138] In some embodiments, the dose and/or release of the product is further based on a measure indicative of activity, e.g., a specific measure of antigen or cell specific activity providing information regarding the risk of an adverse event or toxicity. In some aspects, such a measure is or includes a parameter, as observed or as described herein, that correlates with the risk of an adverse event, such as severe neurotoxicity or a specific grade (e.g., grade 3 neurotoxicity, long-term grade 3 neurotoxicity, grade 4 neurotoxicity, and /or degree 5), or cerebral edema. In some aspects, the parameter is indicative of the modified cell or antigen specific release of a pro-inflammatory cytokine and/or one or a combination of TNFa, IFNg, IL-2 and IL-10.

[0139] In some embodiments, the method further takes into account variability (eg, among cells modified from different individuals and/or to use different feedstocks, such as activation reagents, in the method). For example, in some embodiments, a parameter indicative of cell-specific activity or specific activity is normalized to a similar parameter, which may vary among such individuals or methods, but is not predictive of poor outcome. For example, a parameter indicative of cell-specific activity (e.g., a measure of antigen-specific secretion of a pro-inflammatory cytokine) may be normalized against or relative to another parameter, such as antigen or cell-specific factor release, in some aspects, that does not correlate with an undesirable outcome or is not predicts an undesirable outcome such as severe or some degree neurotoxicity (eg, Grade 3, Grade 3 long-term, Grade 4, and/or Grade 5), or cerebral edema. For example, it has been observed that, in some embodiments, the secretion of cytokines such as IL-5, IL-13, GM-CSF, and IL-6 does not correlate with or predict undesirable outcome. In response to a cell-specific antigen, while it may vary among cells modified from different individuals (eg, variability from patient to patient), variability in this parameter does not generally correlate with risk or predict the risk of an adverse outcome such as severe neurotoxicity or some degree of neurotoxicity (eg, grade 3, long-term grade 3 neurotoxicity, grade 4 and/or grade 5 neurotoxicity), or cerebral edema. In some embodiments, secretion of one or more of these cytokines may serve as a control factor, such as the arithmetic or geometric mean of two or more of IL-5, IL-13, GM-CSF, and IL-6.

[0140] In some aspects, currently available methods are not satisfactory for characterizing both the safety and efficacy of a T cell therapeutic composition. For example, in some cases, current methods use a lower release threshold parameter (eg, an interferon-gamma release rate for a T cell therapeutic composition) that does not include an upper safety limit. In some cases, a T cell therapeutic composition is not released for further use as a therapeutic agent if it does not meet the lower threshold; for example, if a T cell therapeutic composition does not release above a threshold amount of a cytokine in a particular assay, the composition is not released for infusion into the patient. In some aspects, such parameters may include cytokine release, release of cytotoxic granules, and determination of the T cell's activation state (eg, a specific percentage of cells expressing one or more activation markers). In some aspects, currently used parameters include lower limits on the percentages of CAR+ cells and/or cells having a specific phenotype. In some methods, it is possible that while efficacy can be controlled by ensuring that the T cell therapeutic composition has a minimal activity (i.e., releases a minimal amount of cytokine and/or exhibits a certain level of activity in a cytotoxicity assay) or phenotype whether or not a composition is safe to administer is not determined using an upper limit of similar parameters.

[0141] The present embodiments are based on findings that specific phenotypes and/or parameters associated with the function or activity of a T cell therapeutic composition are associated with a risk of toxicity, e.g., severe neurotoxicity, in an individual treated with a T cell composition. . In some aspects, it has been found that the endurance of the injected cells is a trait correlated with the risk of toxicity. In some embodiments, the number or dose of cells administered to an individual that have a phenotype indicative of biological activity, such as cells that do not have an apoptotic phenotype and/or have a phenotype that does not exhibit a parameter indicative of apoptosis (e.g., a parameter indicative of early or late apoptosis, such as detected by annexin V) and/or producing specific cytokines (eg, pro-inflammatory cytokines) may correlate with the risk of toxicity. In some aspects, individuals administered a greater total number of cells, such as based on total CD3+CAR+ cells or CD8+CAR+ cells, or subsets thereof, may be at higher risk of developing severe toxicity than an individual administered a lower total the number of such cells. In some cases, specific dosing regimens that do not take into account the total number of cells with such traits, and/or in which there is a high degree of variability in the phenotype or function of the injected cells among a group of treated individuals, may, in specific aspects, lead to a general risk the occurrence of toxicity in treated individuals.

[0142] In some aspects, it has been found that a lower total dose of injected cells exhibiting such a phenotype may be desirable, for example, to alleviate or minimize the risk of toxicity. However, while such cell traits may affect the risk of toxicity in an individual treated with the cells, administration of too few such cells may reduce the effectiveness of the therapeutic composition. To account for efficacy, it is common for dosing methods to set a lower limit on the activity of a cell composition, e.g., based on cytolytic activity, cytokine production, or other factor associated with the T cell therapeutic composition, e.g., to ensure that enough cells are administered to an individual to achieve effect. The studies described herein, however, show that dosing methods that administer a dose of cells, including a fixed dose of cells that correspond only to a lower level of activity or function, may put an individual at risk of toxicity if such cells have a high level of antigen-specific activity or functions.

[0143] Based on the findings in the present description, the present invention relates to methods, compositions and articles in which a single dose of cells is based on two functions, (A) the number of cells of a particular phenotype, such as a phenotype indicative of biological activity, and (B ) a parameter value that indicates or correlates with the recombinant receptor dependent activity and/or antigen specific activity in the composition. Illustrative variables found in this work to be indicative of A or B are described elsewhere in this specification, including examples. In some embodiments, a B function, such as the production or accumulation of an antigen-specific cytokine, e.g., a pro-inflammatory cytokine, is a function that can often differ and/or be variable among T cell compositions derived from cells derived from individuals, including specific for the patient, risk factors, for example, due to a particular disease or condition of the individual, the age of the individual, the weight of the individual, previous treatment, and other factors that can change or contribute to one or more functional traits of T cells.

[0144] In some embodiments, B is found to be an indicator of the safety of the T cell therapeutic composition. In some cases, if B is too high, a lower dose of cells should be administered to reduce or minimize the risk of toxicity, such as severe neurotoxicity. In some cases, if B is below a threshold level or limit, there may be a larger window for the dose that can be administered so that the target dose of cells can be administered to an individual without a known risk of toxicity or an adverse event, such as severe toxicity. In some aspects, controlling or adjusting the total number of cells or the total number of cells of a particular phenotype (e.g., CD3+CAR+ or CD8+CAR+ or Annexin V-CD8+CAR+) in a T cell therapeutic composition that is administered may standardize or adjust the total number units of activity of administered T cell compositions among treated individuals, thus allowing for functional differences or activity differences from B in T cells among T cell compositions derived from a group of individuals that might otherwise lead to a risk of toxicity, e.g. , severe toxicity, in some individuals.

[0145] In some embodiments, B, which is a recombinant receptor-dependent (eg, CAR-dependent) parameter value, eg, antigen-specific activity, may provide an activity control for a T cell composition. In some aspects, assaying activity based on T cell function from B may provide advantages over existing methods. First, in some aspects, existing activity assays for cell compositions are based on features that correlate with cell efficacy, eg, cytolytic activity and/or IFN-gamma production, as opposed to safety. In addition, such activity assays often only have a lower limit or level of activity within the assay.

[0146] Based on the findings in the present description, among the presented embodiments, there are activity assays, such as assays in which there is a lower specified limit (LSL) and/or an upper specified limit (USL). In some aspects, the USL is based on the B parameter. In some aspects, the USL is provided to test the safety of the T cell composition, for example, in combination with a release assay prior to administering the composition to an individual. As noted above, various clinical criteria and other patient-specific features may influence the level or degree of function of the modified cells. Similarly, in some instances, the overall method, including the reagents used in conjunction with the manufacture or modification of the cell composition and/or freezing of the cells produced by the cell method, may also result in B variability among multiple T cell compositions obtained with using the same method. In some cases, it has been found that freezing cells below a density of less than 15×10 ⁶ cells/ml can lead to increased variability and/or decreased activity of the T cell composition. In some embodiments, the present methods are particularly advantageous for evaluating cell compositions in which the variability of cells having a particular phenotype, such as the A phenotype described below, including a phenotype indicative of biological activity, e.g., annexin V-CD8+CAR+, is different or is likely to differ by more than 20%, more than 30%, more than 40%, more than 50% or more among the set of compositions of T cells obtained by this method, or from the average frequency of the phenotype in the set of compositions T-cells obtained by this method and/or differs or is likely to differ from such an average by no more than one standard deviation.

[0147] In some embodiments of the presented methods and doses, cells are obtained by a method designed to minimize the variability of factors, such as factors observed in this work, correlated (singly or in combination) with the risk of neurotoxicity. In some aspects of such embodiments, dosing may be based on the total number or relative number of modified cells or modified T cells, or T cell population, without increasing the risk of toxicity that may be present when cells are obtained using a method in which such parameters are more variable. In some aspects, such methods include the use of lentiviral vectors, specific ratios of T cell subsets such as CD4 and/or CD8 cells. In some aspects, they involve constructing cells that include a CAR with a 41BB-derived co-stimulatory domain as opposed to a CD28 domain. In some aspects, the method includes controlled ratios of T cell subsets, cytokines, and reagents to provide control of phenotype, function, and metabolic status) and/or controlled and relatively higher overall cell density during cryopreservation (e.g., between 10 and between 60 or between 15 and 60 million cells per ml, including each), storing and/or thawing the sample before administration. In some aspects, a method is used in which the metabolic and functional properties of the T cells are more easily controlled. In some embodiments, such methods are used to provide uniform doses of biologically active cells, even in the context of patient-specific variables that may affect cell health.

[0148] Among the embodiments are those in which the methods obtain a higher frequency of biologically active or healthy cells among CD8+CAR+ cells, as measured by the negativity of various apoptotic markers among the samples obtained using this method. In some aspects, there is a lower degree of variability in this parameter among samples obtained by this method from different individuals. As observed in this work, the number of such cells may correlate with grade 5 neurotoxicity and cerebral edema, while CD3+CAR+ viable cells (not considering the apoptotic state compared to biologically active cells) may not always predict toxicity or clearly define the boundary. security for that risk.

[0149] In some embodiments, using a method with reduced variability in the frequency of such biologically active cells among modified cells reduces the risk that particular patients, such as patients having particular patient-specific risk factors as described (e.g., patients having cells that are less prone to apoptosis or that are healthier) may inadvertently deliver a higher dose than intended of biologically active cells when dosed based on the number of modified T cells overall. In addition, methods with greater control of phenotype and function have been observed to reduce the degree of variability in the ability of cells obtained by this method to produce pro-inflammatory cytokines in an antigen-specific manner. The results herein are consistent with the interpretation that, in particular, in combination with the amounts of biologically active modified cells, a dosing method taking into account such cell-specific activity parameters (e.g., such that a specific target range of such activity - or not more than threshold - presented at a given dose) can be used to provide a dose capable of providing a desired clinical or therapeutic outcome while still remaining within the safety margin or reducing the risk of undesirable toxicity, such as neurotoxicity.

[0150] In some embodiments, the use of a method in which higher frequencies of biologically active modified cells are consistently obtained allows the use of cell doses that are much lower (in terms of numbers of modified, e.g., CAR+ cells) compared to other dosing methods. in which, more frequently, the modified cells are positive for apoptotic markers or are otherwise less healthy. For example, based on the observations herein, and given the observation that available dosing methods generally do not take into account the frequency of apoptotic cells, in some embodiments, the numbers of modified (e.g., CAR+) T cells (e.g., modified CD8+ and/or CD4+ cells) are as low as 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 million cells.

[0151] The present methods provide a release assay that can monitor or evaluate cell compositions to determine if a composition is administered to an individual, whether it is likely to correlate with the risk of toxicity, such as severe neurotoxicity. In some embodiments, the T cell composition is not recommended to be administered to the individual and/or the unit dose to be administered to the individual is changed or adjusted if B exceeds USL. If B is below USL, the product can be released for treatment. In some embodiments, assessing whether B is above LSL and/or below USL may also provide information about whether an individual who has previously received a T cell composition is at risk of developing toxicity, such as severe toxicity. In some embodiments, a sample of the T cell composition administered to an individual is evaluated for B after administration. In some embodiments, the individual to whom the T cell composition has been administered is determined to be at risk for toxicity, such as severe neurotoxicity, if B is equal to or greater than the USL. In such embodiments, the individual to whom the composition has been administered is monitored and/or treated with an agent to reduce or reduce the likelihood of a toxicity outcome, such as neurotoxicity or cytokine release syndrome, following administration of the cell composition, and optionally, prior to the onset of a sign or symptom of a toxicity outcome. .

[0152] In some aspects, the present embodiments are based on observations that the efficacy of adoptive cell therapy may be limited due to the occurrence of toxicity in an individual administered such cells, where the toxicity, in some cases, may be severe. For example, in some instances, administration of a dose of cells expressing a recombinant receptor, eg, CAR, may result in or risk of toxicity, eg, CRS or neurotoxicity. In some cases, while a higher dose of such cells may increase the effectiveness of the treatment, for example, by increasing the effect of the cells, for example, by promoting proliferation and/or persistence, they may also lead to an even higher risk of toxicity or more severe toxicity. . And also, in some cases, individuals with a higher disease burden may also be at higher risk of toxicity or more severe toxicity.

[0153] Specific available methods of treating or alleviating toxicity may not always be completely satisfactory. Many such methods focus on, for example, targeting the downstream effects of toxicity, for example by blocking cytokines, and/or delivering agents such as a high dose of steroids, which can also terminate or impair the function of the injected cells. In addition, such methods often include such interventions only upon detection of physical signs or symptoms of toxicity, which typically include signs or symptoms of moderate or severe toxicity (eg, moderate or severe CRS). Many of these other methods also fail to prevent other forms of toxicity, such as neurotoxicity, that may be associated with adoptive cell therapy. In some cases, this occurs at a time when such symptoms are severe, and thus may require even harsher or more extreme treatments (eg, higher doses or increased frequency of administration) to alleviate or treat the toxicity.

[0154] The use of specific alternative methods does not provide satisfactory solutions to such problems. In some cases, these drugs and therapies (eg, steroids) are themselves associated with toxic side effects. Such side effects may be even greater at a higher dose or frequency at which they must be administered or treated with an agent or therapy to treat or alleviate the severity of toxicity that may result from cell therapy. In addition, in some cases, it is believed that an agent or therapy for the treatment of toxicity may limit the effectiveness of cellular therapy, for example, the effectiveness of a chimeric receptor (for example, CAR) expressed on cells provided as part of cellular therapy (Sentman (2013) Immunotherapy, 5:10).

[0155] The present embodiments provide advantages in that they direct or target the risk of toxicity, such as severe neurotoxicity, in individuals who have been administered a T cell therapeutic composition. In some embodiments, the likelihood of an individual experiencing toxicity, such as severe neurotoxicity, is reduced or prevented by the present embodiments, in which the safety features of the composition are assessed and/or adjusted by dosing in the individual. In some embodiments, the likelihood of an individual experiencing toxicity, such as severe toxicity, is reduced or prevented by monitoring or evaluating the safety of the T cell therapeutic composition prior to release for administration to the individual and/or by administering a single dose of cells containing no more than a target dose of cells or containing or not exceeding the target number of reference units (RU) of cells.

[0156] In some embodiments, the present embodiments are designed to include, or include features that result in a lower or lower risk of toxicity, toxicity outcome, or symptom, toxicity contributing profile, factor, or property, e.g., symptom or outcome associated with or indicative of cytokine release syndrome (CRS) or neurotoxicity, e.g. compared to other methods in which the T cell therapeutic composition was not assessed in a safety assay and/or in which the T cell therapeutic composition was not administered to an individual according to the presented dosing formula, taking into account A and B, and/or not dosed at a target RU amount, such as less than a threshold RU amount, or not dosed at a target cell number, or a cell number within a given target range , in a therapeutic composition of T-cells.

Toxicity and toxicity outcome

[0157] In some aspects, the toxicity outcome of a therapy, such as cell therapy, is or is associated with, or is indicative of, cytokine release syndrome (CRS) or severe CRS (sCRS). CRS, eg sCRS, may occur, in some cases, after adoptive T-cell therapy and administration of other biological products to individuals. 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.

[0158] Typically, CRS is caused by an excessive systemic immune response mediated by, for example, 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 trigger an acute inflammatory response and/or induce organ endothelial damage, which can lead to microvascular leakage, heart failure, or death. Severe, life-threatening CRS can lead to lung infiltration and lung injury, kidney failure, or disseminated intravascular coagulation. Other types of severe, life-threatening toxicity may include cardiac toxicity, respiratory distress, neurological toxicity, and/or liver failure.

[0159] The outcomes, signs and symptoms of CRS are known and include those described herein. In some embodiments, where a particular dosage regimen or administration causes or does not cause a given CRS-associated outcome, sign or symptom, the particular outcomes, signs and symptoms and/or their levels or degrees may be indicated.

[0160] In the context of administration of CAR-expressing cells, CRS, such as severe CRS, typically occurs 6-20 days after infusion of CAR-expressing cells. See Xu et al., Cancer Letters 343 (2014) 172–78. In some cases, CRS occurs less than 6 days or more than 20 days after CAR-T cell infusion. The incidence and timing of CRS may be related to background levels of cytokines 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.

[0161] CRS criteria have been developed that appear to correlate with the onset of CRS to predict which patients are more likely to be at risk for CRS (see Davilla et al. Science translational medicine. 2014;6(224): 224ra25). Factors include fever, hypoxia, hypotension, neurological changes, increased serum levels of pro-inflammatory cytokines, such as a set of seven cytokines (IFNγ, IL-5, IL-6, IL-10, Flt-3L, fractalkine, and GM-CSF), treatment-induced increases in levels of which 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, the criteria reflecting the degrees of CRS are the criteria detailed in Table 1 below.

Table 1: Illustrative grading criteria for CRS Degree Description of symptoms one
Soft Not life-threatening, requires only symptomatic treatment such as antipyretics and antiemetics (eg, fever, nausea, fatigue, headache, myalgia, discomfort) 2
Moderate Requires and responds to moderate intervention:
• Oxygen demand < 40%, or
• Hypotension responding to fluids or low dose of a single vasoconstrictor or
• Grade 2 organ toxicity (as per CTCAE v4.0) 3
heavy Demands and responds to aggressive intervention:
• Oxygen demand ≥ 40%, or
• Hypotension requiring a high dose of a single vasoconstrictor (eg, norepinephrine ≥ 20 mcg/kg/min, dopamine ≥ 10 mcg/kg/min, phenylephrine ≥ 200 mcg/kg/min, or epinephrine ≥ 10 mcg/kg/min), or
• Hypotension requiring multiple vasoconstrictors (eg, vasopressin + one of the above, or combination of vasoconstrictors equivalent to ≥ 20 mcg/kg/min norepinephrine), or
• Grade 3 Organ Toxicity or Grade 4 Transaminitis (CTCAE v4.0) four
life-threatening Dangerous for life:
• Requires artificial ventilation, or
• Grade 4 organ toxicity (excluding transaminitis) 5
Lethal Death

[0162] In some embodiments, an individual is likely to develop "severe CRS"("sCRS") in response to or as a result of receiving cell therapy or administration of a dose of cells for it, if, after administration, the individual shows: (1 a) fever at least 38 degrees Celsius for at least three days; (2) an increase in the level of a cytokine, comprising either (a) a maximum fold change of at least 75 for at least two of the following group of seven cytokines, compared with the level immediately after administration: interferon gamma (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 gamma (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 vasoconstrictor) or hypoxia (PO _{2 <} 90%) or one or more neurological disorder(s) (including changes in mental status, dullness sensitivity and/or convulsions). In some embodiments, severe CRS includes CRS grade 3 or higher, such as those listed in Table 1.

[0163] In some embodiments, outcomes associated with severe CRS or CRS grade 3 or higher, such as grade 4 or higher, such as those listed in Table 1. In some embodiments, they include one or more of: persistent fever eg, fevers at a specified temperature, eg, greater than or about 38 degrees Celsius, for two or more, eg, three or more, eg, four or more days, or for at least three consecutive days; fevers greater than or approximately 38 degrees Celsius; an increase in cytokine levels, such as a maximum fold change, e.g., at least or about 75, compared to pre-treatment levels for at least two cytokines (e.g., at least two from the group consisting of interferon gamma (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 at least for one of these cytokines; and/or at least one clinical sign of toxicity such as hypotension (eg, as measured by at least one intravenous vasoconstrictor); hypoxia (eg, plasma oxygen levels (PO ₂ ) less than or about 90%); and/or one or more neurological disorders (including mental status changes, sensory numbness and seizures). In some embodiments, severe CRS includes CRS requiring management or treatment in an intensive care unit (ICU).

[0164] In some embodiments, severe CRS includes a combination of (1) persistent fever (fever at at least 38 degrees Celsius for at least three days) and (2) a serum CRP level of at least or about 20 mg / dl. In some embodiments, severe CRS includes hypotension requiring the use of two or more vasoconstrictors or respiratory failure requiring mechanical ventilation. In some embodiments, the dose of the vasoconstrictor is increased on the second or subsequent administration.

[0165] In some embodiments, severe CRS or CRS grade 3 includes an increase in alanine aminotransferase, an increase in aspartate aminotransferase, chills, febrile neutropenia, headache, left ventricular dysfunction, encephalopathy, hydrocephalus, and/or tremor.

[0166] In some aspects, the toxicity outcome of a therapy, such as cell therapy, is or is associated with, or is indicative of, neurotoxicity or severe neurotoxicity. In some embodiments, symptoms associated with the clinical risk of neurotoxicity include confusion, delirium, expressive aphasia, numbness, myoclonus, lethargy, altered mental status, convulsions, seizure-like activity, seizures (optionally, as confirmed by electroencephalogram [EEG]) , increased levels of beta-amyloid (Aβ), increased levels of glutamate, and increased levels of oxygen radicals. In some embodiments, neurotoxicity is graded based on severity (e.g., using a 1-5 grade scale (see, e.g., Guido Cavaletti & Paola Marmiroli Nature Reviews Neurology 6, 657-666 (December 2010); US National Cancer Institute – General Criteria for Toxicity Version 4.03 (NCI-CTCAE v4.03).

[0167] In some cases, neurological symptoms may be the earliest symptoms of sCRS. In some embodiments, the neurological symptoms are observed to begin 5-7 days after the cell therapy infusion. In some embodiments, the duration of neurological changes may range from 3 to 19 days. In some cases, recovery of neurological changes occurs after resolution of other sCRS symptoms. In some embodiments, the time or extent of resolution of neurological changes is not accelerated by treatment with anti-IL-6 antibodies and/or steroid(s).

[0168] In some embodiments, the individual appears to develop "severe neurotoxicity" in response to or as a result of receiving cell therapy or administration of a dose of cells for it, if, after administration, the individual shows symptoms that limit self-care (for example, washing , dressing and undressing, eating, using the toilet, taking medication) 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 the peripheral sensory nerves, dysesthesia, such as a distortion of sensory perception resulting in an abnormal and unpleasant sensation, neuralgia, such as intense pain along a nerve or group of nerves, and/or paresthesia, such as functional stimulations of sensory neurons leading to 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 those listed in Table 2. In some embodiments, severe neurotoxicity is considered to be Grade 3 long-term neurotoxicity if symptoms of Grade 3 neurotoxicity last 10 days or longer. In some embodiments, a toxicity, such as neurotoxicity, e.g., grade 3 or 4 neurotoxicity, is considered to be long-term neurotoxicity (e.g., long-term grade 3 neurotoxicity) if symptoms of that grade, e.g., grade 3 or higher neurotoxicity, are present or have been present for at least 10 days, or at least about 10 days, or at least 11 days, or at least about 11 days, or at least 10-11 days, or about 10-11 days. Prolonged Grade 3 neurotoxicity is typically associated with Grade 3 neurotoxicity persisting for more than or on average more than 9 days, 8 days, or 1 week. In some aspects, non-long-term grade 3 neurotoxicity is typically associated with symptoms of grade 3 neurotoxicity not persisting for more than or on average more than 9 days, 8 days, or 1 week, or in some cases less than 6 , 5, 4 or 3 days.

Table 2: Illustrative Grading Criteria for Neurotoxicity Degree Description of symptoms one
Asymptomatic or mild Symptoms of mild or asymptomatic 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 to self-care activities such as washing, dressing and undressing, eating, using the toilet, taking medication four
life-threatening Symptoms that are life-threatening requiring urgent attention 5
Lethal Death

[0169] In some embodiments, the present embodiments, including methods of treatment, uses, products, unit doses, result in a reduction in symptoms associated with neurotoxicity after cell therapy, compared with other embodiments or methods. For example, individuals treated in accordance with the present methods may have reduced symptoms of neurotoxicity such as weakness or numbness of limbs, loss of memory, vision and/or intelligence, uncontrollable obsessive and/or compulsive behavior, hallucinations, headache, cognitive and behavioral disorders, including loss of motor control, cognitive impairment and dysfunction of the autonomic nervous system and sexual dysfunction, compared with individuals who were treated in other ways. In some embodiments, individuals treated according to the present methods may have reduced symptoms associated with peripheral motor neuropathy, peripheral sensory neuropathy, dysesthesia, neuralgia, or paresthesia.

[0170] 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.

[0171] In some embodiments, individuals who are administered one or more unit doses of the presented T cell therapeutic composition or a T cell therapeutic composition formulated for administration in accordance with the presented methods and/or who are dosed in accordance with the presented methods , have a reduced likelihood of experiencing a serious adverse event, such as long-term neurotoxicity of at least grade 3, e.g., grade 4 or grade 5 neurotoxicity. a degree that is less severe than if the individual were being treated by other methods or using other T cell therapeutic compositions, including T cell therapeutic compositions not evaluated according to the methods presented.

II. SIGNS OF CELL COMPOSITIONS

[0172] The methods provided herein relate to features of cells in a T cell therapeutic composition. In some embodiments, the features, alone or in combination, correlate with the risk of an adverse event or toxicity, such as severe toxicity, e.g., at least Grade 3 long-term toxicity, or Grade 4 or Grade 5 toxicity. In some embodiments, the feature is the phenotype of the cells in the composition, also referred to as "A" in the present description, including a phenotype indicative of the biological activity of the cells or cell population. In some embodiments, the feature is a recombinant receptor dependent activity, such as antigen specific activity, also referred to as "B" herein. In some embodiments, the trait of A and/or B cells in a T cell composition is considered in conjunction with the presented methods. In some aspects, feature A and B function is considered in conjunction with the determination of a target number of reference units (RUs), for example, in conjunction with the provision or administration of a single dose of a T cell therapeutic composition. In some aspects, the B feature cut-off value, or Lower Specified Limit (LSL) and Upper Specified Limit (USL) B, is used to assess or determine the safety or likelihood of safety of a T cell therapeutic composition, e.g., in combination with an activity assay or a release assay. . Various exemplary ways are presented in which features A and/B are taken into account or evaluated.

A. Phenotypes of cell compositions

[0173] In some embodiments, the "A" phenotype is the presence or absence of one or more specific molecules, including surface molecules and/or molecules that can accumulate or be produced by cells or a subset of cells in a T cell composition. In some embodiments, the phenotype, either directly or inversely, is indicative of, or indicative of, the biological activity of the cells or cell population in the T cell composition. In some embodiments, the phenotype may include cell activity, such as the production of a factor in response to a stimulus. In specific embodiments, the cell composition assessment is performed to identify, detect, or quantify the phenotype of the cell composition. In specific embodiments, cell composition measurement is performed to identify, detect, or quantify the presence, absence, degree of expression, or level of a specific molecule.

[0174] In some embodiments, the implementation, the phenotype is an indicator of cell viability. In some embodiments, the phenotype is indicative of the absence of apoptosis, the absence of early stages of apoptosis, or the absence of late stages of apoptosis. In some embodiments, the phenotype is the absence of a factor indicative of the absence of apoptosis, early apoptosis, or late stages of apoptosis. In some embodiments, the phenotype is the phenotype of a subpopulation or subgroup of T cells, such as recombinant receptor-expressing T cells (e.g., CAR+ T cells) or CD8+ T cells, naive T cells, or a particular subpopulation of memory T cells, or T cells that have features similar to memory stem cells. In some embodiments, the phenotype is a phenotype of cells that are not activated and/or that lack or have reduced or low expression of one or more activation markers. In some embodiments, the phenotype is a phenotype of cells that are not depleted and/or that lack or have reduced or low expression of one or more markers of depletion.

[0175] In some embodiments, a phenotype is indicated by the presence, absence, or level of expression in a cell of one or more specific molecules, such as certain surface markers indicative of the phenotype, e.g., surface proteins, intracellular markers indicative of the phenotype, or nucleic acids. indicative of the phenotype, or other molecules or factors indicative of the phenotype. In some embodiments, the phenotype is or includes positive or negative expression of one or more specific molecules. In some embodiments, specific molecules include, but are not limited to, a surface marker, such as a membrane glycoprotein or receptor, a marker associated with apoptosis or viability, or a specific molecule indicative of immunocyte status, such as a marker associated with activation, depletion, or maturation. or naive phenotype. In some embodiments, any known method for evaluating or measuring, enumerating and/or quantifying cells based on specific molecules can be used to determine the number of cells of that phenotype.

[0176] In some embodiments, the implementation, the phenotype is or includes the positive or negative expression of one or more specific molecules in the cell. In some embodiments, positive expression is indicated by the detectable amount of the specific molecule in the cell. In specific embodiments, a detectable amount is any detectable amount of a specific molecule in a cell. In specific embodiments, the detectable amount is the amount above the background, eg, background staining, signal, etc., in the cell. In specific embodiments, positive expression is an amount of a specific molecule that does not exceed a threshold, such as a predetermined threshold. Similarly, in specific embodiments, a cell with a negative expression of a specific molecule can be any cell not defined as having a positive expression, or is a cell that lacks a detectable amount of a specific molecule or a detectable amount of a specific molecule is above background. In some embodiments, the cell has a negative expression of a specific molecule if the amount of the specific molecule is below a threshold. One skilled in the art will understand how to define a threshold for positive and/or negative expression of a specific molecule in a conventional manner, and that thresholds can be determined according to specific parameters, for example, but not limited to, assay or detection method, identity of a specific molecule, reagents used for detection, and equipment.

[0177] Examples of methods that can be used to detect a specific molecule and/or analyze the phenotype of cells include, but are not limited to, biochemical analysis; immunochemical analysis; visualization analysis; cytomorphological analysis; molecular analysis such as PCR, sequencing, DNA methylation; proteomic analysis, such as determining the pattern of glycosylation and/or phosphorylation of the protein; genomic analysis; epigenomic analysis; transcriptome analysis; and any combination of them. In some embodiments, the molecular characteristics of the phenotype are analyzed by imaging analysis, PCR (including standard and all PCR variants), microarray (including, but not limited to, DNA microarray, microRNA MMchips, protein microarray, cellular microarray, antibody microarray, and carbohydrate microarray). array), sequencing, biomarker detection, or methods for determining DNA methylation pattern or protein glycosylation. In specific embodiments, the specific molecule is a polypeptide, i. protein. In some embodiments, the specific molecule is a polynucleotide.

[0178] In some embodiments, positive or negative expression of a specific molecule is determined by incubating cells with one or more antibodies or other binders that specifically bind to one or more surface markers expressed or expressed (marker ⁺ ) at a relatively higher level (marker ^high ) on positively or negatively selected cells, respectively. In specific embodiments, positive or negative expression is determined by flow cytometry, immunohistochemistry, or any other suitable method for detecting specific markers.

[0179] In specific embodiments, the expression of a specific molecule is assessed using flow cytometry. Flow cytometry is a laser or resistance-based biophysical technology used in cell counting, cell sorting, biomarker detection, and protein engineering by suspending cells in a fluid stream and passing them through an electronic detection device. It allows simultaneous multi-parameter analysis of physical and chemical characteristics up to thousands of particles per second.

[0180] The data obtained by flow cytometers can be plotted in one dimension to obtain a histogram, or in two-dimensional scatter plots, or even in three dimensions. Regions on these plots can then be subdivided, based on fluorescence intensity, by obtaining a series of subgroup subtractions called "selections". There are methods of specific selection for diagnostic and clinical purposes, especially in relation to immunology. Graphs are often obtained in logarithmic scales. Because the emission spectra of different fluorescent dyes overlap, the signals at the detectors must be compensated electronically as well as computationally. Data collected using a flow cytometer can be analyzed using software such as JMP (statistical software), WinMDI, [10] Flowing software, [11] and web-based Cytobank[12]), Cellcion, FCS Express , FlowJo, FACSDiva, CytoPaint (also known as Paint-A-Gate), [13] VenturiOne, CellQuest Pro, Infinicyt, or Cytospec.

[0181] Flow cytometry is a standard technique in the art, and it is well understood by one skilled in the art how to design or customize methods to detect one or more specific molecules and analyze the data to determine the expression of one or more specific molecules in a population of cells. Standard protocols and methods for flow cytometry are found in Loyd “Flow Cytometry in Microbiology; Practical Flow Cytometry by Howard M. Shapiro; Flow Cytometry for Biotechnology by Larry A. Sklar, Handbook of Flow Cytometry Methods by J. Paul Robinson, et al., Current Protocols in Cytometry, Wiley–Liss Pub, Flow Cytometry in Clinical Diagnosis, v4, (Carey, McCoy, and Keren , eds), ASCP Press, 2007, Ormerod, M.G. (ed.) (2000) Flow Cytometry –A practical approach. 3rd edition. Oxford University Press, Oxford, UK, Ormerod, M.G. (1999) Flow cytometry. 2nd edition. BIOS Scientific Publishers, Oxford., and Flow Cytometry –A basic introduction. Michael G. Ormerod, 2008.

[0182] In some embodiments, cells are sorted by phenotype for further analysis. In some embodiments, cells of different phenotypes within the same cell composition are sorted by fluorescence activated cell sorting (FACS). FACS is a specialized type of flow cytometry that allows a heterogeneous mixture of cells to be sorted into two or more containers, one cell at a time, based on the specific light scattering and fluorescence characteristics of each cell. It is a useful scientific tool because it provides fast, objective, and quantitative detection of fluorescent signals from individual cells, as well as physical isolation of cells of particular interest.

[0183] In some embodiments, the phenotype includes total T cells or total CD3+ T cells. In specific embodiments, a T cell composition, eg, a T cell therapeutic composition containing cells expressing a recombinant receptor or CAR, may include one or more different T cell subtypes. In some embodiments, the phenotype is or includes a T cell subtype identity. Different populations or subtypes of T cells include, but are not limited to, effector T cells, helper T cells, memory T cells, regulatory T cells, CD4+ T cells, and CD8+ T cells. In specific embodiments, a T cell subtype can be identified by detecting the presence or absence of a specific molecule. In specific embodiments, the specific molecule is a surface marker that can be used to identify a T cell subtype.

[0184] In some embodiments, the phenotype is positive or high expression of one or more specific molecules that are surface markers, e.g., CD3, CD4, CD8, CD28, CD62L, CCR7, CD27, CD127, CD45RA, and/or CD45RO. In specific embodiments, the phenotype is a surface marker of T cells or a subpopulation or subgroup of T cells, e.g., based on positive expression of the surface marker for one or more surface markers, e.g., CD3+, CD4+, CD8+, CD28 ⁺ , CD62L ⁺ , CCR7 ⁺ , CD27 ⁺ , CD127 ⁺ , CD4 ⁺ , CD8 ⁺ , CD45RA ⁺ and/or CD45RO ⁺ .

[0185] In specific embodiments, the surface marker shows the expression of a recombinant receptor, for example, CAR. In specific embodiments, the surface marker is the expression of a recombinant receptor, such as CAR, which, in some aspects, can be detected using an antibody, such as an anti-idiotypic antibody. In some embodiments, the surface marker indicative of recombinant receptor expression is a surrogate marker. In some embodiments, the surrogate marker is a protein designed to be co-expressed on the cell surface with a recombinant receptor, such as CAR. In specific embodiments, such a surrogate marker is a surface protein modified to have little or no activity. In specific embodiments, the surrogate marker is encoded by the same polynucleotide that encodes the recombinant receptor used for transduction. Non-limiting examples of a surrogate marker include, but are not limited to, truncated EGFR (EGFRt), truncated HER2 (tHER2), or prostate-specific membrane antigen (PSMA), or a modified form thereof. In some aspects, the marker, e.g. , a surrogate marker, includes all or part (e.g., a truncated form) of CD34, NGFR, CD19, or a truncated CD19, e.g., a truncated non-human CD19, or an epidermal growth factor receptor (e.g., tEGFR). In specific embodiments, the cell having a detectable amount of CAR has a phenotype that is or includes CAR+. In specific embodiments, the cell having a detectable amount of the surrogate marker has a phenotype that is or includes CAR+.

[0186] In specific embodiments, the implementation, the phenotype includes the expression, for example, surface expression, one or more of the surface markers CD3, CD4, CD8 and / or recombinant receptor (for example, CAR) or its surrogate marker, showing or correlated with the expression of the recombinant receptor (for example, CAR).

[0187] In specific embodiments, the implementation, the phenotype is identified by the expression of one or more specific molecules that are surface markers. In specific embodiments, the phenotype is or includes positive or negative expression of CD3, CD4, CD8 and/or a recombinant receptor, eg CAR. In specific embodiments, the recombinant receptor is a CAR. In specific embodiments, the implementation of the phenotype includes CD3+/CAR+, CD4+/CAR+ and/or CD8+/CAR+.

[0188] In some embodiments, the phenotype is viability. In specific embodiments, the phenotype is positive expression of a marker indicating that the cell is undergoing normal functional cellular processes and/or has not or is not undergoing necrosis or programmed cell death. In some embodiments, viability can be assessed by the redox potential of the cell, cell membrane integrity, or mitochondrial activity or function. In some embodiments, viability is the absence of a specific molecule associated with cell death, or the absence of evidence of cell death in the assay.

[0189] In some embodiments, the phenotype is or includes cell viability. In specific embodiments, cell viability can be detected, measured and/or evaluated in a number of ways that are conventional in the art. Non-limiting examples of such viability assays include, but are not limited to, dye uptake assays (eg, calcein AM assays), XTT cell viability assays, and dye exclusion assays (eg, trypan blue, eosin, or propidium dye exclusion assays). Viability assays can be used to determine the number or percentage (eg, frequency) of viable cells in a cell dose, cell composition, and/or cell sample. In specific embodiments, the implementation, the phenotype includes the viability of the cell, along with other features, such as expression of the recombinant receptor.

[0190] In specific embodiments, the phenotype is or includes cell viability, CD3+ viability, CD4+ viability, CD8+ viability, CD3+/CAR+ viability, CD4+/CAR+ viability, CD8+/CAR+ viability, or a combination thereof.

[0191] In specific embodiments, the phenotype is or includes the absence of apoptosis and/or an indication that the cell is undergoing an apoptotic process. Apoptosis is a process of programmed cell death, including a series of stereotyped morphological and biochemical events leading to characteristic cell changes and death. These changes include blistering, cell size reduction, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, and global mRNA cleavage. Apoptosis is a well characterized process and the specific molecules associated with the various steps are well known in the art.

[0192] In some embodiments, the phenotype is the absence of an early stage of apoptosis, and/or the absence of an indicator and/or a specific molecule associated with an early stage of apoptosis. In the early stages of apoptosis, changes in the cell and mitochondrial membrane become apparent. Biochemical changes are also evident in the cytoplasm and nucleus of the cell. For example, early stages of apoptosis can be shown by the activation of specific caspases, for example, 2, 8, 9, and 10. In specific embodiments, the phenotype is the absence of a late stage of apoptosis, and/or the absence of an indicator and/or a specific molecule associated with a late stage. apoptosis. The middle to late stages of apoptosis are characterized by further loss of membrane integrity, chromatin condensation, and DNA fragmentation, and include biochemical events such as activation of caspases 3, 6, and 7.

[0193] In specific embodiments, the implementation, the phenotype is a negative expression of one or more factors associated with programmed cell death. In specific embodiments, the phenotype is a negative expression of one or more factors associated with apoptosis, including pro-apoptotic factors known to initiate apoptosis, e.g., members of the death receptor pathway, activated members of the mitochondrial (intrinsic) pathway, such as members of the Bcl– 2, such as Bax, Bad and Bid, and caspases. In some embodiments, the phenotype is the absence or low amount of a marker of apoptosis. In specific embodiments, the phenotype is a negative expression of an apoptosis marker. In specific embodiments, the phenotype is the absence of an indicator, such as an annexin V molecule, that preferentially binds to cells undergoing apoptosis upon incubation or contact with the cell composition. In some embodiments, the phenotype is or includes the expression of one or more markers that are indicative of an apoptotic state in the cell.

[0194] In some embodiments, the phenotype is negative (or low) expression of a specific molecule that is a marker of apoptosis. Various markers of apoptosis are known to those skilled in the art and include, but are not limited to, an increase in the activity of one or more caspases, ie. activated caspase, increased PARP cleavage, activation and/or translocation of Bcl-2 family proteins, members of the cell death pathway such as Fas and FADD, the presence of a decrease in nuclear size (eg, monitored through a microscope), and the presence of chromosomal DNA fragmentation (eg, the presence of a ladder chromosomal DNA) or apoptosis assays including TUNEL staining and annexin V staining.

[0195] Caspases are enzymes that cleave proteins after an aspartic acid residue, the term is derived from "cysteine-aspartic acid proteases". Caspases are involved in apoptosis, thus activation of caspases such as caspase-3 is indicative of an increase or resumption of apoptosis. In specific embodiments, caspase activation can be detected by methods known to one of skill in the art. In some embodiments, an antibody that specifically binds to an activated caspase (ie, specifically binds to a cleaved polypeptide) can be used to detect caspase activation. In another example, the Fluorochrome Caspase Activity Inhibitor Assay (FLICA) can be used to detect caspase-3 activation by detecting the hydrolysis of acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (Ac-DEVD-AMC) by caspase- 3 (i.e., detecting the release of fluorescent 7-amino-4-methylcoumarin (AMC)). FLICA assays can be used to determine caspase activation by detecting a substrate product processed by multiple caspases (eg, FAM-VAD-FMK FLICA). Other methods include the CASPASE-GLO® (PROMEGA) caspase assays, which use caspase-8 luminogenic tetrapeptide substrate (Z-LETD-aminoluciferin), caspase-9 tetrapeptide substrate (Z-LEHD-aminoluciferin), caspase-3/7 substrate ( Z-DEVD-aminoluciferin), caspase-6 substrate (Z-VEID-aminoluciferin) or caspase-2 substrate (Z-VDVAD-aminoluciferin).

[0196] In specific embodiments, the phenotype is or includes negative expression of active caspase-1, active caspase-2, active caspase-3, active caspase-7, active caspase-8, active caspase-9, active caspase-10, and /or active caspase-13 in the cell. In specific embodiments, the phenotype is or includes active caspase-3-. In some embodiments, a proform (zymogen cleavable) form of caspase, such as any of the above, is also a marker indicative of the presence of apoptosis. In some embodiments, the phenotype is or includes the absence or negative expression of a caspase proform, such as a caspase-3 proform.

[0197] In some embodiments, the apoptotic marker cleaves poly(ADP-ribose) polymerase 1 (PARP). PARP is cleaved by caspase during the early stages of apoptosis. Thus, detection of a cleaved PARP peptide is a marker of apoptosis. In specific embodiments, the phenotype is or includes positive or negative expression of cleaved PARP.

[0198] In some embodiments, the apoptosis marker is a reagent that detects a cell trait associated with apoptosis. In specific embodiments, the reagent is an annexin V molecule. During the early stages of apoptosis, the lipid phosphatidylserine (PS) translocates from the inner to the outer layer of the plasma membrane. PS is normally limited to the inner membrane in healthy and/or non-apoptotic cells. Annexin V is a protein that preferentially binds phosphatidylserine (PS) with high affinity. After conjugation with a fluorescent label or other reporter, annexin V can be used to quickly detect this early indicator of apoptosis on the cell surface. In some embodiments, the presence of PS on the outer membrane may persist in the late stages of apoptosis. Thus, in some embodiments, annexin V staining is indicative of both early and late stages of apoptosis. In specific embodiments, the annexin, e.g., annexin V, is labeled with a detectable label and incubated with, exposed to, and/or contacted with cells from a cell composition to detect cells that are undergoing apoptosis, e.g., by flow cytometry. In some embodiments, fluorescently labeled annexins, eg, annexin V, are used to stain cells for flow cytometry analysis, eg, using the annexin-V/7-AAD assay. Alternative methods useful for detecting apoptosis using annexin include methods and assays that use radioactively labeled annexin V. In specific embodiments, the phenotype is or includes negative staining for annexin, e.g., annexin V–. In specific embodiments, the implementation, the phenotype is or includes the absence of PS on the outer plasma membrane. In specific embodiments, the phenotype is or includes cells that do not bind to annexin, eg, annexin V. In specific embodiments, the cell lacking detectable PS on the outer membrane is annexin V−. In specific embodiments, the cell that does not bind to annexin V- in an analysis, such as flow cytometry, after incubation with labeled annexin V, is annexin V-.

[0199] In specific embodiments, the phenotype is Annexin V-, Annexin V-CD3+, Annexin V-CD4+, Annexin V-CD8+, Annexin V-CD3+/CAR+, Annexin V-CD4+/CAR+, Annexin V-CD8+/CAR+ , activated caspase-3-, activated caspase-3-/CD3+, activated caspase-3-/CD4+, activated caspase-3-/CD8+, activated caspase-3-/CD3+/CAR+, activated caspase-3-/CD4+/CAR+ , activated caspase-3-/CD8+/CAR+ or a combination thereof.

[0200] Specific embodiments provide that cells positive for the expression of an apoptosis marker undergo programmed cell death, have reduced or absent immune function, and if so, have a reduced ability to undergo activation, proliferate, and/or bind to an antigen for initiation, exercising or contributing to an immune response or activity. In specific embodiments, the phenotype is determined by negative expression of activated caspase and/or negative staining for annexin V.

[0201] In specific embodiments, the phenotype is or includes activated caspase-3- (caspase-3-) and/or annexin V-.

[0202] In specific embodiments, the implementation, the phenotype is a negative expression of one or more factors associated with inflammatory cell death. In some embodiments, the inflammatory cell death is pyroptosis. In specific embodiments, pyroptosis occurs or could potentially occur in virus-infected and/or transduced cells, eg, virus-infected and/or transduced CD4+ and/or CD8+ T cells. In some embodiments, pyroptosis is or may be associated with one or more of cell lysis, cell swelling, pore formation, DNA fragmentation, and/or caspase-1 activation. In specific embodiments, pyroptosis does not result in membrane blistering, caspase-3 activation, and/or cytochrome-C release.

[0203] In some embodiments, one or more factors associated with pyroptosis is or includes pattern recognition receptors, inflammasomes, active caspase-1, active IL-1b, and/or active IL-18. In some embodiments, one or more of the factors is or includes activated and/or ligated pattern receptors. In specific embodiments, one or more of the factors is or includes activated and/or ligated TLRs, NOD-like receptors, RIG-I, MDA5, and/or STING. In some embodiments, one or more of the factors is or includes active IL-1b. In specific embodiments, one or more of the factors is or includes active IL-18. In specific embodiments, one or more of the factors is or includes active caspase-1.

[0204] In specific embodiments, the phenotype is or includes activated caspase-1- (caspase-1-). In some embodiments, the phenotype is or includes active IL-1b-. In specific embodiments, the phenotype is or includes active IL-18-.

[0205] In specific embodiments, the inflammatory cell death is or includes necroptosis. In specific embodiments, the implementation, the phenotype is a negative expression of one or more factors associated with necroptosis. In some embodiments, necroptosis is programmed necrotic cell death driven by activation of the RIP family of kinases. In some embodiments, necroptosis occurs or may occur upon binding and/or activation of the TNF receptor family, and/or upon recruitment of pattern-recognizing receptors (i.e., binding of viral components to RIG-I/MDA5 or in related pathways). In some embodiments, production of TNFα during viral infection results in stimulation of its TNFR1 receptor, activation of the TNFR-associated death protein TRADD, which in turn activates RIPK1 to recruit RIPK3 for necrosome formation. In some embodiments, necrosome formation results in MLKL phosphorylation and MLKL oligomerization, allowing MLKL to integrate and enter plasma membranes and organelles. In some embodiments, MLKL integration results in an inflammatory phenotype and release of damage-associated molecular patterns (DAMPs) that elicit immune responses.

[0206] In some embodiments, one or more factors associated with necroptosis are activated and/or ligated pattern recognition receptors, e.g., activated and/or ligated RIG-I/MDA5, activated TNFR1, TRADD, necrosome and/or phosphorylated and/or oligomerized MLKL. In some embodiments, one or more necroptosis-associated factors is or includes an activated RIP kinase, such as RIPK1, RIPK2, RIPK3, RIPK4, and/or RIPK5. In some embodiments, one or more of the factors is an activated RIPK1 and/or RIPK3. In some embodiments, the phenotype is or includes activated RIPK1–, activated RIPK3–, phosphorylated MLKL–, and/or oligomerized MLKL–.

[0207] In specific embodiments, the implementation, the phenotype is a negative expression of one or more factors associated with autophagy. In some embodiments, autophagy is or includes a cell-intrinsic catabolic mechanism that occurs and/or may occur under certain cell culture conditions, such as conditions where critical nutrients are limited and/or in response to certain cytokines. In some embodiments, autophagy occurs independently of caspase activity.

[0208] In some embodiments, autophagy is macro-autophagy, micro-autophagy, and/or chaperone-mediated autophagy. In some embodiments, one or more factors associated with an autophagy marker include AMPK, ULK1, ULK2, and/or other members of the Atg family (e.g., ATG16L), PIK3C3, BECN1, Vps34, Beclin-1, MAP1LC3A, B,C, GABARAP, GABARAPL1, GABARAPL2, UVRAG, IRGM, CLN3, Parkin, p62 and LAMP2, and/or other known factors such as described in Behrends, Nature. 2010 Jull;466(7302):68–76 and Glick et al. J Pathol. 2010 May;221(l):3–12. In some embodiments, the phenotype is AMPK-, ULK1-, ULK2-, PIK3C3-, BECN1-, Vps34-, Beclin-1-, MAP1LC3A-, MAP1LC3B-, MAP1LC3C-, GABARAP-, GABARAPL1-, GABARAPL2-, UVRAG –, IRGM–, CLN3–, Parkin–, p62– and/or LAMP2–.

[0209] Among the phenotypes, there is expression or surface expression of one or more markers, usually associated with one or more subtypes or subpopulations of T cells or their phenotypes. Subtypes and subpopulations of T cells may include CD4+ and/or CD8+ T cells and their subtypes, which may include naive T cells (T _N ), effector T cells (T _EFF ), memory T cells and their subtypes, such as memory T-cells (T _SCM ), memory T-cells (T _CM ), effector memory T-cells (T _EM ), T _EMRA cells or terminally differentiated effector memory T-cells, tumor-infiltrating lymphocytes (TIL), immature T-cells, mature T-cells, helper T-cells, cytotoxic T-cells, mucosal-associated invariant T-cells (MAIT), natural and adaptive regulatory T-cells (T-reg), T-helper cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.

[0210] In some embodiments, the phenotype is or includes the phenotype of memory T cells or a subset of memory T cells. Memory T cells are antigen-specific T cells that have been previously exposed to a related antigen. Memory T cells persist long after infection resolves. Memory T cells multiply rapidly to large numbers of effector T cells after repeated exposure to their cognate antigen, thus providing the immune system with a "memory" against past infections. Memory T cells contain three subtypes: central memory T cells (T _CM cells) and two types of effector memory T cells (T _EM cells and T _EMRA cells). In some embodiments, the phenotype is or includes a memory T cell phenotype (or one or more associated markers), such as T _CM cell, T _EM cell, or T _EMRA cell, stem memory T cell (T _SCM ) or a combination of them. In specific embodiments, the phenotype is or includes the expression of one or more specific marker molecules for memory T cells and/or memory T cells, or subtypes thereof.

[0211] In specific embodiments, the implementation, the phenotype is or includes the expression of one or more specific molecules that are a marker of naive T cells. Naïve T cells include fresh T cells that are formed in the bone marrow and are capable of responding to newly encountered pathogens containing antigens not previously processed by the immune system. After stimulation with their related antigen, some of the activated naive T cells develop into memory cells.

[0212] In some embodiments, the phenotype is or includes a memory T cell or a naive T cell. In specific embodiments, the implementation, the phenotype is a positive or negative expression of one or more specific molecules that are markers of memory. In some embodiments, the memory marker is a specific molecule that can be used to determine the population of memory T cells.

[0213] In some embodiments, the phenotype is or includes the phenotype of one or more markers associated with a non-memory T cell or subtype thereof; in some aspects, it is or includes a phenotype or marker(s) associated with a naive cell. In some embodiments, the phenotype is CCR7 ⁺ /CD27 ⁺ /CD28 ⁺ /CD45RA ⁺ . In specific embodiments, the phenotype is or includes CCR7 ⁺ /CD45RA ⁺ . In some embodiments, the phenotype is or includes the memory T cell phenotype. In specific embodiments, the phenotype is or includes CCR7 ⁺ /CD27 ⁺ /CD28 ⁺ /CD45RA ⁻ . In some embodiments, the phenotype is or includes an effector memory cell. In some embodiments, the phenotype is or includes CCR7 ⁻ /CD27 ⁺ /CD28 ⁺ /CD45RA ⁻ . In specific embodiments, the phenotype is or includes the T _EMRA cell or T _SCM cell phenotype. In specific embodiments, the phenotype is or includes CD45RA ⁺ . In specific embodiments, the phenotype is or includes CCR7 ⁻ /CD27 ⁻ /CD28 ⁻ /CD45RA ⁺ . In some embodiments, the phenotype is or includes one of CD27 ⁺ /CD28 ⁺ , ^CD27− /CD28 ⁺ , CD27 ⁺ /CD28− ^, or ^CD27− / ^CD28− .

[0214] In some embodiments, the phenotype is or includes any of the preceding phenotypic properties and further includes recombinant receptor expression, e.g., a phenotype that is associated with a memory T cell or a memory T cell subtype and that expresses CAR, or a phenotype associated with with a naive cell that expresses CAR. In specific embodiments, the phenotype is or includes the phenotype of a memory T cell or memory T stem cell that expresses CAR. In specific embodiments, the phenotype is or includes the phenotype of an effector memory cell that expresses CAR. In some embodiments, the phenotype is or includes the phenotype of an _EMRA T cell that expresses CAR. In specific embodiments, the phenotype is or includes CAR ⁺ /CCR7 ⁺ /CD27 ⁺ /CD28 ⁺ /CD45RA ⁻ ; CAR ⁺ / ^CCR7− /CD27 ⁺ /CD28 ⁺ / ^CD45RA− ; CAR ⁺ /CCR7 ^- /CD27 ^- /CD28 ^- /CD45RA ⁺ ; CAR ⁺ /CD27 ⁺ /CD28 ⁺ ; CAR ⁺ / ^CD27− /CD28 ⁺ ; CAR ⁺ /CD27 ⁺ / ^CD28− ; or CAR ⁺ / ^CD27− / ^CD28− .

[0215] In specific embodiments, the phenotype is or includes a T cell phenotype that is negative for an apoptosis marker. In specific embodiments, the phenotype is or includes a naive cell that is negative for an apoptosis marker. In some embodiments, the apoptosis marker is activated caspase-3. In some embodiments, the apoptosis marker is positive staining for annexin V.

[0216] In specific embodiments, the phenotype is or includes the phenotype of a memory T cell or subtype thereof negative for an apoptosis marker that expresses CAR. In specific embodiments, the phenotype is or includes the phenotype of a memory T cell or a particular subtype negative for an apoptosis marker that expresses CAR. In specific embodiments, the phenotype is or includes a naive cell that is negative for an apoptosis marker that expresses CAR. In specific embodiments, the phenotype is or includes the phenotype of a memory T cell or T _SCM cell, or a naive cell that is negative for an apoptosis marker that expresses CAR. In specific embodiments, the phenotype is or includes the phenotype of an effector memory cell that is negative for an apoptosis marker that expresses CAR. In specific embodiments, the phenotype is or includes annexin V ⁻ /CAR ⁺ /CCR7 ⁺ /CD27 ⁺ /CD28 ⁺ /CD45RA ⁻ ; annexin V ^- /CAR ⁺ /CCR7 ^- /CD27 ⁺ /CD28 ⁺ /CD45RA ^- ; annexin V ^- /CAR ⁺ /CCR7 ^- /CD27 ^- /CD28 ^- /CD45RA ⁺ ; annexin V ⁻ /CAR ⁺ /CD27 ⁺ /CD28 ⁺ ; annexin V ⁻ /CAR ⁺ /CD27 ⁻ /CD28 ⁺ ; annexin V ⁻ /CAR ⁺ /CD27 ⁺ /CD28 ⁻ ; or annexin V ⁻ /CAR ⁺ /CD27 ⁻ /CD28 ⁻ . In specific embodiments, the phenotype is or includes activated caspase-3 ⁻ /CAR ⁺ /CCR7 ⁺ /CD27 ⁺ /CD28 ⁺ /CD45RA ⁻ ; activated caspase-3 ^- /CAR ⁺ /CCR7 ^- /CD27 ⁺ /CD28 ⁺ /CD45RA ^- ; activated caspase-3 ^- /CAR ⁺ /CCR7 ^- /CD27 ^- /CD28 ^- /CD45RA ⁺ ; activated caspase-3 ^- /CAR ⁺ /CD27 ⁺ /CD28 ⁺ ; activated caspase-3 ^- /CAR ⁺ /CD27 ^- /CD28 ⁺ ; activated caspase-3 ^- /CAR ⁺ /CD27 ⁺ /CD28 ^- ; or activated caspase-3 ^- /CAR ⁺ /CD27 ^- /CD28 ^- .

[0217] In some embodiments, the phenotype is or includes positive or negative expression of a depletion marker. T cell depletion is a state of T cell dysfunction that occurs during a variety of chronic infections and cancers. It is defined by poor effector function, persistent expression of inhibitory receptors, and a transcriptional state that is different from that of functional effector or memory T cells. Depletion prevents optimal control of infection and tumors. In specific embodiments, the phenotype is or includes positive or negative expression of a specific wasting-associated molecule. In specific embodiments, the specific molecule is any molecule associated with depletion or an amount associated with depletion, such as poor effector function or inhibitory receptor expression. In specific embodiments, the phenotype is positive or negative expression of an immune checkpoint inhibitor. In specific embodiments, the depletion marker is CTLA-4, FOXP3, PD-1, TIGIT, LAB-3, 2B4, BTLA, TIM3, VISTA, or CD96, or a combination thereof. In specific embodiments, the phenotype is positive or negative expression of CTLA-4, FOXP3, PD-1, TIGIT, LAB-3, 2B4, BTLA, TIM3, VISTA, or CD96, or a combination thereof. In specific embodiments, the phenotype is positive or negative expression of PD1 and/or FOXP3.

[0218] In specific embodiments, the phenotype is negative expression of a specific molecule associated with T cell activation. In some embodiments, the phenotype is or includes negative expression of one or more specific activation marker molecules. T cell activation refers to the stimulation of a T cell's immune response following stimulation of the latter by an antigen presenting cell (APC). This interaction-based pathway explains the major stimulatory signals that trigger T-cell activation, as well as its downstream pathways. Typically, T cell activation requires two simultaneous signals. The first is the binding of the T-cell receptor (TCR) complex to a major histocompatibility complex (MHC) molecule carrying a peptide antigen. The second is mediated by the binding of the costimulatory CD28 receptor to proteins on the APC surface, such as B7-2 or B7-1. In specific embodiments, the specific molecule is associated with TCR activation, eg, is activated, altered, or expressed as a result of T cell activation. In some embodiments, a specific molecule is associated with activation of the CD28 receptor, for example, a molecule that is activated, altered, or expressed as a result of T cell activation.

[0219] In some embodiments, the phenotype is or includes positive or negative expression of a depletion marker. T cell depletion is a state of T cell dysfunction that occurs during a variety of chronic infections and cancers. It is defined by poor effector function, persistent expression of inhibitory receptors, and a transcriptional state that is different from that of functional effector or memory T cells. Depletion prevents optimal control of infection and tumors. In specific embodiments, the phenotype is or includes positive or negative expression of a specific wasting-associated molecule. In particular embodiments, the specific molecule is any molecule associated with depletion, or an amount associated with depletion, such as poor effector function or inhibitory receptor expression. In specific embodiments, the phenotype is positive or negative expression of an immune checkpoint inhibitor. In specific embodiments, the depletion marker is CTLA-4, FOXP3, PD-1, TIGIT, LAB-3, 2B4, BTLA, TIM3, VISTA, or CD96, or a combination thereof. In specific embodiments, the phenotype is positive or negative expression of CTLA-4, FOXP3, PD-1, TIGIT, LAB-3, 2B4, BTLA, TIM3, VISTA, or CD96, or a combination thereof. In specific embodiments, the phenotype is positive or negative expression of PD1 and/or FOXP3.

[0220] In some embodiments, the phenotype is or includes positive or negative expression of a depletion marker in a CD3+ cell that expresses a recombinant receptor or CAR. In specific embodiments, the phenotype is or includes positive or negative expression of a depletion marker in a CD4+ cell that expresses the recombinant receptor or CAR. In some embodiments, the phenotype is or includes positive or negative expression of a depletion marker and CD3+, and positive expression of a recombinant receptor or CAR. In specific embodiments, the phenotype is or includes positive or negative expression of a depletion marker in a CD4+ cell that expresses the recombinant receptor or CAR. In some embodiments, the phenotype is or includes positive or negative expression of a depletion marker in a CD8+ cell that expresses the recombinant receptor or CAR. In specific embodiments, the depletion marker is one or more of CTLA-4, FOXP3, PD-1, TIGIT, LAB-3, 2B4, BTLA, TIM3, VISTA, or CD96. In specific embodiments, the depletion marker is PD1 and/or FOXP3.

[0221] In specific embodiments, the phenotype is or includes PD1–/CD3+, PD1–/CD4+, PD1–/CD8+, PD1–/CD3+/CAR+, PD1–/CD4+/CAR+, PD1–/CD8+/CAR+, PD1 –/annexin V–, PD1–/annexin V–/CD3+, PD1–/annexin V–/CD4+, PD1–/annexin V–/CD8+, PD1–/annexin V/–CD3+/CAR+, PD1–/annexin V– / CD4+/CAR+, PD1–/annexin V–/CD8+/CAR+, PD1–/activated caspase–3–, PD1–/activated caspase–3–/CD3+, PD1–/activated caspase–3–/CD4+, PD1–/ activated caspase-3-/CD8+, PD1-/activated caspase-3-/CD3+/CAR+, PD1-/activated caspase-3-/CD4+/CAR+, PD1-/activated caspase-3-/CD8+/CAR+ or a combination thereof.

[0222] In specific embodiments, the phenotype is or includes FOXP3–/CD3+, FOXP3–CD4+, FOXP3–/CD8+, FOXP3–/CD3+/CAR+, FOXP3–/CD4+/CAR+, FOXP3–/CD8+/CAR+, FOXP3– /annexin V–, FOXP3–/annexin V–/CD3+, FOXP3–/annexin V–/CD4+, FOXP3–/annexin V–/CD8+, FOXP3–/annexin V/–CD3+/CAR+, FOXP3–/annexin V–/ CD4+/CAR+, FOXP3–/annexin V–/CD8+/CAR+, FOXP3–/activated caspase–3–, FOXP3–/activated caspase–3–/CD3+, FOXP3–/activated caspase–3–/CD4+, FOXP3–/activated caspase-3-/CD8+, FOXP3-/activated caspase-3-/CD3+/CAR+, FOXP3-/activated caspase-3-/CD4+/CAR+, FOXP3-/activated caspase-3-/CD8+/CAR+ or a combination thereof.

[0223] In specific embodiments, the phenotype is negative expression of a specific molecule associated with T cell activation. In some embodiments, the phenotype is or includes negative expression of one or more specific activation marker molecules. T cell activation refers to the stimulation of a T cell's immune response following stimulation of the latter by an antigen presenting cell (APC). This interaction-based pathway explains the major stimulatory signals that trigger T-cell activation, as well as its downstream pathways. Typically, T cell activation requires two simultaneous signals. The first is the binding of the T-cell receptor (TCR) complex to a major histocompatibility complex (MHC) molecule carrying a peptide antigen. The second is mediated by the binding of the co-stimulatory CD28 receptor to proteins on the surface of the APC, such as B7-2 or B7-1. In specific embodiments, a specific molecule associated with TCR activation, for example, is activated, altered, or expressed as a result of T cell activation. In some embodiments, a specific molecule associated with CD28 receptor activation, such as a molecule that is activated, altered, or expressed as a result of T cell activation.

[0224] In specific embodiments, the implementation, the phenotype is or includes negative expression of one or more of the specific molecules that are a marker of activation. In specific embodiments, the activation marker is one or more of CD25, CD26, CD27, CD28, CD30, CD71, CD154, CD40L, CD127, LAG3, Ki67, or a combination thereof. In specific embodiments, the phenotype is negative or positive expression of one or more of CD25, CD26, CD27, CD28, CD30, CD71, CD154, CD40L, CD127, LAG3, or Ki67. In specific embodiments, the phenotype is or includes the expression of CD25, CD127, LAG3, Ki67, or a combination thereof.

[0225] In some embodiments, the phenotype is or includes positive or negative expression of an activation marker in a CD3+ cell that expresses a recombinant receptor or CAR. In specific embodiments, the phenotype is or includes positive or negative expression of an activation marker in a CD4+ cell that expresses the recombinant receptor or CAR. In some embodiments, the phenotype is or includes positive or negative expression of an activation marker in a CD8+ cell that expresses the recombinant receptor or CAR. In specific embodiments, the activation marker is one or more of CD25, CD26, CD27, CD28, CD30, CD71, CD154, CD40L, CD127, LAG3, or Ki67. In specific embodiments, the activation marker is CD25, CD127, LAG3, Ki67, or a combination thereof.

[0226] In specific embodiments, the phenotype is or includes positive or negative expression of an activation marker and CD3+, CD4+, CD8+, CD3+/CAR+, CD4+/CAR+, CD8+/CAR+, annexin V–, annexin V–/CD3+, annexin V –/CD4+, annexin V–/CD8+, annexin V/–CD3+/CAR+, annexin V–/CD4+/CAR+, annexin V–/CD8+/CAR+, activated caspase–3–, activated caspase–3–/CD3+, activated caspase -3-/CD4+, activated caspase-3-/CD8+, activated caspase-3-/CD3+/CAR+, activated caspase-3-/CD4+/CAR+, activated caspase-3-/CD8+/CAR+ or a combination thereof.

[0227] In some embodiments, the phenotype is assessed in response to a stimulus, such as a stimulus that stimulates, triggers, induces, stimulates, or prolongs immunocyte function. In specific embodiments, the cells are incubated in the presence of stimulatory conditions or a stimulant, the phenotype is or includes a response to stimulation. In specific embodiments, the phenotype is or includes the production or secretion of a soluble factor in response to one or more stimulations. In some embodiments, the phenotype is or includes a lack of production or secretion of a soluble factor in response to one or more stimulations. In specific embodiments, the soluble factor is a cytokine.

[0228] In some embodiments, stimulating conditions may include one or more of specific environments, temperature, oxygen content, carbon dioxide content, time, means, such as nutrients, amino acids, antibiotics, ions, and/or stimulating factors such as cytokines, chemokines, antigens, binding patterns, fusion proteins, recombinant soluble receptors, and any other means designed to activate cells. In some embodiments, the cells are stimulated and the phenotype is determined by whether or not a soluble factor, such as a cytokine or chemokine, is produced or secreted. In some embodiments, the stimulation is non-specific, i.e., not antigen-specific stimulation. In some embodiments, cells are incubated in the presence of a stimulating condition or stimulant for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours. hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 18 hours, approximately 24 hours, approximately 48 hours, or for a period of time between 1 hour and 4 hours, between 1 hour and 12 hours, between 12 hours and 24 hours, including each, or for more than 24 hours.

[0229] In some embodiments, cells are stimulated with an agent that is an antigen or an epitope thereof that is specific for the recombinant receptor, or an antibody or fragment thereof that binds and/or recognizes the recombinant receptor, or a combination thereof. In some embodiments, the recombinant receptor is a CAR and the agent is an antigen or epitope thereof that is specific for CARs, or is an antibody or fragment thereof that binds and/or recognizes CARs, or a combination thereof. In specific embodiments, the cells are stimulated by incubating the cells in the presence of target cells with surface expression of an antigen recognized by CAR. In specific embodiments, the recombinant receptor is a CAR and the agent is an antibody or active fragment, variant, or portion thereof that binds the CAR. In specific embodiments, the antibody or active fragment, variant or portion thereof that binds CAR is an anti-idiotypic (anti-ID) antibody.

[0230] In some embodiments, the stimulatory conditions or agents include one or more agents, for example, a ligand capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the agent starts or initiates an intracellular TCR/CD3 signaling cascade in a T cell. Such agents may include antibodies, such as antibodies specific for a component of the TCR and/or a co-stimulatory receptor, for example, an anti-CD3 antibody, an anti-CD28 antibody, for example, bound to a solid support such as a bead, and/or one or more cytokines. In some embodiments, one or more of the agents is PMA and ionomycin.

[0231] In specific embodiments, the implementation, the phenotype is or includes the production or secretion of a cytokine in response to one or more stimulations. The production and/or secretion of cytokines contributes to immune responses and is involved in various processes, including the induction of antiviral proteins and the induction of T cell proliferation. Cytokines are not preformed factors but are rapidly produced and secreted in response to cellular activation. The production or secretion of cytokines can be measured, detected and/or quantified by any suitable method known in the art.

[0232] In specific embodiments, the phenotype is the production of one or more cytokines. In specific embodiments, the production of one or more cytokines is measured, detected and/or quantified by intracellular cytokine staining. Intracellular cytokine (ICS) staining by flow cytometry is a well-suited technique for examining cytokine production at the individual cell level. It detects the production and accumulation of cytokines in the endoplasmic reticulum after cell stimulation, allowing the identification of populations of cells that are positive or negative for the production of a particular cytokine, or the separation of cells with high production and low production based on a threshold. ICS can also be used in combination with other flow cytometry methods for immunophenotyping using cell surface markers or using MHC multimers to assess cytokine production in a specific subset of cells, making it an extremely flexible and versatile method. Other single cell based methods for measuring or detecting cytokine production include, but are not limited to, ELISPOT, limiting dilution, and T cell cloning.

[0233] In some embodiments, the phenotype is the production of a cytokine. In specific embodiments, the phenotype is the absence of cytokine production. In specific embodiments, the phenotype is positive in production or is a high level of cytokine production. In specific embodiments, the phenotype is negative for or low cytokine production. Cytokines may include, but are not limited to, IL-1, IL-1β, IL-2, sIL-2Ra, IL-3, IL-5, IL-6, IL-7, IL-8, IL-10, IL- 12, IL-13, IL 27, IL-33, IL-35, TNF, TNF-alpha, CXCL2, CCL2, CCL3, CCL5, CCL17, CCL24, PGD2, LTB4, interferon gamma (IFN-γ), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage inflammatory protein (MIP)-1a, MIP-1b, Flt-3L, fractalkin and/or IL-5.

[0234] In some embodiments, the phenotype is or includes cytokine production. In specific embodiments, the phenotype is or includes the production of more than one cytokine. In specific embodiments, the phenotype is or includes a lack of production of one or more cytokines. In specific embodiments, the phenotype is or includes the production, or lack thereof, of one or more of IL-2, IL-13, IFN-gamma, or TNF-alpha. In some embodiments, the phenotype is the presence of production, and/or the presence of a high level of cytokine production. In some embodiments, the phenotype is low, reduced, or absent cytokine production.

[0235] In some embodiments, the phenotype is or includes intrinsic (intracellular) cytokine production, for example, as assessed in the presence of a stimulant or under stimulatory conditions where secretion is prevented or inhibited. In specific embodiments, the phenotype is or includes deprivation or absence of intrinsic cytokine production. In specific embodiments, the phenotype is or includes the intrinsic amount of one or more cytokines when the production of more than one cytokine is assessed using an ICS assay. In specific embodiments, the phenotype is or includes an intrinsic level of one or more of IL-2, IL-13, IFN-gamma, or TNF-alpha, as assessed using ICS analysis. In some embodiments, the phenotype is or includes a low intrinsic level or no detectable amount of one or more cytokines, as assessed using an ICS assay. In specific embodiments, the phenotype is or includes a low intrinsic level or no detectable amount of IL-2, IL-13, IFN-gamma, or TNF-alpha, as assessed using ICS analysis.

[0236] Specific embodiments provide that the phenotype may include cytokine production or no or low levels of cytokine production. This may depend on several factors, which include, but are not limited to, the identity of the cytokine, the assay used to detect the cytokine, and the stimulant or condition used in conjunction with the assay. For example, in some embodiments, the phenotype is or includes no or low IL-13 production, as shown by ICS, while, in some embodiments, the phenotype is or includes IFN-gamma production, as shown. through ICS.

[0237] In some embodiments, the phenotype is or includes the production of one or more cytokines and any of CD3+, CD4+, CD8+, CD3+/CAR+, CD4+/CAR+, CD8+/CAR+, annexin V–, annexin V– CD3+, annexin V –CD4+, annexin V– CD8+, annexin V–CD3+/CAR+, annexin V– CD4+/CAR+, annexin V– CD8+/CAR+, activated caspase–3–, activated caspase–3–/CD3+, activated caspase–3–/CD4+ , activated caspase-3-/CD8+, activated caspase-3-/CD3+/CAR+, activated caspase-3-/CD4+/CAR+ or activated caspase-3-/CD8+/CAR+ or a combination thereof. In specific embodiments, the phenotype is or includes the production of one or more cytokines in CD4+/CAR+ and/or CD8+/CAR+.

[0238] In some embodiments, the phenotype is or includes a lack of production of one or more cytokines. In specific embodiments, the phenotype is or includes the absence of production of one or more cytokines and any of CD3+, CD4+, CD8+, CD3+/CAR+, CD4+/CAR+, CD8+/CAR+, annexin V–, annexin V– CD3+, annexin V–CD4+ , annexin V-CD8+, annexin V-CD3+/CAR+, annexin V-CD4+/CAR+, annexin V-CD8+/CAR+, activated caspase-3-, activated caspase-3-/CD3+, activated caspase-3-/CD4+, activated caspase-3-/CD8+, activated caspase-3-/CD3+/CAR+, activated caspase-3-/CD4+/CAR+ or activated caspase-3-/CD8+/CAR+, or a combination thereof.

[0239] In specific embodiments, the phenotype is or includes the presence or absence of an intrinsic level of one or more of IL-2, IL-13, IFN-gamma, or TNF-alpha, as assessed using ICS analysis, and one or more specific markers for a subgroup of cells or cells of a particular cell type. In some embodiments, the phenotype is or includes the production or absence of one or more of IL-2, IL-13, IFN-gamma or TNF-alpha and CD4+/CAR+ and/or CD8+/CAR+. In specific embodiments, the phenotype is or includes IL-2 and CD4+/CAR+ and/or CD8+/CAR+ production. In some embodiments, the phenotype is or includes no or low production of IL-2 and CD4+/CAR+ and/or CD8+/CAR+. In some embodiments, the phenotype is or includes IL-13 and CD4+/CAR+ and/or CD8+/CAR+ production. In some embodiments, the phenotype is or includes IL-13 and CD4+/CAR+ and/or CD8+/CAR+ production. In specific embodiments, the phenotype is or includes no or low production of IL-13 and CD4+/CAR+ and/or CD8+/CAR+. In some embodiments, the phenotype is or includes IFN-gamma and CD4+/CAR+ and/or CD8+/CAR+ production. In specific embodiments, the phenotype is or includes the production of TNF-alpha and CD4+/CAR+ and/or CD8+/CAR+. In specific embodiments, the phenotype is or includes no or low production of TNF-alpha and CD4+/CAR+ and/or CD8+/CAR+.

[0240] Any one or more phenotypes, alone or in combination, can be assessed or determined in accordance with the presented methods. In some embodiments, the phenotype is CD3+, CD3+/CAR+, CD4+/CAR+, CD8+/CAR+, or a combination thereof.

[0241] In specific embodiments, the phenotype is or includes CD3+. In specific embodiments, the phenotype is or includes CD3+/CAR+. In some embodiments, the phenotype is or includes CD8+/CAR+. In specific embodiments, the phenotype is or includes

[0242] In specific embodiments, the phenotype is or includes annexin-/CD3+/CAR+. In some embodiments, the phenotype is or includes annexin-/CD4+/CAR+ In specific embodiments, the phenotype is annexin-/CD8+/CAR.

[0243] In specific embodiments, the phenotype is or includes no or low levels of intracellular IL-2 and CD4+/CAR+. In specific embodiments, the phenotype is the absence or low level of intracellular IL-13 and CD4+/CAR+. In some embodiments, the phenotype is the absence or low level of intracellular expression of IL-13 and CD8+/CAR+ cells. In specific embodiments, the phenotype is the absence or low level of intracellular TNF-alpha CD4+/CAR+.

[0244] In specific embodiments, the phenotype is or includes CD8+/CAR+. In specific embodiments, the phenotype is or includes annexin-/CD8+/CAR+.

[0245] In specific embodiments, a number, multiple, or fraction of cells of a particular phenotype from a cell composition is determined, measured, obtained, detected, observed, and/or identified. In some embodiments, the cell composition is a T cell composition. In specific embodiments, the cell composition comprises cells expressing a recombinant receptor, such as CAR. In specific embodiments, the cell composition is a T cell therapeutic composition containing cells expressing the recombinant receptor, which can be administered to an individual to treat a disease or condition. In specific embodiments, the number of phenotype cells is the total number of phenotype cells in the cell composition. In specific embodiments, the number of cells of a phenotype is the total number of cells of that phenotype present in a dose of the cell composition. In specific embodiments, the number of cells of that phenotype is the number of cells of that phenotype present in a sample of the cell composition. In some embodiments, the number of phenotype cells can be expressed as the frequency, ratio, and/or percentage of phenotype cells present in a cell composition or in a dose or sample thereof.

[0246] In some embodiments, the cell composition is obtained from an individual, and the number, multiple, or fraction of cells of a phenotype is the number of cells of that phenotype present in the cell composition, normalized by the individual's body weight. In specific embodiments, the phenotype score is the number of cells of that phenotype per pound of an individual's body weight. In specific embodiments, the phenotype score is the number of cells of that phenotype per 1 lb (0.4 kg), 2 lb (0.8 kg), 3 lb (1.2 kg), 4 lb (1.6 kg), 5 pounds (2.0 kg), 10 pounds (4.1 kg), or 20 pounds (8.2 kg) of an individual's body weight. In some embodiments, the phenotype score is the number of cells of that phenotype per kg of an individual's body weight. In specific embodiments, the phenotype score is the number of cells of that phenotype per kg, 2 kg, 3 kg, 4 kg, 5 kg, 10 kg, or 20 kg of an individual's body weight.

[0247] In specific embodiments, the number, multiple, or fraction of cells of the phenotype is converted, for example, to narrow the range of corresponding values of the number, multiple, or fraction. In some embodiments, a transformation is any application of a deterministic mathematical function to each data point of a data set such that each data point x is replaced by a transformed value y=f(x), where f is a function. In general, transformations can be applied in such a way that the data appears to more closely match the assumptions of the statistical survey method to be used, or to improve the interpretability or appearance of the graphs. In most cases, the function that is used to transform data is reversible, and is usually continuous. A transformation is usually applied to a set of comparable measurements. Examples of suitable transformations include, but are not limited to, log and square root transformations, inverse transformations, and power transformations. In specific embodiments, the number, multiple, or proportion of cells of the phenotype is converted by logarithmic transformation. In particular embodiments, the logarithmic transformation is decimal log (log ₁₀ (x)), natural log (ln(x)) or binary log (log ₂ (x)).

B. Antigen-Specific or Recombinant Receptor-Dependent Activity

[0248] Specific embodiments provide that the recombinant receptor dependent activity, e.g., CAR dependent activity or "B", is an activity that occurs in a cell expressing the recombinant receptor that does not and/or cannot occur in the cell that does not express the recombinant receptor. In some embodiments, the recombinant receptor-dependent activity is an activity that depends on the activity or presence of the recombinant receptor. The recombinant receptor dependent activity can be any cellular process that is directly or indirectly affected by the expression and/or presence of the recombinant receptor or a change in activity, such as receptor stimulation, for the recombinant receptor. In some embodiments, the recombinant receptor dependent activity may include, but is not limited to, cellular processes such as cell division, DNA replication, transcription, protein synthesis, membrane transport, protein translocation, and/or secretion, or it may be a function immunocyte, for example, cytolytic activity. In specific embodiments, recombinant receptor-dependent activity can be measured by conformation change of the CAR receptor, phosphorylation of an intracellular signaling molecule, protein degradation, transcription, translation, protein translocation, and/or production and secretion of a factor such as a protein or growth factor, cytokine . In specific embodiments, the recombinant receptor is a CAR.

[0249] In specific embodiments, the recombinant receptor-dependent activity, eg, CAR-dependent activity, is a measure of a factor, eg, amount or concentration, or change in amount or concentration after stimulation of the cell composition. In specific embodiments, the factor may be a protein, a phosphorylated protein, a cleaved protein, a translocated protein, a protein in an active conformation, a polynucleotide, an RNA polynucleotide, mRNA, and/or shRNA. In specific embodiments, the measure may include, but is not limited to, an increase or decrease in kinase activity, protease activity, phosphatase activity, cAMP production, ATP metabolism, translocation, e.g., protein nuclear localization, increase in transcription activity, increase in translation activity, production and/or or soluble factor secretion, cellular uptake, ubiquitinylation and/or protein degradation.

[0250] In specific embodiments, the factor is a soluble factor that is secreted, such as a hormone, growth factor, chemokine, and/or cytokine.

[0251] In some embodiments, the recombinant receptor activity, eg, CAR-dependent activity, is a response to stimulation. In specific embodiments, the cells are incubated in the presence of a stimulating condition or stimulant, and the activity is or includes at least one aspect of a response to the stimulus. The response may include, but is not limited to, an intracellular signaling event, such as increased activity of a receptor molecule, increased kinase activity for one or more kinases, increased transcription of one or more genes, increased protein synthesis for one or more proteins, and/or an intracellular signaling, such as increased protein kinase activity. In some embodiments, the response or activity is associated with immune activity, and may include, but is not limited to, the production and/or secretion of a soluble factor, such as a cytokine, an increase in antibody production, and/or an increase in cytolytic activity.

[0252] In specific embodiments, the response to stimulation from the cell composition is assessed by measuring, detecting, or quantifying the response to the stimulus, i.e., at least one activity initiated, triggered, maintained, prolonged, and/or evoked by the stimulus. In specific embodiments, the cells are stimulated and the response to stimulation is an activity that is specific to cells expressing the recombinant receptor. In specific embodiments, the activity is a recombinant receptor-specific activity and the activity occurs in cells expressing the recombinant receptor but does not occur, or only minimally occurs, in cells not expressing the receptor. In specific embodiments, the recombinant receptor is a CAR. In some embodiments, the activity is a CAR dependent activity.

[0253] The conditions used to stimulate cells, such as immunocytes or T cells, may include one or more of specific environments, temperature, oxygen content, carbon dioxide content, time, means, such as nutrients, amino acids, antibiotics, ions and/or stimulatory factors such as cytokines, chemokines, antigens, binding patterns, fusion proteins, recombinant soluble receptors, and any other means designed to activate cells. In some embodiments, cells are stimulated and activity is determined by whether or not a soluble factor, such as a cytokine or chemokine, is produced or secreted. In some embodiments, the stimulation is non-specific, i.e., not antigen-specific stimulation.

[0254] In some embodiments, the activity is specific to cells expressing the recombinant receptor. In some embodiments, activity that is specific to cells expressing the recombinant receptor does not occur in cells that do not express the recombinant receptor. In specific embodiments, the recombinant receptor is a CAR and the activity is a CAR dependent activity. In specific embodiments, the activity is not present in cells that do not express the recombinant receptor under the same conditions that the activity is present in cells expressing the recombinant receptor. In specific embodiments, the CAR dependent activity is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or about 99% less than CAR dependent activity in CAR cells under the same conditions.

[0255] In some embodiments, the activity is specific for cells expressing a recombinant receptor, e.g., CAR, and the activity is obtained by stimulation with an agent or under stimulatory conditions that are specific for cells expressing the recombinant receptor. In some embodiments, the recombinant receptor is a CAR, and the CAR-specific stimulation stimulates, triggers, initiates, and/or prolongs activity in CAR+ cells, but does not stimulate, trigger, initiate, and/or prolong activity in CAR– cells. In some embodiments, the CAR dependent activity is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or about 99% less in CAR– cells than in CAR+ cells after stimulation through a CAR-specific stimulus.

[0256] In specific embodiments, the activity is a recombinant receptor-dependent, eg, CAR-dependent, activity stimulated by an agent that is specific for the recombinant receptor. In some embodiments, a recombinant receptor-specific agent, eg, a CAR-specific agent, is an antigen or epitope thereof that is bound and/or recognized by a recombinant receptor, eg, CAR. In some embodiments, the recombinant receptor-specific agent is an antibody or active fragment thereof that binds and/or recognizes the recombinant receptor. In some embodiments, the agent is an anti-idiotypic antibody, or an active fragment, variant, or portion (anti-ID) thereof, that binds a recombinant receptor. In specific embodiments, the recombinant receptor-specific agent is a cell that expresses an antigen on its surface. In some embodiments, the recombinant receptor-dependent activity is stimulated by an antigen or epitope thereof bound and/or recognized by the recombinant receptor. In specific embodiments, the recombinant receptor-dependent activity is stimulated by an antibody or active fragment thereof that binds and/or recognizes the recombinant receptor. In specific embodiments, the recombinant receptor dependent activity is stimulated by an anti-ID.

[0257] In some embodiments, the activity is measured in a cell composition containing cells expressing a recombinant receptor, for example, CAR, and the measurement is compared to one or more controls. In specific embodiments, the control is a similar or identical cell composition that has not been stimulated. For example, in some embodiments, activity is measured in a cell composition after or during incubation with the agent, and the resulting measurement is compared to a control activity measurement for a similar or identical cell composition that was not incubated with the agent. In some embodiments, the activity is a recombinant receptor dependent activity and both the cell composition and the control cell composition contain cells expressing the recombinant receptor. In some embodiments, the activity is a recombinant receptor dependent activity and the control is selected from a similar cell composition that does not contain cells expressing the recombinant receptor, such as CAR+ cells. Thus, in some embodiments, a cell composition that contains recombinant receptor-expressing cells and a control cell composition that does not contain recombinant receptor-expressing cells are contacted with a specific agent that stimulates the recombinant receptor. In specific embodiments, the control is an indicator for the same cell composition that expresses the recombinant receptor that is obtained prior to any stimulation. In specific embodiments, a benchmark is obtained to determine the background signal, and the benchmark is subtracted from the activity score. In some embodiments, the activity score in the cell composition is divided by the control score to obtain a value that is the ratio of activity to control level.

[0258] In specific embodiments, the activity is or includes the production and/or secretion of a soluble factor. In some embodiments, the activity is a recombinant receptor, eg, CAR, dependent activity that is or includes the production and/or secretion of a soluble factor. In specific embodiments, the soluble factor is a cytokine or chemokine.

[0259] Suitable methods for measuring the production or secretion of a soluble factor are known in the art. The production and/or secretion of a soluble factor can be measured by determining the concentration or amount of the extracellular level of the factor, or by determining the level of transcriptional activity of the gene encoding the factor. Suitable methods include, but are not limited to, assays such as immunoassay, aptamer-based assay, histological or cytological assay, mRNA expression level assay, ELISA, immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, flow cytometry, surface plasmon resonance (SPR), chemiluminescence analysis, flow immunoassay, inhibition or avidity assay, protein microarray analysis, high performance liquid chromatography (HPLC), Meso Scale Discovery (MSD) electrochemiluminescence, and bead-based multiplex immunoassays (MIA). In some embodiments, a suitable method may use a detectable binding reagent that specifically binds a soluble factor.

[0260] In specific embodiments, the measurement of the soluble factor is a measurement by ELISA (enzyme-linked immunosorbent assay). ELISA is a plate assay method designed for the detection and quantification of substances such as peptides, cytokines, antibodies and hormones. In ELISA, the soluble factor must be immobilized on a solid surface and then complexed with the antibody bound to the enzyme. Detection is carried out by assessing the activity of the conjugated enzyme by incubation with the substrate to obtain a detectable signal. In some embodiments, CAR-dependent activity is measured using an ELISA assay.

[0261] In some embodiments, the recombinant receptor dependent activity is the secretion or production of a soluble factor. In specific embodiments, production or secretion is stimulated in a cell composition comprising recombinant receptor expressing cells, eg CAR expressing cells, by a recombinant receptor specific agent, eg a CAR+ specific agent. In some embodiments, the recombinant receptor-specific agent is an antigen or an epitope thereof that is specific for the recombinant receptor; a cell, eg a target cell, that expresses an antigen; or an antibody or portion or variant thereof that binds and/or recognizes a recombinant receptor; or a combination of them. In specific embodiments, the recombinant receptor-specific agent is a recombinant protein containing an antigen or epitope thereof that is bound or recognized by the recombinant receptor.

[0262] In specific embodiments, recombinant receptor-dependent production and/or secretion of a soluble factor is measured by incubating a cell composition containing cells expressing a recombinant receptor, e.g., CAR, with a recombinant receptor-specific agent, e.g., a CAR+ specific agent. In specific embodiments, the soluble factor is a cytokine or chemokine. In some embodiments, cells from a cell composition containing recombinant receptor-expressing cells are incubated in the presence of a recombinant receptor-specific agent for a period of time, and soluble factor production and/or secretion is measured at one or more time points during incubation. In some embodiments, cells are incubated with a CAR-specific agent for up to or about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours, approximately 48 hours, or within a period of time between 1 hour and 4 hours, between 1 hour and 12 hours, between 12 hours and 24 hours, each included, or for more than 24 hours, and the amount of a soluble factor such as a cytokine is detected.

[0263] In some embodiments, the recombinant receptor-specific agent is a target cell that expresses an antigen recognized by the recombinant receptor. In some embodiments, the recombinant receptor is a CAR and cells of the cell composition are incubated with target cells at a ratio of total cells, CAR+ cells, CAR+/CD8+ cells, or annexin-/CAR+/CD8+ cells from the cell composition to target cells of approximately 10 :1, approximately 5:1, approximately 4:1, approximately 3:1, approximately 2:1, approximately 1:1, approximately 1:2, approximately 1:3, approximately 1:4, approximately 1:5, approximately 1 :6, about 1:7, about 1:8, about 1:9 or about 1:10, or in a range between any of the above, for example, in a ratio between 10:1 and 1:1, 3:1 and 1: 3, or 1:1 and 1:10, each included.

[0264] In some embodiments, between about 1x102 and about 1x104, between about 1x103 and about 1x105, between about 1x104 and about 1x106, between about 1x105 and about 1x 107, between about 1x106 and about 1x108, between about 1x107 and about 1x109, or between about 1x108 and about 1x1010 cells from the cell composition, including each, are incubated with a recombinant receptor specific agent , for example, with a CAR+ specific reagent. In some embodiments, between about 1x106 and about 1x107 the cells of the cell composition, inclusive, are incubated with a recombinant receptor specific agent, eg, a CAR+ specific reagent. In specific embodiments, approximately 2.5 x 106 cells of the composition are incubated with a recombinant receptor specific agent. In particular embodiments, between about 1x102 and about 1x104, between about 1x103 and about 1x105, between about 1x104 and about 1x106, between about 1x105 and about 1x107, between about 1x106 and about 1x108, between about 1x107 and about 1x109, or between about 1x108 and about 1x1010 recombinant receptor-expressing cells, CAR+ cells, CAR+/CD8+ cells, or annexin–/CAR+ The /CD8+ cells from the cell composition, including each, are incubated with a CAR+ specific agent.

[0265] In some embodiments, cells from the cell composition are incubated with a recombinant receptor specific agent, eg, a CAR+ specific agent, in a volume of cell medium. In specific embodiments, cells are incubated with a recombinant receptor-specific agent in a volume of at least or about 1 µl, at least or about 10 µl, at least or about 25 µl, at least or about 50 µl, at least or about 100 µl, at least or about 500 µl, at least or about 1 ml, at least or about 1.5 ml, at least or about 2 ml, at least or about 2.5 ml, at least or about 5 ml, at least or about 10 ml, at least or about 20 ml, at least or about 25 ml, at least or about 50 ml, at least or about 100 ml, or more than 100 ml. In specific embodiments, cells are incubated with a CAR+ specific agent in a volume ranging between about 1 µl and about 100 µl, between about 100 µl and about 500 µl, between about 500 µl and about 1 ml, between about 500 µl and about 1 ml, between about 1 ml and about 10 ml, between about 10 ml and about 50 ml, or between about 10 ml and about 100 ml, each included. In specific embodiments, the cells are incubated with the recombinant receptor specific agent in a volume between about 100 µl and about 1 ml, inclusive. In specific embodiments, the cells are incubated with a recombinant receptor specific agent in a volume of approximately 500 µl.

[0266] In some embodiments, cells from the cell composition are incubated with a CAR+ specific agent in an amount between about 1 fmol and about 1 pmol, between about 1 pmol and about 1 nmol, between about 1 nmol and about 1 µmol, between about 1 µmol and about 1 mmol, or between about 1 mmol and 1 mol, each included. In specific embodiments, the cells of the cell composition are incubated with a CAR+ specific agent at a concentration between about 1 pM and about 1 pM, between about 1 pM and about 1 nM, between about 1 nM and about 1 µM, between about 1 µM and about 1 mM. , or between approximately 1 mm and 1 M, each including. Illustrative units include, but are not limited to, pg/ml, pg/(ml/hour), pg(ml x cell), pg/(ml x hour x cell), and pg/(ml x hour x 10 ⁶ cells).

[0267] In specific embodiments, a measure of recombinant receptor specific activity, e.g., CAR+ specific activity, is the amount or concentration, or the relative amount or concentration, of a soluble factor in the T cell composition at a time point during or after incubation. In specific embodiments, the benchmark is subtracted from the score, or the score is normalized to the benchmark. In some embodiments, the control value is the value for the same cell composition obtained prior to incubation. In specific embodiments, a control score is a score obtained from an identical control cell composition that has not been incubated with a recombinant receptor-specific stimulant. In specific embodiments, a control is a score obtained at an identical time point during incubation with a recombinant receptor-specific agent for a cell composition not containing recombinant receptor-positive cells.

[0268] In some embodiments, the indicator is a normalized ratio of an amount or concentration compared to a control. In specific embodiments, the rate is the amount or concentration of soluble factor per amount of time, such as per minute or per hour. In some embodiments, the indicator is the amount or concentration of soluble factor per cell or group or reference number of cells, e.g., per 100 cells, per ¹⁰³ cells, per ¹⁰⁴ cells, per ¹⁰⁵ cells, per ¹⁰⁶ cells, etc. .d. In specific embodiments, the indicator is the amount or concentration of soluble factor per amount of time, per cell, or per reference number of cells. In some embodiments, the indicator is the amount or concentration of soluble factor per cell that expresses the recombinant receptor, CAR+ cell, CAR+/CD8+ cell, or annexin-/CAR+/CD8+ cell from the cell composition. In specific embodiments, the score is the amount or concentration of soluble factor per amount of time (e.g., per minute or hour) per cell that expresses the recombinant receptor, CAR+ cell, CAR+/CD8+ cell, or annexin-/CAR+/CD8+ cell from the composition cells. In some embodiments, the measure is the amount or concentration of soluble factor per amount of time per amount or concentration of the recombinant receptor or CAR+ specific agent. In some embodiments, the score is the amount or concentration of soluble factor per cell or group, or a reference number of cells per amount or concentration of CAR+ specific agent. In specific embodiments, the metric is the amount or concentration of soluble factor per amount of time, per amount or concentration of recombinant receptor or CAR+ specific agent, per cell, or per reference number of cells. In some embodiments, the score is the amount or concentration of a soluble factor per amount or concentration of a recombinant receptor or CAR+ specific agent, per cell that expresses the recombinant receptor, CAR+ cell, CAR+/CD8+ cell, or annexin–/CAR+/CD8+ cell from cell compositions. In specific embodiments, the score is the amount or concentration of soluble factor per amount of time, per amount or concentration of recombinant receptor or CAR+ specific agent, per number of CAR+ cells, CAR+/CD8+ cells, or annexin-/CAR+/CD8+ cells from the cell composition.

[0269] In specific embodiments, the recombinant receptor or CAR dependent activity is the production or secretion of two or more soluble factors. In specific embodiments, the recombinant receptor or CAR dependent activity is the production or secretion of two, three, four, five, six, seven, eight, nine, ten, or more than ten soluble factors. In some embodiments, scores for two, three, four, five, six, seven, eight, nine, ten, or more than ten soluble factors are combined into an arithmetic or geometric mean. In specific measurements, a measure of recombinant receptor activity is the composite secretion of two, three, four, five, six, seven, eight, nine, ten, or more than ten soluble factors.

[0270] In specific embodiments, the recombinant receptor dependent activity score is converted, for example, by a logarithmic transformation. In specific embodiments, the recombinant receptor activity score is converted to decimal log (log ₁₀ (x)), natural log (ln(x)) or binary log (log ₂ (x)). In some embodiments, the recombinant receptor dependent activity score is a composite measure of the production or secretion of two or more soluble factors. In some embodiments, two or more measures of production or secretion of soluble factors are combined into a composite measure. In specific embodiments, the recombinant receptor dependent activity score is converted to normalize to a reference score. In specific embodiments, the recombinant receptor dependent activity score is converted to normalize to a reference score.

[0271] In specific embodiments, the soluble factor is a cytokine. Cytokines are a large group of small signaling molecules that function extensively in cellular communication. Cytokines are most commonly associated with various immunomodulatory molecules, including interleukins, chemokines, and interferons. Alternatively, cytokines can be characterized by their structure, which classifies them into four families, a four-alpha-helix family including the IL-2 subfamily, the IFN subfamily, and the IL-10 subfamily; the IL-1 family, the IL-17 family, and cysteine node cytokines, including members of the transforming growth factor beta family. In some embodiments, the CAR-dependent activity is the production or secretion of one or more soluble factors, including interleukins, interferons, and chemokines. In specific embodiments, the CAR-dependent activity is the production or secretion of one or more of an IL-2 family member, an IFN subfamily member, an IL-10 subfamily member; a member of the IL-1 family, a member of the IL-17 family, a cysteine-knotted cytokine, and/or a member of the transforming growth factor beta family.

[0272] In specific embodiments, the recombinant receptor or CAR dependent activity is the production and/or secretion of one or more of IL-1, IL-1β, IL-2, sIL-2Ra, IL-3, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL 27, IL-33, IL-35, TNF, TNF-alpha, CXCL2, CCL2, CCL3, CCL5, CCL17 , CCL24, PGD2, LTB4, interferon gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein (MIP)-1a, MIP-1b, Flt-3L, fractalkine and/or IL-5 . In specific embodiments, the CAR-dependent activity is the production or secretion of a Th17 cytokine. In some embodiments, the Th17 cytokine is GMCSF. In some embodiments, the CAR-dependent activity includes the production or secretion of a Th2 cytokine, where the Th2 cytokine is IL-4, IL-5, IL-10, or IL-13.

[0273] In specific embodiments, the recombinant receptor or CAR dependent activity is the production or secretion of a pro-inflammatory cytokine. Pro-inflammatory cytokines play a role in initiating the inflammatory response and in regulating host defenses against pathogens mediated by the innate immune response. Pro-inflammatory cytokines include, but are not limited to, interleukins (IL), interleukin-1-beta (IL-1), interleukin-3 (IL-3), interleukin-5 (IL-5), interleukin-6 (IL-6) , interleukin-13 (IL-13), tumor necrosis factor (TNF), CXC chemokine ligand 2 (CXCL2), CC chemokine ligand 2 (CCL2), CC chemokine ligand 3 (CCL3), CC chemokine ligand 5 ( CCL5), 17 CC chemokine ligand (CCL17), 24 CC chemokine ligand (CCL24), prostaglandin D2 (PGD2) and leukotriene B4 (LTB4), as well as IL-33.). In some embodiments, the CAR-dependent activity is the production and or secretion of a member of the interleukin and/or TNF family. In specific embodiments, the CAR-dependent activity is the production and/or secretion of IL-1, IL-6, IL-8, and IL-18, TNF-alpha, or a combination thereof.

[0274] In specific embodiments, the CAR-specific activity is IL-2 secretion, IFN-gamma, TNF-alpha, or a combination thereof.

[0275] In specific embodiments, the activity is the cytolytic (cytotoxic) activity of the T cell composition. In specific embodiments, the activity is a recombinant receptor dependent, eg CAR, cytolytic activity. In some embodiments, recombinant receptor-dependent cytolytic activity is assessed by exposing, incubating, and/or contacting cells expressing the recombinant receptor, or a cell composition containing cells expressing the recombinant receptor, with a target cell that expresses the antigen and/or or an epitope bound and/or recognized by the recombinant receptor. Cytolytic activity can be measured by direct or indirect measurement of the number of target cells over time. For example, target cells can be incubated with a detectable marker prior to incubation with cells expressing the recombinant receptor, for example, with a marker that is detectable when the target cell is lysed, or a detectable marker that is detectable in viable target cells. These readings provide direct or indirect target cell count and/or target cell death and can be measured at various time points during the assay. A decrease in the number of target cells and/or an increase in target cell death indicates the cytolytic activity of the cells. Suitable methods for performing cytolytic assays are known in the art and include, but are not limited to, chromium-51 release assays, assays using non-radioactive chromium, flow cytometry assays that use fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE), PKH -2 and PKH-26.

[0276] In specific embodiments, dependent on a recombinant receptor, e.g., CAR, cytolytic activity is measured by incubating a cell composition containing cells expressing the recombinant receptor with target cells expressing an antigen or epitope thereof bound or recognized by the recombinant receptor. In specific embodiments, the recombinant receptor is a CAR. In some embodiments, cells from the cell composition are incubated with target cells in a ratio of about 10:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1: 2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10, or in a ratio between 10:1 and 1:1, 3:1 and 1:3 or 1:1 and 1:10, each included. In some embodiments, cells from the cell composition are incubated with target cells in a ratio of CAR+ cells, CAR+/CD8+ cells, or annexin-/CAR+/CD8+ cells from the cell composition to target cells of about 10:1, about 5:1, about 4:1, approximately 3:1, approximately 2:1, approximately 1:1, approximately 1:2, approximately 1:3, approximately 1:4, approximately 1:5, approximately 1:6, approximately 1:7, approximately 1:8, about 1:9, or about 1:10, or in a ratio between 10:1 and 1:1, 3:1 and 1:3, or 1:1 and 1:10, each included.

[0277] In specific embodiments, the cells of the cell composition are incubated with target cells for up to or about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 8 hours, about 12 hours, approximately 18 hours, approximately 24 hours, approximately 48 hours, or more than 48 hours. In some embodiments, cell compositions are incubated for approximately 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In some embodiments, between about 1x102 and about 1x104, between about 1x103 and about 1x105, between about 1x104 and about 1x106, between about 1x105 and about 1x107, between about 1x106 and about 1x108, between about 1x107 and about 1x109, or between about 1x108 and about 1x1010 cells from the cell composition, including each, are incubated with target cells. In particular embodiments, between about 1x102 and about 1x104, between about 1x103 and about 1x105, between about 1x104 and about 1x106, between about 1x105 and about 1x107, between about 1x106 and about 1x108, between about 1x107 and about 1x109, or between about 1x108 and about 1x1010 CAR+ cells, CAR+/CD8+ cells, or annexin–/CAR+/CD8+ cells from a cell composition , including each, are incubated with target cells.

[0278] In some embodiments, the activity score is compared to a control. In specific embodiments, the control is a target cell culture that has not been incubated with the cell composition. In some embodiments, the control is a measure of a control cell composition containing no CAR+ cells that is incubated with the target cells at the same ratio.

[0279] In specific embodiments, the indicator in the analysis of cytolytic activity is the number of target cells that are viable, at a time point during or at the end of incubation. In specific embodiments, the indicator is the amount of a target cell death marker, eg, chromium-51, released during incubation. In some embodiments, the index is the level of target cell death, which is determined by subtracting the number of target cells in co-incubation, at a given time point, from the number of target cells in a control that was incubated separately. In some embodiments, the score is the percentage of target cells remaining at the time point compared to the original number of target cells. In specific embodiments, the indicator is the number of cells destroyed over a period of time. In specific embodiments, the metric is the number of cells killed per cell of the cell composition. In some embodiments, the indicator is the number of cells killed per cell, or the number of cells killed per set or reference number of cells, for example, but not limited to, the number of target cells killed per 100 cells, per 10 ³ cells, per 10 ⁴ cells, per ¹⁰⁵ cells, per ¹⁰⁶ cells, per ¹⁰⁷ cells, per ¹⁰⁸ cells, per ¹⁰⁹ cells or per ¹⁰¹⁰ cells from the composition. In specific embodiments, the score is the number of cells killed per CAR+ cell, CAR+/CD8+ cell, or annexin–/CAR+/CD8+ cell, or a reference or set number thereof, from a cell composition. In specific embodiments, the indicator is the number of cells killed over a period of time per cell of the cell composition. In specific embodiments, the score is the number of cells killed over a period of time per CAR+ cells, CAR+/CD8+ cells, or annexin-/CAR+/CD8+ cells from a cell composition.

[0280] In some embodiments, the recombinant receptor dependent activity is not a cytolytic activity. In specific embodiments, the CAR+ dependent activity is not a cytolytic activity.

Reference standards

[0281] Specific embodiments provide that recombinant receptor-dependent activity and/or CAR+-dependent activity can be normalized to a reference measurement (i.e., a benchmark). In particular embodiments, the benchmark is a predetermined metric, or value thereof, for activity. In some embodiments, the benchmark is the mean or mode for a plurality of cell compositions originating from different individuals. In some embodiments, the cell compositions of the plurality are from different individuals and contain cells expressing the same recombinant receptor. In specific embodiments, the reference standard is derived from the normalized or subtracted scores for the control groups in the same manner as the recombinant receptor dependent activity score. In specific embodiments, the reference score is derived from scores obtained from different cell compositions obtained under the same assay conditions as the recombinant receptor activity score. In specific embodiments, the recombinant receptor dependent activity score is normalized to a reference score.

[0282] In specific embodiments, the benchmark is the mean or mode of the scores for a plurality of cell compositions. In specific embodiments, a benchmark is obtained from a cell composition that contains cells expressing the same CAR as at least a portion of the cells in the cell compositions of the plurality express. In specific embodiments, the benchmark is the mean or median of the measurements obtained for two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, more than twenty-five, more than thirty, more than forty, or more than fifty cell compositions.

[0283] In specific embodiments, a measure of recombinant receptor-specific or CAR-specific activity is obtained from a cell composition derived from a human individual with a disease or condition and compared against a benchmark. In specific embodiments, a reference value is obtained for cell compositions that are T cell therapeutic compositions. In specific embodiments, the T cell therapeutic compositions are cell compositions that are administered therapeutically to human subjects, eg, in clinical trials. In some embodiments, safety and efficacy outcomes after administration of reference T cell compositions are known. In specific embodiments, the individual has a disease or condition expressing an antigen or associated with an antigen.

[0284] In some embodiments, each of the reference T cell compositions was administered to an individual. In specific embodiments, administration of the reference T cell compositions to individuals is monitored and determined to obtain an acceptable safety profile after administration to the individual. In specific embodiments, administration of the T cell reference compositions did not result in any severe toxicity. In specific embodiments, administration of the reference T cell compositions did not result in any severe neurotoxicity. In specific embodiments, the reference T cell compositions are all compositions associated with a neurotoxicity score of Grade 4 or less, Grade 3 or less, Grade 2 or less, Grade 1 or less, or Grade 0. In some embodiments, the reference compositions cells are associated with acceptable safety profiles. In specific embodiments, the acceptable safety profile is no observed grade 1 or higher neurotoxicity, observed grade 2 or higher neurotoxicity, observed grade 3 or higher neurotoxicity, or grade 4 or higher neurotoxicity. In specific embodiments, the reference T cell compositions are associated with acceptable safety profiles with no observable grade 3 or higher neurotoxicity. In specific embodiments, the reference T cell compositions are associated with acceptable safety profiles with no observable grade 3 or higher neurotoxicity.

[0285] In specific embodiments, for each of the reference T cell therapeutic compositions, the desired efficacy was observed or determined as a result after administration to the individual. In specific embodiments, the individual has a disease or condition expressing an antigen or associated with an antigen, such as those receiving the reference T cell compositions. In specific embodiments, for each of the reference T cell therapeutic compositions, a complete response (CR) was observed or determined as a result.

[0286] In some embodiments, the measure of recombinant receptor dependent activity is a measure of the production or secretion of one or more soluble factors. In specific embodiments, the soluble factors are one or more cytokines. In specific embodiments, the CAR-specific activity is the secretion of two or more cytokines. In specific embodiments, a recombinant receptor-dependent secretion score of two or more cytokines is obtained for the cell composition and normalized to reference scores for the respective cytokines. In some embodiments, two or more normalized measurements are normalized to reference values. In some embodiments, the normalized scores are combined into a single composite value.

[0287] In specific embodiments, the CAR-specific activity is IL-2 secretion, IFN-gamma, TNF-alpha, or combinations thereof. In specific embodiments, a measure of CAR-dependent secretion of IL-2, IFN-gamma, TNF-alpha, or combinations thereof, is obtained from the cell composition and normalized to reference values for the respective cytokines. In some embodiments, scores for two or more of IL-2, IFN-gamma, and TNF-alpha are normalized to reference scores. In some embodiments, the normalized scores are combined into a single value.

[0288] Specific embodiments provide that when a reference score is obtained for reference compositions associated with acceptable safety profiles and/or desired efficacy, a normalized score close to 1 has a high probability of also being associated with acceptable safety profiles and/or desired efficacy . Particular embodiments provide that a score for a T cell composition has a high probability of being associated with acceptable safety profiles and/or desired efficacy if the score to benchmark ratio is about or about 1:1.

[0289] Specific embodiments provide that observed CAR+ dependent secretion of particular cytokines is not indicative of outcome or likelihood, or does not correlate with outcome or likelihood, or does not significantly correlate with outcome or likelihood of an adverse event or toxicity. For these cytokines, the CAR-dependent secretion score does not predict the safety or efficacy of therapeutic administration of a T cell composition. In some embodiments, measures of CAR-dependent cytokine secretion that do not correlate with safety or efficacy, or are not indicative of safety or efficacy, are measured and compared to a benchmark, or normalized to a benchmark derived from measurements of the respective cytokines obtained from cell compositions. derived from other individuals. In some embodiments, CAR-dependent cytokine secretion scores for cytokines that do not correlate with safety or efficacy, or are not indicative of safety or efficacy, are compared to corresponding benchmarks to determine if the cell composition elicits different responses to CAR-dependent stimulation. . In some embodiments, secretion is not considered to be correlated with safety or efficacy or indicative of safety or efficacy. Specific embodiments contemplate that normalization of one or more measures of CAR-dependent cytokine secretion for IL-4, IL-5, IL-10, or IL-13, or a composite value thereof, can be used to determine or validate the variability and reliability of measures obtained for cell compositions.

III. METHODS FOR EVALUATION OF THE THERAPEUTIC COMPOSITION OF T-CELLS

[0290] The present invention relates to methods comprising characterizing or evaluating the specific activity and/or safety and/or activity and/or efficacy of a therapeutic composition, in some embodiments, and a T cell therapeutic composition before or during or after administration one or more unit doses of the T cell therapeutic composition to an individual suffering from a disease or condition. In some embodiments, the present methods evaluate the risk of an adverse event following administration of a T cell composition described herein. In some embodiments, the T cell composition is not released for subsequent administration to an individual if the composition is determined to be outside a certain safety range. In some aspects, the methods provided include a release assay that can evaluate cell compositions to determine, among other aspects, if the composition is administered to an individual, whether it is likely to correlate with a risk of toxicity, such as severe neurotoxicity, and/or whether it is likely to be efficient. In some embodiments, the T cell composition is not recommended for administration to the individual, and/or the unit dose to be administered to the individual is changed or adjusted if B exceeds the USL. If B is below USL, the product can be released for treatment.

[0291] In some embodiments, the sample to be evaluated using the methods described herein is selected from or obtained from a T cell composition containing T cells derived from an individual having a disease or condition and transduced with a nucleic acid encoding a recombinant a receptor, optionally a chimeric antigen receptor (CAR), that specifically binds to an antigen associated with a disease or condition. In some aspects, the assays described herein determine B for a cell composition, where B is a parameter value, or a multiple thereof, or a transformation that indicates the degree of or correlates with the degree of recombinant receptor dependent activity in the composition. The present invention also relates to a method for assaying a therapeutic T cell composition comprising evaluating a sample from a T cell composition containing T cells containing a recombinant receptor that specifically binds to an antigen associated with a disease or condition by the activity of the cell composition based on B where B is a parameter value, or a multiple thereof, or a transformation that indicates the degree of or correlates with the degree of activity in a given composition. In some embodiments, the present methods further evaluate the activity of the cell composition based on B and/or evaluate whether the composition is above the LSL and/or below the USL for B.

[0292] In some embodiments, if B is greater than USL, the unit dose to be administered to the individual is changed or adjusted, where the adjusted unit dose contains a target number of cells or a target number of reference units (RU) from a T cell composition, where the RU in the composition is determined according to the formula: RU=A x B, where A is the number of cells, or its multiple, proportion or transformation, of cells of a certain phenotype present in a given composition, or represents the average or weighted average of the number of cells, or its multiple, proportion or transformation , two or more phenotypes in a given composition; and B is a parameter value, or a fraction thereof, or a multiple, or a transformation that indicates the degree of or correlates with the degree of recombinant receptor dependent, optionally CAR dependent, activity in the composition.

[0293] In some embodiments, the parameter is a measure of one or more factors, or a normalized value thereof. In some embodiments, a production indicator is measured in an intracellular cytokine staining assay comprising incubating a sample from a T cell composition with a polyclonal agent, an antigen specific agent, or a recombinant receptor binding agent. In some embodiments, the indicator is determined in an assay comprising culturing or incubating for a fixed time, optionally 24 hours, a given composition or a sample thereof in the presence of an antigen, antigen-expressing cells, and/or an agent that specifically binds to a recombinant receptor, optional, CAR. In some aspects, the assay is an ELISA.

[0294] In some embodiments, a factor score is a score or level indicative of concentration, relative concentration, amount or relative amount of factor, amount or relative amount of factor per unit of cells administered from a given composition, amount or relative amount of factor per unit of administered cells from this composition per unit of time, optionally one hour. In some embodiments, one or more of the factors is one or a combination of soluble factors, optionally one or a combination of cytokines, chemokines, or soluble receptors, optionally soluble costimulatory receptors.

[0295] In some embodiments, the parameter is a score for one or more factors, or a normalized value thereof. In some embodiments, the recombinant receptor dependent activity is indicative of the production or accumulation of one or more of IL-1, IL-1β, IL-2, sIL-2Ra, IL-3, IL-5, IL-6, IL- 7, IL-8, IL-10, IL-12, IL-13, IL 27, IL-33, IL-35, TNF, TNF-alpha, CXCL2, CCL2, CCL3, CCL5, CCL17, CCL24, PGD2, LTB4 , interferon gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein (MIP)-1a, MIP-1b, Flt-3L, fractalkine and/or IL-5. In specific embodiments, the CAR-dependent activity is the production or secretion of a Th17 cytokine. In some embodiments, the Th17 cytokine is GMCSF. In some embodiments, the recombinant receptor dependent activity is indicative of the production or accumulation of a Th2cytokine, where the Th2cytokine is IL-4, IL-5, IL-10, or IL-13. In specific embodiments, B is indicative of the production and/or secretion of a pro-inflammatory cytokine such as interleukins (IL), interleukin-1-beta (IL-1), interleukin-3 (IL-3), interleukin-5 (IL-5 ), interleukin-6 (IL-6), interleukin-13 (IL-13), tumor necrosis factor (TNF), CXC chemokine ligand 2 (CXCL2), CC chemokine ligand 2 (CCL2), CC chemokine ligand 3 (CCL3), CC-chemokine ligand 5 (CCL5), CC-chemokine ligand 17 (CCL17), CC-chemokine ligand 24 (CCL24), prostaglandin D2 (PGD2) and leukotriene B4 (LTB4), as well as IL-33. In some embodiments, the recombinant receptor dependent activity is indicative of the production or accumulation of an interleukin and/or a member of the TNF family such as IL-1, IL-6, IL-8 and IL-18, TNF-alpha, or combinations thereof. In specific embodiments, the recombinant receptor dependent activity is indicative of the production or accumulation of IL-2, IFN-gamma, TNF-alpha, or a combination thereof. In some embodiments, one or more of the factors is one or a combination of pro-inflammatory cytokines, Th2 cytokines, and Th17 cytokines. In some embodiments, one or more of the factors is one or a combination of IL-2, IFN-gamma, TNF-alpha, IL4, IL-5, IL-10, IL-13, GM-CSF, sCD137, MIP1a, and M1Pb . In some embodiments, one or more of the factors is one or a combination of IL-2, IFN-gamma, TNF-alpha, and IL-10.

[0296] In some embodiments, one or more of the factors is one or a combination of soluble factors, optionally one or one or a combination of cytokines, chemokines, or soluble receptors, optionally soluble costimulatory receptors. In some embodiments, the parameter is the arithmetic mean or geometric mean of the scores for two or more factors. In some embodiments, the parameter is an arithmetic or geometric mean, optionally, of an amount or concentration, for at least two of TNF-alpha, IFN-gamma and IL-2 or TNF-alpha, IFN-gamma and IL-2 . In some embodiments, the parameter is a normalized score value, where the normalization is compared to a reference factor score.

[0297] In some embodiments, A is the number of cells, or a multiple thereof, or proportion, or transformation, of a given phenotype present in a given composition. In some aspects, A is the average or weighted average of the number of cells, or a multiple, proportion or conversion thereof, of two or more phenotypes in a given composition, where optionally two or more phenotypes include a first CD8+ phenotype and a second CD4+ phenotype.

[0298] In some embodiments, the target number of reference RUs is equal to or less than a threshold number of RUs, where the adjusted unit dose does not contain more than a threshold RU. In some aspects, the target number of units is less than a threshold number of units, optionally a safe number of reference units, where the safe number of reference units is, in relation to a group of individuals analyzed after treatment with a therapeutic composition of T-cells containing T- cells expressing the recombinant receptor, optionally CAR, the lowest number of drug reference units administered to an individual among those individuals in the group who continued to develop an adverse event. In some aspects, the adverse event is a serious adverse event, optionally severe neurotoxicity, optionally a grade equal to or greater than grade 4 or grade 5, or at least long-term grade 3 neurotoxicity. In some aspects, the target number of reference units is less than, than a safe number of reference units, by an amount corresponding to a safety factor and/or by an amount in the range from 1.5- to 3-fold, optionally approximately 2-fold, or by an amount that is a multiple of the standard deviation in a group of individuals, which no adverse event occurred, optionally, neurotoxicity grade 0-2, optionally, where the multiple is in the range from 1.5- to 3-fold. In some embodiments, the target number of reference units is less than the safe number of reference units by an amount corresponding to a safety factor. In specific embodiments, the safety factor is in the range of 0.5-fold to 10-fold, 1.0-5.0-fold, or 1.5- to 3-fold, and/or is approximately or at least 1x, 1.5x, 2x, 2.5x, or 3x the safe amount. In particular embodiments, the safety factor is less than the safety amount. In some embodiments, the margin of safety is, in relation to a group of individuals analyzed after treatment using a T cell therapeutic composition containing T cells expressing a recombinant receptor, such as CAR, a multiple of the standard deviation of the mean, for example, the mean dose of the reference units administered to individuals in a group in which no adverse event occurred, eg, individuals in a group experiencing neurotoxicity grade 2 or less. In some embodiments, the safety factor is in the range of 0.5-fold to 10-fold, 1.0-5.0-fold, or 1.5- to 3-fold, and/or is approximately, or at least 1x, 1.5x, 2x, 2.5x, or 3x the standard deviation. In some embodiments, the number or dose of reference units that are below a safe amount by an amount corresponding to a safety factor includes amounts or doses of reference units that are associated with a response, such as CR, for example, in relation to a group of individuals who continued to develop response, for example, a CR.

[0299] In some aspects, the target number of reference units is equal to or greater than the reference effective number of reference units, where the reference effective amount is, in relation to a group of individuals analyzed after treatment using a therapeutic composition of T cells containing a recombinant receptor, optionally, CAR, the number of drug units administered to one or more individuals in the group for which the desired therapeutic outcome is indicated, optionally a partial response or complete response (CR).

[0300] In some aspects, the adjusted unit dose is less than, optionally, less than 1.5-fold, less than 2-fold, less than 3-fold, less than 4-fold, the average unit dose for a group of individuals subjected to treatment with a T cell therapeutic composition comprising T cells expressing a recombinant receptor, optionally a CAR.

[0301] In some embodiments, a sample from the T cell composition, optionally a cryopreserved sample, is evaluated after administration of the T cell composition to an individual. In some embodiments, if B is greater than USL, the individual is determined to be at risk of toxicity. In some embodiments, if B is greater than USL, the individual administered the composition is monitored and/or treated with an agent to alleviate or reduce the likelihood of a toxicity outcome or cytokine release syndrome following administration of the cell composition, and optionally, prior to the onset of a sign or symptom of a toxicity outcome. .

[0302] The present invention also relates to a method for assessing the risk of toxicity of a T cell therapeutic composition, comprising: (a) evaluating a sample from a T cell composition administered to an individual by the number of reference units (RU) within a given range, where the composition T cells contains T-cells containing a recombinant receptor that specifically binds to an antigen associated with a disease or condition, where RU in this composition is determined by the formula: RU=A x B, where: A is the number of cells, or a multiple of it, the proportion or transformation, cells of a certain phenotype present in the composition, or is the average or weighted average of the number of cells, or a multiple, proportion or transformation, of two or more phenotypes in the composition; and B is a parameter value, or a fraction thereof, or a multiple, or a transformation that indicates the degree of or correlates with the degree of recombinant receptor dependent, optionally CAR dependent, activity in the composition; and (b) comparing the amounts of RU to reference safe amounts of RU, where the comparison indicates whether or not the individual is at risk of experiencing an adverse event, optionally a serious adverse event, optionally, severe neurotoxicity of grade equal to or greater than grade 4 or grade 5, or at least a measure of grade 3 long-term neurotoxicity. In some aspects, A is the number of cells, or a multiple of it, or the fraction or transformation of a given phenotype present in a given composition. In some aspects, A is the average or weighted average of the number of cells, or a multiple, proportion or conversion thereof, of two or more phenotypes in a given composition, where optionally, two or more phenotypes include a first phenotype, including CD8+, and a second phenotype, including CD4+. In some aspects, the safe reference amount of RU is, in relation to a group of individuals analyzed after treatment with a T cell therapeutic composition containing T cells expressing a recombinant receptor, optionally CAR, the smallest number of reference drug units administered to an individual, among those individuals in the group who continued to develop an adverse event. In some aspects, if the comparison indicates an amount of RU above a reference safe amount of RU, the individual administered the composition is monitored and/or treated with an agent to alleviate or reduce the likelihood of a toxicity or cytokine release syndrome outcome following administration of the cell composition, and optionally, up to the occurrence of a sign or symptom of the outcome of toxicity.

[0303] The present invention also relates to a method for preparing a therapeutic composition containing a single dose of a T cell composition, comprising: (a) analyzing a T cell composition containing T cells derived from an individual having a disease or condition and transduced with a nucleic acid , encoding a recombinant receptor, optionally a chimeric antigen receptor (CAR), that specifically binds to an antigen associated with a disease or condition, where the assay determines B for a cell composition, where B is a parameter value, or a multiple thereof, or a transformation that indicates the degree or correlates with the degree of recombinant receptor-dependent, optionally CAR-dependent, activity in the composition; and (b) filling a container, e.g., an ampoule, with all or part of the composition and optionally another solution to obtain a unit dose of the T cell composition, where the unit dose contains the target number of reference units (RU) from the T cell composition, where RU in a given compositions are determined by the formula: RU=A x B, where A is the number of cells, or a multiple, proportion or transformation, of cells of a certain phenotype present in a given composition, or represents the average or weighted average of the number of cells, or its multiple, proportion or transformation of two or more phenotypes in a given composition;

[0304] The present invention also relates to a method for preparing a therapeutic composition containing a single dose of a T cell composition, comprising filling a container, for example, an ampoule, with all or part of the T cell composition containing T cells containing a recombinant receptor that specifically binds to an antigen associated with the disease or condition, and optionally another solution, to produce a unit dose of a T cell composition, where the unit dose contains the target number of reference units (RU) from the T cell composition, where the RU in the composition is determined by the formula: RU =A x B, where A is the number of cells, or a multiple, proportion or transformation, of some phenotype present in a given composition, or is the average or weighted average of the number of cells, or its multiple, proportion or transformation, of two or more phenotypes in this composition; and B is a parameter value, or a multiple thereof, or a transformation that indicates the degree of or correlates with the degree of recombinant receptor dependent, optionally CAR dependent, activity in the composition. In some aspects, A is the number of cells, or a multiple thereof, or proportion, or transformation, of a given phenotype present in a given composition. In some aspects, A is the average or weighted average of the number of cells, or a multiple, proportion or conversion thereof, of two or more phenotypes in a given composition, where optionally, two or more phenotypes include a first phenotype, including CD8+, and a second phenotype, including CD4+.

[0305] In some embodiments, A is the number of cells of a certain phenotype present in a given composition, and B is a parameter value that indicates the degree of or correlates with the degree of recombinant receptor dependent activity in the composition. In some embodiments, A and/or B is a quantity or value transform, respectively, where the transform includes a log transform, a power transform, or a logit transform. In some embodiments, A is the number of cells of some phenotype present in a given composition, and B is a multiple or transformation of a parameter value indicative of the degree of or correlated with the degree of CAR-dependent activity in a given T cell composition, optionally, where B represents the logarithmic transformation of the value. In some embodiments, the logarithmic transformation is decimal log (log10(x)), natural log (ln(x)) or binary log (log2(x)).

[0306] In some embodiments, A is the number of viable cells in the composition and/or is the number of cells that are not apoptotic, do not have a factor indicative of early apoptosis or apoptosis, are not in the early stages of apoptosis, or are not in the late stages of apoptosis, and/or is the number of cells in a certain state of differentiation, and/or is the number of cells with memory cells/stem cells, or is its multiple or transformation. In some embodiments, the phenotype includes the absence of a factor indicative of apoptosis or one or more stages of the apoptotic cascade or pathway, optionally, expression of an apoptosis marker. In some embodiments, the phenotype includes negative expression of an apoptosis marker, optionally an early apoptosis or late apoptosis marker. In some embodiments, the apoptosis marker is a surface phosphatidylserine and/or detected using annexin V, or is an active caspase or a caspase proform, optionally an active caspase 3 or a caspase 3 proform. In some embodiments, the phenotype includes annexin-. In some embodiments, A is total T cells, total CD3+ cells, total CD4+ or CD8+ cells, total CD3+CAR+ cells, total CD8+CAR+ cells, total CD4+ CAR+, or total live or viable cells from any of the above, or its multiple or converted value. In some embodiments, A is total CD3+ cells, total CD8+, total CD3+CAR+ cells, total CD8+CAR+ cells, or total live or viable cells from any of the above, or a multiple or converted value thereof. In some embodiments, A is the total number of apoptosis marker negative (–) cells that are CD3+ CAR+ cells, the total number of apoptosis marker negative (–) cells that are CD4+ CAR+, the total number of apoptosis marker negative (–) cells representing CD8+ CAR+ cells, or its multiple or converted value, where the apoptosis marker is annexin V or caspase. A is the total number of annexin-CD3+ CAR+ cells, the total number of annexin-CD4+ CAR+ cells, the total number of annexin-CD8+ CAR+ cells, or its multiple or converted value. In some embodiments, A is the total number of annexin-CD3+ CAR+ cells or the total number of annexin-CD8+ CAR+ cells.

[0307] In some embodiments, the phenotype includes positive expression of a surface marker that is one or more of CD3, CD4, or CD8, and/or includes positive expression of a recombinant receptor, optionally CAR, or a surrogate recombinant receptor expression marker. In some aspects, the phenotype is CD3+ CAR, CD4+/CAR+, CD8+/CAR+. In some embodiments, the phenotype includes a measure of the production of one or combination of cytokines, optionally non-antigen or recombinant receptor specific and/or polyclonal production, where one or more cytokines is IL-2, IL-13, IL-17, IFN- gamma or TNF-alpha. In some embodiments, the production index is measured in an assay, optionally an intracellular cytokine staining assay, comprising incubating a sample from a T cell composition with a polyclonal agent, an antigen specific agent, or a recombinant receptor binding agent, optionally CAR. In some embodiments, the agent is or contains PMA and ionomycin, or is or contains a T cell receptor or a T cell receptor complex agonist. In some embodiments, the phenotype includes negative expression of an activation marker, where the activation marker is selected from CD25, CD127, LAG3, Ki67, and combinations thereof. In some embodiments, the phenotype includes negative expression of a depletion marker, where the depletion marker is a PD1 or FOXP3 gene product, or a combination thereof. In some embodiments, the phenotype includes a naïve or memory cell phenotype, optionally wherein the memory cell phenotype includes an effector memory T cell phenotype, a central memory T cell phenotype, or a CD45RA-expressing effector memory T cell phenotype (Temra).

[0308] In some embodiments, the recombinant receptor-dependent activity is indicative of the production or accumulation of a pro-inflammatory cytokine, optionally one or a combination of TNF-alpha, IFN-gamma, IL-2, and IL-10. In some embodiments, the reference score is the average of scores among a plurality, optionally at least 10, at least 15, at least 20, of reference chimeric antigen receptor (CAR) T cell therapeutic compositions in which : (i) for each of the reference T cell therapeutic compositions, an acceptable safety profile was observed or determined as a result after administration to an individual, optionally where the individual has a disease or condition with antigen expression or association with an antigen; and/or (ii) for each of the reference T cell therapeutic compositions, the desired efficacy is observed or determined as a result after administration to an individual, optionally where the individual has a disease or condition with antigen expression or association with an antigen.

[0309] In some specific embodiments, the implementation, the parameter is a normalized value of the indicator, where the normalization is carried out in comparison with the reference indicator of the factor. In some embodiments, the reference score is the average of scores among a plurality, optionally at least 10, at least 15, at least 20, of reference chimeric antigen receptor (CAR) T cell therapeutic compositions in which: (i) for each of the reference T cell therapeutic compositions, an acceptable safety profile was observed or determined as a result after administration to an individual, optionally where the individual has a disease or condition with antigen expression or association with an antigen; and/or (ii) for each of the reference T cell therapeutic compositions, the desired efficacy is observed or determined as a result after administration to an individual, optionally where the individual has a disease or condition with antigen expression or association with an antigen. In some embodiments, the acceptable safety profile is no observable grade 2 or higher neurotoxicity, or no grade 3 or higher neurotoxicity.

[0310] In some embodiments, the acceptable safety profile is no observable grade 3 or higher neurotoxicity. In some embodiments, the effectiveness is a partial response or is a complete response (CR). In some embodiments, the benchmark is the score, in the same assay, of a factor in a reference T cell composition that is prepared in the same manner as the T cell therapeutic composition but does not express a recombinant receptor, optionally a CAR, does not specifically recognize antigen and/or does not express any recombinant receptor, optionally any CAR. In some embodiments, this parameter is normalized to control for patient-specific variability in a score of one or more factors. In some embodiments, the parameter is a normalized value of a factor score, compared to the same score in the same assay, for a control factor, where the control factor level in the T cell therapeutic composition is known to be absent or observed to be absent of a feature or correlation, or significant correlation with the outcome of an adverse event or toxicity, or their likelihood or risk, where the outcome of an adverse event or toxicity is not necessarily severe neurotoxicity. In some embodiments, the control factor is or contains a factor that is not statistically correlated and/or correlated with the occurrence of an adverse event among a plurality of individuals who continued to develop an adverse event after administration of the T cell composition, optionally, the control factor is one or a combination of IL-5, IL-13, GM-CSF and IL-6, optionally wherein the control factor score is the arithmetic or geometric mean of two or more of the above.

[0311] In some embodiments, the implementation, the parameter does not include cytolytic activity or its indicator. In some embodiments, the parameter does not include a recombinant receptor dependent or antigen specific cytolytic activity or a measure thereof.

[0312] In some specific embodiments, the phenotype is CD8+ CAR+ cells or annexin- CD8+ CAR+ cells; and the parameter is a score for a pro-inflammatory cytokine, which is optionally one or a combination of TNF-alpha, IL-2, and IFN-gamma, or a normalized value thereof.

[0313] In some embodiments, the adverse event is a grade 4 or 5 neurotoxicity, and the threshold: is or is about 1.75×10 ⁷ if A is negative (-) for an apoptosis marker and CD8+CAR+, and B is TNF-alpha or its normalized value; is or is approximately 2.19×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha or a normalized value thereof; is or is approximately 1.25×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma or a normalized value thereof; is or is approximately 1.56×10 ⁷ if A is CD8+CAR+ and B is IFN-gamma or its normalized value; is or is approximately 1.5×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IL-2 or a normalized value thereof; is or is approximately 1.88×10 ⁷ if A is CD8+CAR+ and B is IL-2 or its normalized value; is or is approximately 1.5×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IFN-gamma or a normalized value thereof; is or is about 1.88×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof; is or is approximately 2.0×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IL-2 or a normalized value thereof; is or is about 2.5×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha and IL-2 or its normalized value; is or is approximately 1.25×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma and IL-2 or a normalized value thereof; is or is about 1.56×10 ⁷ if A is CD8+CAR+ and B is IFN-gamma and IL-2 or its normalized value; is or is approximately 1.75×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha, IFN-gamma and IL-2 or a normalized value thereof; is or is approximately 2.19×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma and IL-2 or its normalized value, optionally, where the apoptosis marker is annexin V or active caspase 3.

[0314] In some embodiments, the adverse event is a grade 4 or 5 neurotoxicity and the target reference unit range: is between or about 2.0×10 ⁵ and 1.75×10 ⁷ inclusive, if A is negative (–) for apoptosis marker and CD8+CAR+, and B is TNF-alpha or its normalized value; is between or approximately between 2.5×10 ⁵ and 2.19×10 ⁷ inclusive if A is CD8+CAR+ and B is TNF-alpha or a normalized value thereof; is between or approximately between 4×10 ⁵ and 1.25×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma or a normalized value thereof; is between or approximately between 5x10 ⁶ and 1.56x10 ⁷ inclusive if A is CD8+CAR+ and B is IFN-gamma or a normalized value thereof; is between or approximately between 2.0×10 ⁵ and 1.5×10 ⁷ inclusive if A is negative (–) for the apoptosis marker CD8+CAR+ and if B is IL-2 or a normalized value thereof; is between or approximately between 2.5x10 ⁵ and 1.88x10 ⁷ inclusive if A is CD8+CAR+ and B is IL-2 or a normalized value thereof; is between or approximately between 3.0×10 ⁵ and 1.5×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IFN-gamma or its normalized value; is between or approximately between 3.75×10 ⁵ and 1.88×10 ⁷ inclusive if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof; is between or approximately between 3.0×10 ⁵ and 2.0×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IL-2 or its normalized value; is between or approximately between 3.75×10 ⁵ and 2.5×10 ⁷ inclusive if A is CD8+CAR+ and B is TNF-alpha and IL-2 or a normalized value thereof; is between or approximately between 4.0×10 ⁵ and 1.25×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma and IL-2 or its normalized value; is between or approximately between 5.0×10 ⁵ and 1.56×10 ⁷ inclusive if A is CD8+CAR+ and if B is IFN-gamma and IL-2 or its normalized value; is between or approximately between 4.0×10 ⁵ and 1.75×10 ⁷ inclusive if A is negative (–) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha, IFN-gamma and IL –2 or its normalized value; is between or approximately between 5.0×10 ⁵ and 2.19×10 ⁷ inclusive if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma and IL-2 or its normalized value, optionally, where the apoptosis marker is annexin V or active caspase 3.

[0315] In some embodiments, the adverse event is at least a long-term grade 3 neurotoxicity, and the threshold: is or is about 1.0×10 ⁶ if A is negative (-) for apoptosis marker and CD8+CAR+ , and B is TNF-alpha or its normalized value; is or is approximately 1.25×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha or a normalized value thereof; is or is approximately 2.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma or a normalized value thereof; is or is about 2.5×10 ⁶ if A is CD8+CAR+ and B is IFN-gamma or its normalized value; is or is approximately 3.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IL-2 or a normalized value thereof; is or is approximately 3.75×10 ⁶ if A is CD8+CAR+ and B is IL-2 or its normalized value; is or is approximately 1.5×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IFN-gamma or a normalized value thereof; is or is approximately 1.88×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof; is or is approximately 2.5×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IL-2 or a normalized value thereof; is or is approximately 3.12×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha and IL-2; or its normalized value is or is approximately 3.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma and IL-2 or its normalized value; is or is approximately 3.75×10 ⁶ if A is CD8+CAR+ and B is IFN-gamma and IL-2 or its normalized value; is or is approximately 2.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha, IFN-gamma and IL-2 or a normalized value thereof; is or is approximately 2.5×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma and IL-2 or its normalized value, optionally, where the apoptosis marker is annexin V or active caspase 3.

[0316] In some embodiments, the adverse event is at least Grade 3 long-term neurotoxicity, and the range of target reference units: is between or about between 3.0×10 ⁵ and 1.0×10 ⁶ , inclusive, if A is negative (-) for apoptosis marker and CD8+CAR+, and B is TNF-alpha or its normalized value; is between or approximately between 3.75×10 ⁵ and 1.25×10 ⁶ inclusive if A is CD8+CAR+ and B is TNF-alpha or a normalized value thereof; is between or approximately between 4×10 ⁵ and 2.0×10 ⁶ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma or a normalized value thereof; is between or approximately between 5×10 ⁶ and 2.5×10 ⁶ inclusive if A is CD8+CAR+ and B is IFN-gamma or a normalized value thereof; is between or approximately between 2.0×10 ⁵ and 3.0×10 ⁶ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IL-2 or a normalized value thereof; is between or approximately between 2.5×10 ⁵ and 3.75×10 ⁶ inclusive if A is CD8+CAR+ and B is IL-2 or a normalized value thereof; is between or approximately between 3.0×10 ⁵ and 1.5×10 ⁶ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IFN-gamma or its normalized value; is between or approximately between 3.75×10 ⁵ and 1.88×10 ⁶ inclusive if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof; is between or approximately between 3.0×10 ⁵ and 2.5×10 ⁶ inclusive if A is negative (–) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IL-2 or its normalized value; is between or approximately between 3.75×10 ⁵ and 3.12×10 ⁶ inclusive if A is CD8+CAR+ and B is TNF-alpha and IL-2 or a normalized value thereof; is between or approximately between 4.0×10 ⁵ and 3.0×10 ⁶ inclusive if A is negative (–) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma and IL-2 or its normalized value; is between or approximately between 5.0×10 ⁵ and 3.75×10 ⁶ inclusive if A is CD8+CAR+ and B is IFN-gamma and IL-2 or its normalized value; is between or approximately between 4.0×10 ⁵ and 2.0×10 ⁶ inclusive if A is negative (–) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha, IFN-gamma and IL –2 or its normalized value; is between or approximately between 5.0×10 ⁵ and 2.5×10 ⁶ inclusive if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma and IL-2 or a normalized value thereof, optional where the apoptosis marker is annexin V or active caspase 3.

[0317] In some embodiments, which can be combined with any other embodiment described herein, the recombinant receptor is a CAR. In some embodiments, the CAR comprises an extracellular antigen recognition domain that specifically binds to an antigen and an intracellular signaling domain containing ITAM, where optionally, the intracellular signaling domain comprises an intracellular domain from the CD3-zeta (CD3ζ) chain; and/or wherein the CAR further comprises a co-stimulatory signaling region, which optionally comprises a signaling domain from CD28 or 4-1BB. In some embodiments, which can be combined with any other embodiment, the T cells are CD4+ or CD8+. In some embodiments, the T cells are primary T cells, optionally autologous or allogeneic for the individual.

IV. TREATMENT METHODS USING T-CELL THERAPEUTIC COMPOSITION

[0318] The present invention provides methods comprising administering to an individual suffering from a disease or condition one or more unit doses of a T cell therapeutic composition. In some embodiments, one or more unit doses contains a target reference number of cell composition units based on A (number of cells of a certain phenotype) and/or B (a parameter value associated with the degree of recombinant receptor dependent activity, such as CAR dependent activity e.g. antigen-specific activity).

A. Diseases and conditions and routes of administration

[0319] In some embodiments, a dose of recombinant receptor-expressing cells is administered to an individual to treat or prevent diseases, conditions, and disorders, including cancer. In some embodiments, cells, populations, and compositions are administered to an individual or patient having a particular disease or condition being treated, for example, through adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, cells and compositions, such as modified compositions and compositions at the end of the production cycle after incubation and/or other processing steps, are administered to an individual, such as an individual having a disease or condition, or at risk of a disease or condition. In some aspects, the methods thus provide treatment, for example, alleviation of one or more symptoms of a disease or condition, for example, by reducing the tumor burden in a cancer expressing an antigen recognized by a modified T cell.

[0320] Methods for introducing cells for adoptive cell therapy are known and can be used in combination with the presented methods and compositions. For example, adoptive T cell therapy methods are described, for example, in US Patent Application Publication No. 2003/0170238 from Gruenberg et al; US Patent No. 4690915 from 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.

[0321] The disease or condition being treated can be any disease or condition in which antigen expression is associated with the etiology and/or is involved in the etiology of the disease, condition or disorder, e.g. causes, aggravates or is otherwise involved in such disease , condition or violation. Exemplary diseases and conditions may include diseases or conditions associated with malignancy or cell transformation (eg, cancer), an autoimmune or inflammatory disease, or an infectious disease, eg, caused by a bacterial, viral, or other pathogen. Exemplary antigens, including antigens associated with various diseases and conditions that can be treated, are described above. In specific embodiments, the chimeric antigen receptor or transgenic TCR specifically binds to an antigen associated with a disease or condition.

[0322] Among the diseases, conditions and disorders are tumors, including solid tumors, hematological malignancies and melanomas, and including localized and metastatic tumors, infectious diseases such as infection with a virus or other pathogen, for example, HIV, HCV, HBV, CMV, and parasitic disease, and autoimmune and inflammatory diseases. In some embodiments, the disease or condition is a tumour, cancer, cancer, neoplasia, or other proliferative disease or disorder. Such diseases include, but are not limited to, leukemia, lymphoma, e.g., chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, indolent B -cell lymphoma, B-cell malignancies, colon, lung, liver, breast, prostate, ovary, skin, melanoma, bone and brain malignancies, ovarian cancer, epithelial malignancies, renal cell carcinoma, adenocarcinoma pancreas, Hodgkin's lymphoma, cervical carcinoma, colorectal cancer, glioblastoma, neuroblastoma, Ewing's sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma and/or mesothelioma. In some embodiments, the individual has acute lymphoblastic leukemia (ALL). In some embodiments, the individual has non-Hodgkin's lymphoma.

[0323] 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 or inflammatory disease or condition, such as arthritis, e.g., rheumatoid arthritis (RA), type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroiditis , Grave's disease, Crohn's disease, multiple sclerosis, asthma, and/or a disease or condition associated with transplantation.

[0324] In some embodiments, the antigen associated with the disease or disorder is selected from the group consisting of orphan tyrosine kinase receptor RORl, tEGFR, Her2, Ll-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen , antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, 0EPHa2, ErbB2, 3 or 4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL –22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, cell adhesion molecules L1, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligands, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate-specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, aphrin B2, CD123, CS-1, c-Met, GD-2 , and MAGE A3, CE7, Wilms tumor antigen 1 (WT-1), cyclin such as cyclin A1 (CCNA1), and/or biotinylated molecules and/or molecules expressed by HIV, HCV, HBV and or other pathogens.

[0325] In some embodiments, the antigen is or includes an αvβ6 integrin (avb6 integrin), B-cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), cancer-testicular antigen, cancer-testicular antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, chemokine ligand 1 with C-C motif (CCL-1 ), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD138, CD171, epidermal growth factor protein (EGFR), epidermal growth factor truncated protein (tEGFR), receptor epidermal growth factor mutated type III (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin A2 receptor (EPHa2), estrogen receptor, Fc receptor-like protein 5 (FCRL5 ; also known as Fc receptor homologue 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), folate binding protein (FBP), p alpha folate receptor, GD2 ganglioside, O-acetylated GD2 (OGD2), GD3 ganglioside, glycoprotein 100 (gp100), G protein-coupled receptor 5D (GPCR5D), Her2/neu (erb-B2 receptor tyrosine kinase), Her3 (erb-B3) ), Her4 (erb–B4), erbB dimers, melanoma-associated human high molecular weight antigen (HMW–MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA–A1), human leukocyte antigen A2 (HLA–A2), IL-22 alpha receptor (IL-22Ra), IL-13 alpha 2 receptor (IL-13Ra2), kinase insert domain-containing receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope from L1-CAM, member A of family 8 containing leucine-rich repeats (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, mesothelin, c-Met, mouse cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 D member ligands (NKG2D), melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, preferably expressed melanoma antigen (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblastic cell glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor antigen 1 (WT-1), pathogen-specific antigen, or antigen associated with universal labeled and/or biotinylated molecules and/or molecules expressed by HIV, HCV, HBV or other pathogens. The antigens targeted by the receptors, in some embodiments, include antigens associated with B cell cancer, such as any of a number of known B cell markers. In some embodiments, the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig-kappa, Ig-lambda, CD79a, CD79b, or CD30.

[0326] 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 the individual to be treated with cell therapy, or from a sample obtained from such an individual. . Thus, in some aspects, cells are obtained from an individual, such as a patient in need of treatment, and the cells, after isolation and processing, are administered to the same individual.

[0327] In some embodiments, cell therapy, e.g., adoptive T-cell therapy, is performed via allogeneic transfer, in which cells are isolated and/or otherwise obtained from an individual other than the individual to be treated with cell therapy or ultimately to be conducting cell therapy, for example, the first individual. In such embodiments, the cells are then administered to another individual, such as a second individual, of the same species. In some embodiments, the first and second individuals are genetically identical. In some embodiments, the first and second individuals are genetically similar. In some embodiments, the second individual expresses an HLA of the same class or supertype as the first individual.

[0328] Cells can be administered by any suitable means, e.g., by bolus infusion, by injection, e.g., intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, vitreous injection, septal puncture injection, subscleral injection, intrachoroidal injection , intracameral injection, subconjunctival injection, subtenon injection, retrobulbar injection, peribulbar injection, or posterior parascleral delivery. In some embodiments, they are administered via 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, a given dose is administered via a single bolus of cells. In some embodiments, it is administered via multiple bolus injections of cells, eg over a period of no more than 3 days, or via continuous infusion of cells.

[0329] For the prevention or treatment of diseases, the appropriate dose may depend on the type of diseases being treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are administered for prophylactic or therapeutic purposes, prior therapy, the individual's history and response to cells, and the decisions of the treating therapist. The compositions and cells, in some embodiments, are suitably administered to an individual at one time or over a series of treatments.

[0330] In some embodiments, the cells are administered as part of a combination treatment, e.g., simultaneously or sequentially, in any order, with another therapeutic intervention, e.g., an antibody or modified cell or receptor, or an agent such as a cytotoxic or drug . Cells, in some embodiments, are administered in conjunction with one or more additional drugs or in combination with another therapeutic intervention, either simultaneously or sequentially, in any order. In some contexts, cells are co-administered with another therapy close enough in time such that cell populations enhance the effect of one or more additional drugs, or vice versa. In some embodiments, the cells are administered prior to one or more additional drugs. In some embodiments, the cells are administered after one or more additional drugs. In some embodiments, 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.

[0331] In some embodiments, the methods include administering a chemotherapeutic agent, eg, a preparatory chemotherapeutic agent, eg, to reduce tumor burden prior to administration.

[0332] Pretreatment of individuals with immunodepleting (eg, antilymphocyte) therapies may improve the effects of adoptive cell therapy (ACT) in some aspects.

[0333] Thus, in some embodiments, the methods include administering a pretreatment agent, such as an anti-lymphocyte or chemotherapeutic agent, eg, cyclophosphamide, fludarabine, or combinations thereof, to an individual prior to initiation of cell therapy. For example, the pretreatment agent may be administered to the individual at least 2 days before, for example, at least 3, 4, 5, 6, or 7 days before the start of cell therapy. In some embodiments, the individual is administered the pretreatment agent no more than 7 days before, for example, no more than 6, 5, 4, 3, or 2 days before the start of cell therapy. Exemplary dosing regimens for pretreatment are described in International Patent Application No. WO2016/191756.

[0334] In some embodiments, the individual is pretreated with cyclophosphamide at a dose between or between about 20 mg/kg and 120 mg/kg, such as between or between about 40 mg/kg and 80 mg/kg, each including. In some aspects, the individual is pretreated with or with about 60 mg/kg of cyclophosphamide. In some embodiments, the individual is pretreated with cyclophosphamide at a dose of between or between about 200 mg/m2 and 4440 mg/m2, such as between or between about 200 mg/m2 and 500 mg/m2, each including. In some cases, the individual is pretreated with 300 mg/m2 cyclophosphamide, in some cases daily, for example 300 mg/m2/day. In some cases, the individual is pretreated with or using approximately 500 mg/m2 cyclophosphamide, in some cases daily, eg 500 mg/m2/day. In some cases, the individual is pretreated with or using approximately 2220-4440 mg/m2 of cyclophosphamide. In some aspects, the individual is pretreated with or using about 200 mg/m2/day, about 250 mg/m2/day, about 300 mg/m2/day, about 350 mg/m2/day, about 400 mg/m2/day 24 hours, approximately 450 mg/m2/day, approximately 500 mg/m2/day, approximately 550 mg/m2/day, approximately 600 mg/m2/day, approximately 650 mg/m2/day, approximately 700 mg/m2/day, about 800 mg/m2/day, about 900 mg/m2/day, or about 1000 mg/m2/day. In some embodiments, cyclophosphamide may be administered in a single dose or may be administered in multiple doses, such as daily, every second day, or every third day. In some embodiments, the implementation, cyclophosphamide is administered once a day for one or two, or three or more days. In some embodiments, the doses described herein are daily doses, eg, mg/m2/day administered daily for three consecutive days.

[0335] In some embodiments, where the antilymphocyte agent contains fludarabine, the individual is administered fludarabine at a dose between or between about 1 mg/m2 and 100 mg/m2, such as between or between about 10 mg/m2 and 75 mg/m2, 15 mg/m2 and 50 mg/m2, 20 mg/m2 and 30 mg/m2, or 24 mg/m2 and 26 mg/m2, each included. In some cases, the individual is preconditioned with or using about 10 mg/m2/day, about 15 mg/m2/day, about 20 mg/m2/day, about 25 mg/m2/day, about 30 mg/m2 /day, approximately 35 mg/m2/day, approximately 40 mg/m2/day, approximately 45 mg/m2/day, approximately 50 mg/m2/day, approximately 55 mg/m2/day, approximately 60 mg/m2/day , approximately 65 mg/m2/day, approximately 70 mg/m2/day, approximately 75 mg/m2/day, approximately 80 mg/m2/day, approximately 85 mg/m2/day, approximately 90 mg/m2/day, approximately 95 mg/m2/day, approximately 100 mg/m2/day, approximately 200 mg/m2/day, or approximately 300 mg/m2/day. In some cases, the individual is administered 25 mg/m2 of fludarabine. In some cases, the individual is administered 30 mg/m2 of fludarabine. In some embodiments, fludarabine may be administered in a single dose or may be administered in multiple doses, such as daily, every second day, or every third day. In some embodiments, fludarabine is administered daily, eg, for 1 to 5 days, eg, 3 to 5 days. In some embodiments, the doses described herein are daily doses, eg, mg/m2/day administered daily for three consecutive days.

[0336] In some embodiments, the antilymphocyte agent comprises 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, such as those described above, and fludarabine, at any dose or schedule, such as those described above. For example, in some aspects, the individual is administered 60 mg/kg (~2 g/m2) of cyclophosphamide and 3-5 doses of 25 mg/m2 of fludarabine prior to the first or subsequent dose. Dosing

[0337] The pharmaceutical composition, in some embodiments, contains cells in amounts effective to treat or prevent a disease or condition, such as a therapeutically effective or prophylactically effective amount. In some embodiments, the composition contains cells in an amount effective to reduce the burden of the disease or condition.

[0338] In some embodiments, the present invention relates to methods comprising administering to an individual one or more unit doses of a T cell therapeutic composition, wherein each unit dose contains either (i) a target amount of total recombinant receptor-expressing (e.g., CAR-expressing) cells, or a target number or total number of recombinant receptor-expressing (eg, CAR-expressing) cells of a particular phenotype; or (ii) a target number of reference units (RU) from the T cell composition that is within the given or target RU range. In some embodiments, the unit dose does not contain more than a threshold RU, eg, more than a safe number of reference units.

[0339] In some embodiments, the present invention relates to methods of treatment comprising administering to an individual suffering from a disease or condition, a single dose of a T cell composition containing cells containing a recombinant receptor, such as a chimeric antigen receptor (CAR), that specifically binds with an antigen associated with a disease or condition, where the unit dose contains the target number of reference units (RU) within a given range. RU in a given composition is determined by the formula RU=A x B, where A is the number of cells, or a multiple thereof, the proportion or transformation, of some phenotype present in a given composition, or is the average or weighted average of the number of cells, or a multiple thereof, proportion, or conversion, of two or more phenotypes in a given composition; and B is a parameter value, or a multiple thereof, or a transformation that indicates the degree of or correlates with the degree of recombinant receptor dependent, optionally CAR dependent, activity in the composition. Illustrative features associated with A and B are described herein along with a reference for determining or providing a unit dose of cells for administration.

[0340] In some embodiments, A is the number of cells, or a multiple thereof, or proportion, or transformation, of a given phenotype present in a given composition, such as a phenotype that is or includes CD8+ cells, or another phenotype as described in the present description. In some embodiments, A is the average or weighted average of the number of cells, or a multiple thereof, proportion or conversion, of two or more phenotypes in a given composition. In one example, two or more phenotypes include a first CD8+ phenotype and a second CD4+ phenotype.

[0341] In some embodiments, a target number of units contained in a unit dose for administration that is less than a threshold number of reference units, e.g., a predetermined threshold, as identified or known to be indicative of the risk of an adverse event, such as severe toxicity , such as at least Grade 3 or greater long-term toxicity, or Grade 4 or Grade 5 toxicity. using a T cell therapeutic composition, and typically a T cell therapeutic composition prepared, for example, modified, maintained in culture, cultured, activated, cryopreserved, using the same or similar methods. In some embodiments, the plurality of individuals includes at least 10 individuals, e.g., at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60 , at least 70, at least 80, at least 90, at least 100 or more individuals.

[0342] In some embodiments, the safe number of reference units is, in relation to the group of individuals analyzed after treatment using a therapeutic T cell composition containing T cells expressing a recombinant receptor, such as CAR, the smallest number of reference units of the drug, administered to an individual, among those individuals in the group who continued to develop an adverse event. In some embodiments, the adverse event is a serious adverse event, optionally severe neurotoxicity, optionally a grade equal to or greater than grade 4 or grade 5, or at least long-term grade 3 neurotoxicity.

[0343] In some embodiments, the target number of reference units is less than the safe number of reference units by an amount corresponding to a safety factor and/or by an amount in the range of 1.5- to 3-fold, optionally, approximately 2 - a multiple, or a multiple of the standard deviation in a group of individuals who did not experience an adverse event, optionally, neurotoxicity of degree 0-2, optionally, where the multiple is in the range from 1.5- to 3-fold.

[0344] In some embodiments, the target number of reference units is equal to or greater than the reference effective number of reference units that can be determined for a group or plurality of individuals who have similarly been treated with a T cell therapeutic composition, and typically a T cell therapeutic composition. cells obtained, for example, modified, maintained in culture, cultured, activated, cryopreserved, using the same or similar methods. In some embodiments, the plurality of individuals includes at least 10 individuals, e.g., at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60 , at least 70, at least 80, at least 90, at least 100 or more individuals. In some embodiments, the effective number of reference units is, in relation to a group of individuals analyzed after treatment with a therapeutic composition of T cells containing a recombinant receptor, optionally CAR, the number of drug units administered to one or more individuals among the group in which shows the desired therapeutic outcome, optionally partial response or complete response (CR).

[0345] In some embodiments, the present invention relates to methods comprising administering to an individual suffering from a disease or condition, a single dose of a T cell composition containing cells containing a recombinant receptor, such as a chimeric antigen receptor (CAR), that specifically binds to an antigen associated with a disease or condition, where either a target number of total CD8+ cells expressing the recombinant receptor is administered from a therapeutic composition, and/or a single dose of such cells is administered, in which a target number of reference units is administered within a given range, as described above. In some cases, the target number of reference units that is administered is equal to or less than a threshold amount of RU, such that a single dose does not contain more than the threshold RU. In some aspects, the target number of total recombinant receptor CD8+ cells that are administered are CD8+ that are negative (-) for an apoptosis marker and CD8+, optionally, where the apoptosis marker is annexin V or active caspase 3.

[0346] In some embodiments, the methods provided include administering a dose containing a number of cells, such as a dose that provides a target number of cells. In some embodiments, the dose, such as the target number of cells, is between or between about 5.0×10 ⁶ and 2.25×10 ⁷ , 5.0×10 ⁶ and 2.0×10 ⁷ , 5.0× 10 ⁶ and 1.5×10 ⁷ , 5.0×10 ⁶ and 1.0×10 ⁷ , 5.0×10 ⁶ and 7.5×10 ⁶ , 7.5×10 ⁶ and 2.25×10 ⁷ , 7.5×10 ⁶ and 2.0×10 ⁷ , 7.5×10 ⁶ and 1.5×10 ⁷ , 7.5×10 ⁶ and 1.0×10 ⁷ , 1.0×10 ⁷ and 2.25×10 ⁷ , 1.0×10 ⁷ and 2.0×10 ⁷ , 1.0×10 ⁷ and 1.5×10 ⁷ , 1.5×10 ⁷ and 2.25×10 ⁷ , 1.5×10 ⁷ and 2.0×10 ⁷ , 2.0×10 ⁷ and 2.25×10 ⁷ each included. In some embodiments, such a dose, such as such a target number of cells, refers to the total number of cells expressing the recombinant receptor in the administered composition. In some embodiments, such a dose, such as such a target number of cells, refers to the total number of cells expressing the recombinant receptor that are CD8+ or that are negative (-) for an apoptosis marker and CD8+. In some embodiments, the apoptosis marker is annexin V or active caspase 3.

[0347] In some embodiments, the unit dose of cells contains a cell number, such as a target cell number, between at least or at least about 5×10 ⁶ , 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9x10 ⁶ , 10x10 ⁶ , and approximately 15x10 ⁶ recombinant receptor-expressing cells, such as recombinant receptor-expressing cells that are CD8+, or that are negative (–) for apoptosis marker and CD8+, optionally where the marker is apoptosis is annexin V or active caspase 3.

[0348] In some embodiments, the unit dose of cells contains a cell number, such as a target cell number, between at least or at least about 5.55×10 ⁶ , 6.66×10 ⁶ , 7.77× 10 ⁶ , 8.99×10 ⁶ , 1.0×10 ⁷ , 1.1×10 ⁷ , and approximately 1.67×10 ⁷ recombinant receptor-expressing cells, optionally, recombinant receptor-expressing cells that are CD8+ or are negative (-) for an apoptosis marker and CD8+, for example, where the apoptosis marker is annexin V or active caspase 3.

[0349] In some embodiments, the unit dose of cells contains a cell number, such as a target cell number, between at least or at least about 6.25×10 ⁶ , 7.5×10 ⁶ , 8.75× 10 ⁶ , 1.0 x 10 ⁷ , 1.13 x 10 ⁷ , 1.25 x 10 ⁷ , and approximately 1.9 x 10 ⁷ recombinant receptor-expressing cells, optionally recombinant receptor-expressing cells that are CD8+ or are negative (-) for an apoptosis marker and CD8+, for example, where the apoptosis marker is annexin V or active caspase 3.

[0350] In some embodiments, the dose of cells from a single dose contains a number of cells, such as a target number of cells, between at least or at least about between at least or at least about 7.14×10 ⁶ , 8, 5×10 ⁶ , 1.0×10 ⁷ , 1.14×10 ⁷ , 1.29×10 ⁷ , 1.42×10 ⁷ , and approximately 2.14×10 ⁷ recombinant receptor-expressing cells, optionally recombinant receptor-expressing cells cells that are CD8+, or that are negative (-) for the apoptosis marker and CD8+, optionally, where the apoptosis marker is annexin V or active caspase 3.

[0351] In some embodiments, the unit dose of cells contains a cell number, such as a target cell number, between at least or at least about 5×10 ⁶ , 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9x10 ⁶ , 10x10 ⁶ and approximately 15x10 ⁶ recombinant receptor expressing cells that are negative (-) for the apoptosis marker and CD8+, optionally, where the apoptosis marker is annexin V or active caspase 3.

[0352] In some embodiments, the unit dose of cells contains a cell number, such as a target cell number, between at least or at least about 6.25×10 ⁶ , 7.5×10 ⁶ , 8.75× 10 ⁶ , 1.0×10 ⁷ , 1.13×10 ⁷ , 1.25×10 ⁷ , and approximately 1.9×10 ⁷ recombinant receptor-expressing CD8+ cells.

[0353] In some embodiments, the method includes administering to an individual suffering from a disease or condition, a single dose of a T cell composition containing cells containing a recombinant receptor, such as a chimeric antigen receptor (CAR), that specifically binds to an antigen associated with the disease or a state where the unit dose contains a target number of reference units (RU) within a given range. In some embodiments, the target number of reference units is less than a threshold, eg, less than a reference security level. In some aspects, the adverse event is a serious adverse event, such as severe neurotoxicity equal to or greater than grade 4 or grade 5, or at least long-term neurotoxicity of grade 3. In some embodiments, the threshold is less than the reference safety level. , by the amount corresponding to the safety factor, or at least 2 times.

[0354] In some embodiments, the adverse event is a grade 4 or 5 neurotoxicity and the threshold: is or is about 1.75×10 ⁷ if A is negative (-) for an apoptosis marker and CD8+CAR+, and B is TNF-alpha or its normalized value; is or is approximately 2.19×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha or a normalized value thereof; is or is approximately 1.25×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma or its normalized value; is or is approximately 1.56×10 ⁷ if A is CD8+CAR+ and B is IFN-gamma or its normalized value; is or is approximately 1.5×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IL-2 or a normalized value thereof; is or is approximately 1.88×10 ⁷ if A is CD8+CAR+ and B is IL-2 or its normalized value; is or is approximately 1.5×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IFN-gamma or a normalized value thereof; is or is about 1.88×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof; is or is approximately 2.0×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IL-2 or a normalized value thereof; is or is approximately 2.5×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha and IL-2 or its normalized value; is or is approximately 1.25×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma and IL-2 or a normalized value thereof; is or is about 1.56×10 ⁷ if A is CD8+CAR+ and B is IFN-gamma and IL-2 or its normalized value; is or is approximately 1.75×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha, IFN-gamma and IL-2 or a normalized value thereof; is or is approximately 2.19×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma, and IL-2 or a normalized value thereof. In some embodiments, the apoptosis marker is annexin V or active caspase 3.

[0355] In some embodiments, the adverse event is a grade 4 or 5 neurotoxicity and the target reference unit range: is between or about 2.0×10 ⁵ and 1.75×10 ⁷ , inclusive, if A is negative (–) for apoptosis marker and CD8+CAR+, and B is TNF-alpha or its normalized value; is between or approximately between 2.5×10 ⁵ and 2.19×10 ⁷ inclusive if A is CD8+CAR+ and B is TNF-alpha or a normalized value thereof; is between or approximately between 4×10 ⁵ and 1.25×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma or a normalized value thereof; is between or approximately between 5×10 ⁶ and 1.56×10 ⁷ inclusive if A is CD8+CAR+ and B is IFN-gamma or a normalized value thereof; is between or approximately between 2.0×10 ⁵ and 1.5×10 ⁷ inclusive if A is negative (–) for the apoptosis marker CD8+CAR+ and if B is IL-2 or a normalized value thereof; is between or approximately between 2.5x10 ⁵ and 1.88x10 ⁷ inclusive if A is CD8+CAR+ and B is IL-2 or a normalized value thereof; is between or approximately between 3.0×10 ⁵ and 1.5×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IFN-gamma or its normalized value; is between or approximately between 3.75×10 ⁵ and 1.88×10 ⁷ inclusive _if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof; is between or approximately between 3.0×10 ⁵ and 2.0×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IL-2 or its normalized value; is between or approximately between 3.75×10 ⁵ and 2.5×10 ⁷ inclusive if A is CD8+CAR+ and B is TNF-alpha and IL-2 or a normalized value thereof; is between or approximately between 4.0×10 ⁵ and 1.25×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma and IL-2 or its normalized value; is between or approximately between 5.0×10 ⁵ and 1.56×10 ⁷ inclusive if A is CD8+CAR+ and if B is IFN-gamma and IL-2 or its normalized value; is between or approximately between 4.0×10 ⁵ and 1.75×10 ⁷ inclusive if A is negative (–) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha, IFN-gamma and IL –2 or its normalized value; is between or approximately between 5.0×10 ⁵ and 2.19×10 ⁷ inclusive if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma and IL-2 or a normalized value thereof. In some embodiments, the apoptosis marker is annexin V or active caspase 3.

[0356] In some embodiments, the adverse event is at least a long-term grade 3 neurotoxicity, and the threshold: is or is about 1.0x10 ⁶ if A is negative (-) for apoptosis marker and CD8+CAR+ , and B is TNF-alpha or its normalized value; is or is approximately 1.25×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha or a normalized value thereof; is or is approximately 2.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma or its normalized value; is or is approximately 2.5×10 ⁶ if A is CD8+CAR+ and B is IFN-gamma or its normalized value; is or is approximately 3.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IL-2 or a normalized value thereof; is or is approximately 3.75×10 ⁶ if A is CD8+CAR+ and B is IL-2 or its normalized value; is or is approximately 1.5×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IFN-gamma or a normalized value thereof; is or is about 1.88×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof; is or is approximately 2.5×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IL-2 or a normalized value thereof; is or is approximately 3.12×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha and IL-2; or its normalized value; is or is approximately 3.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma and IL-2 or a normalized value thereof; is or is approximately 3.75×10 ⁶ if A is CD8+CAR+ and B is IFN-gamma and IL-2 or its normalized value; is or is approximately 2.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha, IFN-gamma and IL-2 or a normalized value thereof; is or is about 2.5×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma, and IL-2 or a normalized value thereof. In some embodiments, the apoptosis marker is annexin V or active caspase 3.

[0357] In some embodiments, the adverse event is at least Grade 3 long-term neurotoxicity, and the target reference unit range is between or about 3.0x10 ⁵ and 1.0x10 ⁶ , inclusive, if A is negative (-) for apoptosis marker and CD8+CAR+, and B is TNF-alpha or its normalized value; is between or approximately between 3.75×10 ⁵ and 1.25×10 ⁶ inclusive if A is CD8+CAR+ and B is TNF-alpha or a normalized value thereof; is between or approximately between 4×10 ⁵ and 2.0×10 ⁶ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma or a normalized value thereof; is between or approximately between 5×10 ⁶ and 2.5×10 ⁶ inclusive if A is CD8+CAR+ and B is IFN-gamma or a normalized value thereof; is between or approximately between 2.0×10 ⁵ and 3.0×10 ⁶ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IL-2 or a normalized value thereof; is between or approximately between 2.5×10 ⁵ and 3.75×10 ⁶ inclusive if A is CD8+CAR+ and B is IL-2 or its normalized value; is between or approximately between 3.0×10 ⁵ and 1.5×10 ⁶ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IFN-gamma or its normalized value; is between or approximately between 3.75×10 ⁵ and 1.88×10 ⁶ inclusive if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof; is between or approximately between 3.0×10 ⁵ and 2.5×10 ⁶ inclusive if A is negative (–) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IL-2 or its normalized value; is between or approximately between 3.75×10 ⁵ and 3.12×10 ⁶ inclusive if A is CD8+CAR+ and B is TNF-alpha and IL-2 or a normalized value thereof; is between or approximately between 4.0×10 ⁵ and 3.0×10 ⁶ inclusive if A is negative (–) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma and IL-2 or its normalized value; is between or approximately between 5.0×10 ⁵ and 3.75×10 ⁶ inclusive if A is CD8+CAR+ and B is IFN-gamma and IL-2 or its normalized value; is between or approximately between 4.0×10 ⁵ and 2.0×10 ⁶ inclusive if A is negative (–) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha, IFN-gamma and IL –2 or its normalized value; is between or approximately between 5.0×10 ⁵ and 2.5×10 ⁶ inclusive if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma and IL-2 or a normalized value thereof. In some embodiments, the apoptosis marker is annexin V or active caspase 3.

[0358] In specific embodiments, cells, or individual populations of cell subtypes, are administered to an individual in the range of from about one million to about 100 billion cells and/or in a number of cells per kilogram of body weight, such 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 in the range determined by any two of the preceding values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells current, approximately 90 million cells, approximately 10 billion cells, approximately 25 billion cells, approximately 50 billion cells, approximately 75 billion cells, approximately 90 billion cells, or in the range defined by any two of the preceding values), and, in some cases, approximately 100 million cells to approximately 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 cells, approximately 30 billion cells, approximately 45 billion cells) or any amount within these ranges and/or per kilogram of body weight. Doses may be varied depending on the indications specific to the disease or disorder and/or the patient and/or other treatment.

[0359] In some embodiments, for example, when the individual is a human, the dose comprises less than about 5 x 10 ⁸ total recombinant receptor (e.g., CAR) expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs), e.g. , ranging from approximately 1×10 ⁶ to 5×10 ⁸ such cells, for example, 2×10 ⁶ , 5×10 ⁶ , 1×10 ⁷ , 5×10 ⁷ , 1×10 ⁸ , or 5×10 ⁸ such total cells, or in the range between any two of the previous values.

[0360] In some embodiments, the dose of genetically modified cells contains from or about 1x10 ⁵ to 5x10 ⁸ total CAR expressing T cells, 1x10 ⁵ to 2.5x10 ⁸ total CAR expressing T cells , 1×10 ⁵ to 1×10 ⁸ total CAR expressing T cells, 1×10 ⁵ to 5×10 ⁷ total CAR expressing T cells, 1×10 ⁵ to 2.5×10 ⁷ total CAR expressing T cells , 1×10 ⁵ to 1×10 ⁷ total CAR expressing T cells, 1×10 ⁵ to 5×10 ⁶ total CAR expressing T cells, 1×10 ⁵ to 2.5×10 ⁶ total CAR expressing T cells , 1×10 ⁵ to 1×10 ⁶ total CAR expressing T cells, 1×10 ⁶ to 5×10 ⁸ total CAR expressing T cells, 1×10 ⁶ to 2.5×10 ⁸ total CAR expressing T cells , 1×10 ⁶ to 1×10 ⁸ total CAR expressing T cells, 1×10 ⁶ to 5×10 ⁷ total CAR expressing T cells, 1×10 ⁶ to 2.5×10 ⁷ total CAR expressing T cells , 1×10 ⁶ to 1×10 ⁷ total CAR expressing T cells, 1×10 ⁶ to 5×10 ⁶ total CAR expressing T cells, 1×10 ⁶ to 2.5×10 ⁶ total CAR expressing T cells, 2.5×10 ⁶ to 5×10 ⁸ total CAR expressing T cells, 2.5×10 ⁶ up to 2.5×10 ⁸ total CAR expressing T cells, 2.5×10 ⁶ to 1×10 ⁸ total CAR expressing T cells, 2.5×10 ⁶ to 5×10 ⁷ total CAR expressing T cells, 2 .5×10 ⁶ to 2.5×10 ⁷ total CAR expressing T cells, 2.5×10 ⁶ to 1×10 ⁷ total CAR expressing T cells, 2.5×10 ⁶ to 5×10 ⁶ total expressing CAR T cells, 5×10 ⁶ to 5×10 ⁸ total expressing CAR T cells, 5×10 ⁶ to 2.5×10 ⁸ total CAR expressing T cells, 5×10 ⁶ to 1×10 ⁸ total expressing CAR T cells, 5×10 ⁶ to 5×10 ⁷ total expressing CAR T cells, 5×10 ⁶ to 2.5×10 ⁷ total CAR expressing T cells, 5×10 ⁶ to 1×10 ⁷ total expressing CAR 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 tot total CAR expressing T cells, 1×10 ⁷ to 5×10 ⁷ total CAR expressing T cells, 1×10 ⁷ to 2.5×10 ⁷ total CAR expressing T cells, 2.5×10 ⁷ to 5× 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, 2.5 ×10 ⁷ to 5 × 10 ⁷ total CAR expressing T cells, 5 × 10 ⁷ to 5 × 10 ⁸ total CAR expressing T cells, 5 × 10 ⁷ to 2.5 × 10 ⁸ total CAR expressing T cells, 5 x10 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 2.5×10 ⁸ to 5×10 ⁸ total CAR expressing T cells.

[0361] In some embodiments, the dose of genetically modified cells contains at least or at least about 1x10 ⁵ CAR expressing cells, at least or at least about 2.5x10 ⁵ CAR expressing cells, at least or at least about 5×10 ⁵ CAR expressing cells, at least or at least about 1×10 ⁶ CAR expressing cells, at least or at least about 2.5×10 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 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 approx. 2.5 x 10 ⁸ expressing CAR cells, or at least or at least about 5 x 10 ⁸ expressing CAR cells.

[0362] In some embodiments, cell therapy comprises administering a dose containing a number of cells from or about 1×10 ⁵ to 5×10 ⁸ total recombinant receptor-expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMC) , from or about 5x10 ⁵ to 1x10 ⁷ total recombinant receptor-expressing cells, total T-cells, or total peripheral blood mononuclear cells (PBMC), or from or about 1x10 ⁶ to 1x10 ⁷ total recombinant receptor-expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMC), including each. In some embodiments, cell therapy comprises administering a dose of cells containing a cell count of at least or approximately at least 1×10 ⁵ total recombinant receptor-expressing cells, total T cells, or total peripheral blood mononuclear cells (PBMC), for example, at least or at least 1×10 ⁶ , at least or approximately at least 1×10 ⁷ , at least or approximately at least 1×10 ⁸ such cells. In some embodiments, the amount 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, cell therapy comprises administering a dose containing a cell count of 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+ cells expressing the recombinant receptor, or from or about 1x10 ⁶ to 1x10 ⁷ CD3+ or CD8+ total T cells or CD3+ or CD8+ expressing the recombinant receptor cells, including each. In some embodiments, the cell therapy comprises administering a dose containing a cell count 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 about 1×10 ⁶ to 1×10 ⁷ total CD3+/CAR+ or CD8+/CAR+ cells, each included.

[0363] In some embodiments, the T cells from the dose include CD4+ T cells, CD8+ T cells, or CD4+ and CD8+ T cells.

[0364] In some embodiments, for example, when the individual is a human, CD8+ T cells in a dose, including a dose including CD4+ and CD8+ T cells, comprises between about 1×10 ⁶ and 5×10 ⁸ , inclusive, total recombinant receptor (e.g., CAR) expressing CD8+ cells, e.g., in the range from about 5x10 ⁶ to 1x10 ⁸ such cells, 1x10 ⁷ , 2.5x10 ⁷ , 5x10 ⁷ , 7.5 ×10 ⁷ , 1×10 ⁸ or 5×10 ⁸ total such cells, or in the range between any two of the previous values. In some embodiments, a plurality of doses are administered to a patient, and each of the doses or the total dose may be between any of the preceding values. In some embodiments, the cell dose comprises administering from or about 1×10 ⁷ to 0.75×10 ⁸ total recombinant CD8+ receptor-expressing T cells, 1×10 ⁷ to 2.5×10 ⁷ total recombinant CD8+ T receptor-expressing -cells, from or about 1×10 ⁷ to 0.75×10 ⁸ total recombinant CD8+ receptor-expressing T cells, including each. In some embodiments, the dose of cells comprises administering or administering approximately 1×10 ⁷ , 2.5×10 ⁷ , 5×10 ⁷ 7.5×10 ⁷ , 1×10 ⁸ , or 5×10 ⁸ total recombinant CD8+ receptor-expressing T cells.

[0365] In some embodiments, a dose of cells, e.g., expressing a recombinant T cell receptor, is administered to an individual in the form of a single dose or is administered only once over a period of two weeks, one month, three months, six months, 1 year, or more .

[0366] In the context of adoptive cell therapy, administering a given "dose" includes administering a given number or amount 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 includes administering a given amount or amount cells in the form of a fractional dose, presented in a variety of individual compositions or infusions, over a specified period of time, which is not more than 3 days. Thus, in some contexts, a dose is a single or continuous administration of a specified number of cells given or initiated at a single time point. 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 through multiple infusions over a period of one day.

[0367] Thus, in some aspects, cells from a dose are administered in a single pharmaceutical composition. In some embodiments, cells from a dose are administered in a plurality of compositions containing cells from the first dose together.

[0368] The term "fractional dose" refers to a dose that is divided in such a way that it is administered over more than one day. This type of dosage is included in the present methods and is considered to be a single dose.

[0369] Thus, the dose, in some aspects, can be administered in the form of a fractional dose. For example, in some embodiments, the dose may be administered to an individual over 2 days or over 3 days. Illustrative split dosing 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 first 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 fractional dose is not spread over more than 3 days.

[0370] In some embodiments, the cells are administered at the desired dose, which, in some aspects, includes the desired dose or number of cells or cell type(s) and/or the desired ratio of cell types. Thus, cell dosing in some embodiments is based on the target total cell count or target reference number of units and the desired ratio of individual populations or subtypes, such as the ratio of CD4+ to CD8+. In some embodiments, cell dosing is based on the desired target number of total cells or target reference number of units in individual populations or for individual cell types. In some embodiments, dosing is based on a combination of such features, for example, the desired total number of cells, the target number of reference units, the desired ratio, and the desired total number of cells in individual populations.

[0371] In some embodiments, populations or subtypes of cells, such as CD8 ⁺ and CD4 ⁺ T cells, are administered at the desired dose or within tolerable differences in the desired dose of total cells, for example, 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 individual to whom the cells are administered, eg, cells/kg. In some aspects, the desired dose is or exceeds the minimum number of cells or the minimum number of cells per unit of body weight. In some aspects, among total cells administered at the desired dose, individual populations or subtypes are present in the desired ratio, or approximately in the desired output ratio (such as the ratio of CD4+ to CD8+), for example, within certain tolerable differences or errors of such ratio.

[0372] In some embodiments, the cells are administered at the desired dose or within tolerable differences in the desired dose of one or more of the individual cell populations or subtypes, such as the desired dose of CD4+ cells and/or the desired dose of CD8+ cells. In some aspects, the desired dose is the desired number of cells from a subtype or population, or the desired number of such cells per unit body weight of the individual to which the cells are administered, eg, cells/kg. In some aspects, the desired dose is or is greater than the minimum number of cells from a population or subtype, or the minimum number of cells from a population or subtype, per unit body weight.

[0373] Thus, in some embodiments, dosing is based on the desired fixed dose of total cells and the desired ratio, and/or is based on the desired fixed dose of one or more, e.g., each, of the individual subtypes or subpopulations. Thus, in some embodiments, dosing is based on the desired fixed dose, which may be or exceed the minimum T cell dose and/or be or exceed the maximum dose, and in some aspects, the desired ratio of CD4 ⁺ to CD8 ⁺ cells, and/or based on a desired fixed dose of CD4 ⁺ and/or CD8 ⁺ cells.

[0374] In some embodiments, the target dose or dose includes or includes about or is less than or about 5x10 ⁶ CD8+CAR+ cells, includes or includes about, or is less than or about 7x10 ⁶ CD8+ CAR+ cells or CD3+CAR+ cells, comprises or is less than or less than or about 8×10 ⁶ CD8+ CAR+ cells or CD3+CAR+ cells, comprises or includes less than or less than or about 8×10 ⁶ CD8+ CAR+ cells or CD3 +CAR+ cells, includes or is less than or less than or less than or less than 9x10 ⁶ CD8+ CAR+ cells or CD3+CAR+ cells, includes or includes about, or is less than or about 10x10 ⁶ CD8+ CAR+ cells, includes or includes about, or less than or less than or about 11×10 ⁶ CD8+ CAR+ cells or CD3+CAR+ cells, includes or includes about, or less than or about 12 ×10 ⁶ CD8+ CAR+ cells, comprises or includes less than or less than or less than or less than 13×10 ⁶ CD8+ CAR+ cells or CD3+CAR+ cells, includes or includes less than or less than or approximately 14×10 ⁶ CD8+ CAR+ cells, includes or includes about, or is less than or about 15x10 ⁶ CD8+ CAR+ cells or CD3+CAR+ cells, includes or includes about, or is less than or about 20x10 ⁶ or 30x10 ⁶ or 40x10 ⁶ or 50×10 ⁶ CD8+ CAR+ cells or CD3+CAR+ cells. In some aspects of such embodiments, or about, or at least or about 70%, 75%, 80%, 85%, or 90% of the CD8+CAR+ cells from such a dose exhibit one or more properties or phenotypes indicative of cell health or biologically active cells with CAR, such as the absence of an apoptosis marker, and/or in the method used to obtain cells, achieve the percentage of such cells among CD8+CAR+ cells at a dose in the range of 70-90% or an average of 80% fold. In some aspects, the variability in the frequency of biologically active or functional or non-apoptotic cells among doses is less than a threshold value and/or the frequency is not more than 1, 5, 10 or 20% fold variability outside the 70-80% range.

[0375] In some embodiments, cells are administered at a desired output ratio or within a tolerable range of a desired output ratio of multiple populations or subtypes of cells, 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., the ratio of CD4 ⁺ to CD8 ⁺ cells) is between 1:5 or about 1:5 and 5:1 or about 5:1 (or more than about 1:5 and less than about 5:1), or between 1:3 or about 1:3 and 3:1 or about 3:1 (or more than about 1:3 and less than about 3:1), e.g. between 2:1 or about 2:1 and 1:5 or about 1:5 (or more than about 1:5 and less than about 2:1, each included, or for example, is or is 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 in the range of about 1%, about 2%, about 3%, about 4%, about 5%, about 10% , approx. about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any amount within these ranges.

B. Patient-specific and/or clinical factors

[0376] In some of the embodiments presented, a dose of cells according to the methods provided herein may be administered to an individual with a patient-specific and/or clinical risk factor, such as any described herein. In some embodiments, among such treated individuals, the method does not result in a significant difference, does not result in a significant difference, or does not result in a difference above a threshold level or degree, frequency of occurrence, or risk of toxicity, e.g., severe adverse side effect, CRS, and/ or neurotoxicity, or their degree, compared with the general group of individuals treated in accordance with this method, or individuals who do not have such a patient-specific or clinical risk factor. In specific embodiments, the difference for individuals with such a risk factor, compared to the total number of individuals or individuals without a risk factor, is no more than 1%, 5%, 10%, 15%, 20%, 25%, or 30% , 35% or 40%. In some embodiments, the toxicity is CRS. In specific embodiments, the toxicity is CRS grade 2 or higher, grade 3 or higher, grade 4 or higher, or grade 5. In specific embodiments, the toxicity is neurotoxicity. In specific embodiments, the toxicity is Grade 2 or greater, Grade 3 or greater, Grade 4 or greater, or Grade 5 neurotoxicity. In some embodiments, the toxicity is or is associated with cerebral edema.

[0377] In specific embodiments, a dose of cells according to the methods provided herein is administered to an individual without a patient-specific and/or clinical factor, such as any described herein. In some embodiments, a reduced and/or lower dose of cells is administered to an individual with a patient-specific and/or clinical factor. In specific embodiments, the reduced and/or lower dose is or is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60 %, 70%, 80%, 90%, or 95% less than the dose administered to an individual without a patient-specific and/or clinical factor. In specific embodiments, there is no significant difference in the incidence of toxicity, e.g., severe adverse side effect, CRS and/or neurotoxicity, between individuals without a patient-specific and/or clinical factor dosed with cells and those with a patient-specific and /or clinical factor, which is injected with a lower dose of cells.

[0378] In some embodiments, the patient-specific and/or clinical factor is the number of prior cycles of therapy, eg, one or more cycles of therapy prior to administration of the T cell therapeutic composition. In some embodiments, prior cycles of therapy have been for the same disease and/or condition as the T cell therapeutic composition. In specific embodiments, the patient-specific and/or clinical factor is less than ten prior therapy cycles, nine prior therapy cycles, eight prior therapy cycles, seven prior therapy cycles, six prior therapy cycles, five prior therapy cycles, four prior cycles of therapy, three previous cycles of therapy, two previous cycles of therapy, or less than one previous cycle of therapy. In some embodiments, the patient-specific and/or clinical factor is less than three previous cycles of therapy. In specific embodiments, the patient-specific and/or clinical factor is two or less cycles of prior therapy.

[0379] In specific embodiments, the patient-specific and/or clinical factor is age, eg, the age of the individual at the start of administration of the T cell therapeutic composition. In specific embodiments, the patient-specific and/or clinical factor is young and/or relatively young age. In some embodiments, the patient-specific and/or clinical factor is age less than 65 years, 60 years, 55 years, 50 years, 45 years, 40 years, 35 years, 30 years, 25 years, or 20 years. In specific embodiments, the patient-specific and/or clinical factor is less than 30 years of age.

[0380] In specific embodiments, the patient-specific and/or clinical factor is the ratio of CD4:CD8 T cells in a post-apheresis sample from an individual. In some embodiments, the patient-specific and/or clinical factor is the ratio of CD4:CD8 T cells in the apheresis sample below a certain threshold. In specific embodiments, the patient-specific and/or clinical factor is a CD4:CD8 T cell ratio below 3:1, 2:1, 1.5:1, 1:1, 0.9:1, 0.8 :1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1 or 0.1:1. In specific embodiments, the ratio of CD4:CD8 T cells in the apheresis sample is a patient-specific and/or clinical factor when the dose administered is based on the total number of T cells or the total number of T cells expressing the recombinant receptor. In specific embodiments, the patient-specific and/or clinical factor is the ratio of CD4:CD8 T cells in the apheresis sample below 1:5, below 1:1, or below 0.5:1.

[0381] In specific embodiments, the patient-specific and/or clinical factor is the weight of the individual, eg body weight. In specific embodiments, the weight of the individual is the weight of the individual at the time of administration of the T cell therapeutic composition. In specific embodiments, the patient-specific and/or clinical factor is over 100 pounds (41.0 kg), over 125 pounds (51.2 kg), over 150 pounds (61.4 kg), over 175 pounds ( 71.7 kg), over 200 pounds (81.9 kg), over 225 pounds (92.1 kg), over 250 pounds (102.4 kg), over 275 pounds (112.6 kg), over 300 pounds ( 122.9 kg), over 350 lb (143.3 kg), or over 400 lb (163.8 kg) In specific embodiments, the patient-specific and/or clinical factor is over 50 kg, 60 kg, 70 kg , 80 kg, 90 kg, 100 kg, 125 kg, 150 kg, 175 kg, or 200 kg. In some embodiments, the patient-specific and/or clinical factor is a weight greater than the average weight among a group of individuals. In specific embodiments, the patient-specific and/or clinical factor is weight above the mean, median, and/or mode of weight among a group of individuals. In some embodiments, a group of individuals are treated with the same therapeutic cell composition.

[0382] In specific embodiments, the patient-specific and/or clinical factor is platelet count. In some embodiments, the patient-specific and/or clinical factor is a platelet count of less than or about less than 500,000, less than 450,000, less than 400,000, less than 350,000, less than 300,000, less than 250,000, less than 200,000, less than less than 180,000, less than 160,000, less than 140,000, less than 120,000, less than 100,000, less than 75,000, less than 50,000, or less than 25,000. In some embodiments, the patient-specific and/or clinical factor is a platelet count of less than 120000.

[0383] In specific embodiments, the patient-specific and/or clinical factor is the presence of leukemia. In some embodiments, the patient-specific or clinical factor is the presence of B-cell leukemia. In specific embodiments, the leukemia is acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), or acute myeloid leukemia (AML). In specific embodiments, the patient-specific and/or clinical factor is the presence of acute lymphocytic leukemia (ALL).

[0384] In specific embodiments, the patient-specific and/or clinical factor is a high disease load, eg, a high disease load prior to administration of the T cell therapeutic composition. In specific embodiments, the patient-specific and/or clinical factor is a high disease burden immediately before or within 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks , one month, two months, three months, four months, five months, six months, or more than six months prior to the administration of the T cell therapeutic composition. In some embodiments, the high disease burden is determined based on the percentage of bone marrow blasts. In specific embodiments, the high disease burden is greater than or equal to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of blast cells. In some embodiments, the patient-specific and/or clinical factor is the disease load of more than 5% of blast cells immediately prior to, or within one month prior to, administration of the T cell therapeutic composition. In specific embodiments, the patient-specific and/or clinical factor is a high disease load, such as sum product diameters (SPD) or lactate dehydrogenase levels.

[0385] In specific embodiments, the patient-specific and/or clinical factor is a high tumor burden, eg, a high disease burden prior to administration of the T cell therapeutic composition. In some embodiments, the tumor burden indicative factor is a measure of the volume of the tumor(s). In some embodiments, the volume score is a measure of the lesion(s), such as tumor size, tumor diameter, tumor volume, tumor mass, tumor burden, or bulk tumor volume, tumor-associated edema, tumor-associated necrosis, and/or amount or degree of metastases. In some embodiments, the tumor volume index is a two-dimensional measurement. For example, in some embodiments, the area of the lesion(s) is calculated as the product of the longest diameter and the longest perpendicular diameter for all measurable tumors. In some cases, the tumor volume index is a one-dimensional measurement. In some cases, the size of measurable lesions is estimated as the longest diameter. In some embodiments, sum of product diameters (SPD), longest tumor diameter (LD), sum of longest tumor diameters (SLD), necrosis, tumor volume, necrosis volume, necrosis–tumor ratio (NTR), peritumoral edema (PTE) are measured. ), and edema-tumor ratio (ETR).

[0386] Exemplary methods for measuring and evaluating tumor burden include those described, for example, in 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 index is the sum of products of diameters (SPD), measured by determining the sum of products of the largest perpendicular diameters for all measurable tumors. In some aspects, the tumor or lesion is measured in one direction using the longest diameter (LD) and/or by determining the sum of the longest tumor diameters (SLD) for all measurable lesions. In some embodiments, the tumor volume score is a volumetric measure 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 index is a volumetric quantification of tumor-associated edema, such as peritumoral edema (PTE) and/or edema-tumor ratio (ETR). In some embodiments, the measurement can be performed using imaging techniques such as computed tomography (CT), positron emission tomography (PET), and/or magnetic resonance imaging (MRI) of the individual.

[0387] In some embodiments, the volume measure is the SPD and, in some cases, the occurrence of toxicity, such as CRS or NT, correlates with an SPD value greater than a threshold value. In some embodiments, the volume index is SPD and ^the threshold is or is about 30 per cm2, is or is about 40 per ^cm2 , is or is about 50 per cm2, is or is about 60 per ^{cm2 .} or is or is approximately 70 per cm ² . In some embodiments, the volume index is SPD and ^the threshold is or is about 30 per cm2, is or is about 40 per ^cm2 , is or is about 50 per cm2, is or is about 60 per ^{cm2 .} or is or is approximately 70 per cm ² . In specific embodiments, the patient-specific and/or clinical factor is an SPD of greater than 30/cm2, greater ^than 40/cm2, greater ^than 50/cm2, greater ^than 60/cm2, or greater ^than 70/cm ² .

[0388] In some embodiments, a measure of tumor volume is determined at a screening step, such as a routine evaluation or blood draw to confirm and/or identify a condition or disease in an individual.

[0389] In specific embodiments, the patient-specific and/or clinical factor is the level, amount, and/or concentration of an inflammatory marker. In some embodiments, the inflammatory marker is or includes the level or presence of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), albumin, ferritin, β2-microglobulin (β2-M), or lactate dehydrogenase (LDH) detected and valued. In some embodiments, the inflammatory marker is assessed using an immunoassay. For example, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunoassay (EIA), radioimmunoassay (RIA), surface plasmon resonance (SPR), Western blotting, horizontal flow analysis, immunohistochemistry, protein array, or immuno-PCR (iPCR) can be used to detection of an inflammatory marker. In some embodiments, the presence, level, amount, and/or concentration of an inflammatory marker is indicative of tumor burden, eg, high tumor burden. In some cases, analysis or evaluation of an inflammatory marker is performed using flow cytometry. In some cases, the reagent is a soluble protein that binds to an inflammatory marker. In some examples, the reagent is a protein that is associated with C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), albumin, ferritin, β2-microglobulin (β2-M), or lactate dehydrogenase (LDH).

[0390] In some embodiments, the patient-specific and/or clinical factor is a level, amount, or concentration of an inflammatory marker greater than or greater than about, or greater than or equal to 5%, 10%, 15%, 20%, 25 %, 30%, 40%, 50%, 75%, 100%, 150%, 1x, 2x, 3x, 4x, 5x the median, median, or mean, and/or 3, 2.5, 2, 1.5, 1, 0.8, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 standard deviations above the median or level mean, the amount or concentration of an inflammatory marker measured in samples obtained from a group of individuals prior to administration of the T cell therapeutic composition, where each individual in the group did not continue to develop Grade 2 or greater toxicity, Grade 3 or greater, Grade 3 or greater long-term toxicity, Grade 4 or higher, or grade 5 toxicity following administration of the T cell therapeutic composition. In some embodiments, the toxicity is neurotoxicity.

[0391] In some embodiments, the biomarker, such as an inflammatory marker, is or includes C-reactive protein (CRP). In some embodiments, CRP is assessed using an in vitro enzyme-linked immunosorbent assay to quantify human CRP for a sample, such as serum, plasma, or blood. In some examples, CRP is detected using a human enzyme-linked immunosorbent assay (ELISA). In some embodiments, the biomarker, such as an inflammatory marker, is or includes an erythrocyte sedimentation rate (ESR). In some embodiments, the ESR is estimated by measuring the distance (in millimeters per hour) that red cells fall after separation from plasma in a vertical pipette or tube. In some embodiments, the biomarker is or includes albumin. In some aspects, albumin is assessed using a colorimetric test or in vitro enzyme-linked immunosorbent assay. In some examples, albumin is detected using a human enzyme-linked immunosorbent assay (ELISA). In some embodiments, the biomarker, such as an inflammatory marker, is or includes ferritin or β2-microglobulin. In some embodiments, ferritin or β2-microglobulin is assessed using an immunoassay or detected using an ELISA. In some aspects, the biomarker, eg, an inflammatory marker, is or includes lactate dehydrogenase (LDH), and the LDH is assessed using a colorimetric test or an in vitro enzyme-linked immunosorbent assay.

[0392] In some embodiments, the patient-specific and/or clinical factor is the level, concentration, and/or amount of LDH. In some embodiments, the occurrence of toxicity, such as CRS or NT, correlates with an LDH value above a threshold. 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. In specific embodiments, the patient-specific and/or clinical risk factor is at least 300 units per liter, at least 400 units per liter, at least 500 units per liter, or at least 600 units per liter.

[0393] In some embodiments, the level, concentration, and/or amount of LDH is a surrogate for disease burden, e.g., for tumors or malignancies, and may be useful in assessing the potential risk of neurotoxicity and/or risk-adapted dosing, or adjustments in the treatment of specific individuals. In some aspects, LDH levels may be assessed alone and/or in combination with another pre-treatment parameter, such as another measure or indicator of disease burden, e.g. on MRI, a volumetric indicator of disease burden, such as any described herein. In some aspects, one or more parameters are assessed that are indicative of disease burden, and in some contexts, may indicate the presence, absence, or risk of neurotoxicity following T cell therapy. In some aspects, one or more parameters include LDH and/or a tumor volumetric index. In some embodiments, the parameter is SPD and/or LDH.

[0394] In some embodiments, the patient-specific and/or clinical factor is the exposure to transitional chemotherapy prior to administration of the T cell therapeutic composition.

[0395] In specific embodiments, the patient-specific and/or clinical factor is a pre-treatment using anti-lymphocyte therapy, for example, prior to the administration of the T cell therapeutic composition. In some embodiments, the antilymphocyte therapy is or includes chemotherapy. In specific embodiments, the antilymphocyte therapy is or includes administration. In specific embodiments, the patient-specific and/or clinical factor is a pretreatment with fludarabine and/or cyclophosphamide prior to administration of the T cell therapeutic composition. In specific embodiments, the patient-specific and/or clinical factor is a pretreatment with cyclophosphamide prior to administration of the T cell therapeutic composition. In some embodiments, the patient-specific and/or clinical factor is a pretreatment with fludarabine and cyclophosphamide prior to administration of the T cell therapeutic composition.

[0396] In specific embodiments, the patient-specific and/or clinical factor is a molecular subtype of the disease. In specific embodiments, the patient-specific and/or clinical factor is a molecular subtype of leukemia. In specific embodiments, the patient-specific and/or clinical factor is a molecular subtype of ALL. In specific embodiments, the patient-specific and/or clinical factor is an ALL molecular subtype that is not a Philadelphia chromosome (Ph+) positive or Philadelphia chromosome (Ph)-like ALL molecular subtype, such as a non-Ph molecular subtype. In specific embodiments, non-Ph molecular subtypes of ALL include, but are not limited to, subtypes associated with TCF3-PBX1 fusion, ETV6-RUNX1 fusion, EP300-ZNF384 fusion, KMT2A-AFF1 fusion, hyperploidy, or dic(9;20) chromosome abnormality. ), for example, dic(9;20)(p13,2;q11,2).

[0397] In specific embodiments, the Philadelphia chromosome contains a translocation, t(9;22)(q34;ql 1), resulting in a new chimeric gene and protein, fusion of the BCR gene on chromosome 22 with the gene encoding Abelson's tyrosine kinase (ABL1 ), on chromosome 9. In specific embodiments, the Philadelphia-like (Ph-like) ALL subtype is characterized by associated gene expression patterns, variously referred to as the "R8 cluster group" profile, "Philadelphia-like (Ph) subtype" , "Ph-like", "BCR-ABLl-like" profile or "activated tyrosine kinase gene expression profile". These gene expression profiles show high similarity to the gene expression profiles measured for Ph+ individuals with ALL, despite the fact that, in some embodiments, Ph-like individuals do not have a Philadelphia chromosome translocation or BCR-ABL1 fusion transcript. Methods and methods for identifying and/or defining Ph+ and/or a Ph-like ALL subtypes are described (see, e.g., Roberts et al., N Engl J Med (2014) 371(11): 1005–1015; Roberts et al. Cancer Cell (2012) 14;22(2):153–66 Perez–Andreu et al Nature Genetics (2013) 45(12): 1494–1498 Yap et al Leuk Lymphoma (2017) 58(4): 950–958 Roberts et al J Clin Oncol (2017) 35(4): 394–401 Harvey et al Blood (2013) 122:826 Harvey et al Blood (2010) 116(23): 4874 –4884; and application Pct No. WO 2013/090419, the contents of which are hereby incorporated by reference).

[0398] In some embodiments, the patient-specific and/or clinical factor is the level, amount, or concentration of a cytokine in a blood, serum, or plasma sample prior to administration of the T cell therapeutic composition. In some embodiments, the cytokine is an interleukin, such as interleukin-15 (IL-15). In specific embodiments, the patient-specific and/or clinical factor is an increased level, amount, or concentration of IL-15 in the blood sample prior to administration of the T cell therapeutic composition. In specific embodiments, the patient-specific and/or clinical factor is an IL-15 level, amount or concentration greater than or equal to 1 pg/mL, 5 pg/mL, 10 pg/mL, 15 pg/mL, 20 pg /ml, 25 pg/ml, 30 pg/ml, 35 pg/ml, 40 pg/ml, 45 pg/ml, 50 pg/ml, 100 pg/ml, 150 pg/ml, 200 pg/ml, 250 pg /ml, or 300 pg/ml in a blood, serum or plasma sample prior to administration of the T cell therapeutic composition. In specific embodiments, the patient-specific and/or clinical factor is an IL-15 level, amount, or concentration greater than or equal to 30 pg/mL in a blood, serum, or plasma sample prior to administration of the T cell therapeutic composition.

[0399] In specific embodiments, the patient-specific and/or clinical factor is a level, amount, or concentration of IL-15 that is greater than or greater than about, or greater than or equal to 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, 1x, 2x, 3x, 4x, 5x the mean, median, or mean, and/or 3, 2.5, 2, 1.5, 1, 0.8, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 standard deviations above the median or level mean , amount or concentration of IL-15 in blood samples obtained from a group of individuals prior to administration of a therapeutic composition of T-cells containing a dose of cells modified using a recombinant receptor, where each individual from the group did not continue to develop toxicity of grade 2 or higher, grade 3 or greater, Grade 3 or greater long-term toxicity, Grade 4 or greater, or Grade 5 toxicity following administration of a T cell therapeutic composition. In some embodiments, the toxicity is neurotoxicity.

[0400] In specific embodiments, the patient-specific and/or clinical factor is the pharmacokinetic property of the cell composition after administration to the individual, eg, is the in vivo expansion of recombinant receptor-expressing cells, eg, CAR-T cells. In some embodiments, the pharmacokinetic parameter may include exposure, amount, concentration, persistence, and proliferation. In some cases, pharmacokinetics can be assessed by measuring parameters such as the maximum (peak) plasma concentration (C _max ), peak time (i.e. when the maximum plasma concentration occurs (C _max ); T _max ), minimum plasma concentration (i.e. minimum plasma concentration between doses of a drug, e.g. CAR+ T cells; C _min. ), half-life (T _1/2 ) and area under the curve (i.e. obtained when plotted against the plasma drug concentration of CAR+ T cells; AUC) after administration. Plasma concentration of a particular drug, e.g. CAR+ T cells, after administration can be measured using any method known in the art suitable for assessing drug concentrations, e.g. CAR+ T cells, in blood samples, or any methods described in the present description. For example, nucleic acid based methods such as quantitative PCR (QPCR) or flow cytometry based methods, or other assays such as immunoassay, ELISA, or chromatography/mass spectrometry based assays can be used.

[0401] In some embodiments, the pharmacokinetics (PK) of the administered cells, e.g., CAR compositions⁺ T cells are determined to assess the availability, eg bioavailability, of injected cells. In some embodiments, the administered cell pharmacokinetic parameters determined include maximum (peak) plasma concentrations (C_Max.), such as C_Max. CD3⁺CAR⁺ cells, CD4⁺CAR⁺ cells and or CD8⁺CAR⁺ T-cells; point in time at which they reach C_Max. (T_Max.), such as T_Max. CD3⁺CAR⁺ cells, CD4⁺CAR⁺ cells and or CD8⁺CAR⁺ T cells and/or area under the curve (AUC) such as AUC_0–28 CD3⁺CAR⁺ cells, CD4⁺CAR⁺ cells and or CD8⁺CAR⁺ T cells. In some embodiments, the pharmacokinetic parameter is the peak concentration of CD3⁺ CAR⁺ T-cells (C_Max. CD3⁺ CAR⁺ T cells) or CD8 concentration⁺ CAR⁺ T-cells (C_Max. CD8⁺ CAR⁺ T cells). In some embodiments, the pharmacokinetic parameter is AUC_0–28, CD3⁺ CAR⁺ T cells, (AUC0–28 CD3⁺ CAR⁺ T cells) or AUC_0–28, CD8⁺ CAR⁺ T cells, (AUC0–28 CD8⁺ CAR⁺ T cells).

[0402] Exposure, e.g., number or concentration of cells, e.g. , T cells, administered for T cell therapy, indicative of proliferation and/or persistence, can be indicated in relation to the maximum numbers or concentrations of cells exposed to the individual, duration of presence detectable cells or cells above a certain number or percentage, area under the curve (AUC) of the number or concentration of cells versus time, and/or combinations and indicators thereof. Such outcomes can be assessed using known methods such as qPCR to detect the copy number of a nucleic acid encoding a recombinant receptor compared to the total amount of nucleic acid or DNA in a particular sample, e.g. blood, serum, plasma, or tissue, such as a tissue sample. , and/or flow cytometry assays detecting cells expressing the receptor, typically using antibodies specific for the receptor. Cell-based assays can also be used to detect the number or percentage or concentration of functional cells, such as cells capable of binding and/or neutralizing and/or inducing responses, e.g., cytotoxic responses, against cells associated with a disease or condition, or expressing an antigen recognized by the receptor.

[0403] In some embodiments, exposure may be indicated as the area under the drug concentration versus time (AUC) curve, as determined by post-dose pharmacokinetic analysis, e.g., CAR+ T cells. In some cases, AUC is expressed in cells*day/μl, for cells administered in cell therapy, or in their respective units. In some embodiments, the AUC is measured as the average AUC in a patient population, such as a sample of the patient population, eg, the average AUC for one or more patient(s). In some embodiments, systemic exposure refers to area under the curve (AUC) over a specified time period, e.g., day 0 to day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28 days or more, or weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, or months 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 48 or more. In some embodiments, AUC is measured as AUC from day 0 to day 28 (AUC _0-28 ) after drug administration, e.g., CAR+ T cells, including all measured data and data extrapolated from measured pharmacokinetic (PK) parameters, such as as the mean AUC for a patient population, such as a sample of a patient population. In some embodiments, to determine exposure over time, e.g. AUC for a certain period of time, e.g. AUC _0-28 , a drug concentration versus time curve is obtained using multiple measurements or parameter estimation, e.g. cell concentrations, with over time, for example, measurements taken every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, or 28 days, or more.

[0404] In some embodiments, the presence and/or amount of recombinant receptor-expressing cells (eg, CAR-expressing cells administered for T cell therapy) in an individual following T cell administration is determined. In some aspects, nucleic acid-based methods such as quantitative PCR (QPCR) are used to evaluate the number of cells expressing a recombinant receptor (e.g., CAR-expressing cells introduced for T-cell therapy) in a blood or serum sample, or an organ or tissue (eg, from a disease site, eg, in a tumor sample) of an individual. In some aspects, persistence is quantified as the number of copies of a DNA or plasmid encoding a receptor, e.g., CAR, per microgram of DNA, or as the number of receptor-expressing, e.g., CAR-expressing cells per microliter of sample, e.g., blood or serum, or per total the number of peripheral blood mononuclear cells (PBMC) or leukocytes or T-cells per microliter of sample. In some embodiments, the primers or probe used for qPCR or other nucleic acid based methods are specific for binding, recognition and/or amplification of nucleic acids encoding the recombinant receptor and/or other components or elements of the plasmid and/or vector, including regulatory elements, such as promoters, transcriptional and/or post-transcriptional regulatory elements or response elements, or markers, such as surrogate markers. In some embodiments, the primers may be specific for regulatory elements, such as the North American woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0405] In some embodiments, cells are detected in an individual at or at least 2, 3, 4, 5, 6, 7, 14, 15, 27, or 28 days after administration of T cells, e.g., expressing CAR T -cells. In some aspects, cells are detected after or at least 2, 4, or 6 weeks after, or after 3, 6, or 12, 18, or 24 or 30, or 36 months, or 1, 2, 3, 4, 5 , or more years, after the introduction of T-cells, for example, expressing CAR T-cells. In some embodiments, cells expressing the receptor are detectable in serum, plasma, blood, or tissue, for example, in a tumor sample, of an individual, for example, by said method, such as a QPCR or flow cytometric detection method.

[0406] In some embodiments, the receptor-expressing cells, eg , CAR-expressing cells, proliferate in an individual after administration of T cells, eg, CAR-expressing T cells. In some aspects, increased cell exposure to an individual includes increased cell proliferation. As shown herein, increased proliferation that occurs early, e.g., within 7 days, e.g., 4-7 days, following cell therapy is associated with, correlates with, and/or may be indicative of a risk or likely risk of toxicity. In some embodiments, the present invention relates to methods in which the pharmacokinetic properties of administered recombinant receptor-expressing cells are monitored in individuals and, if early or rapid multiplication is detected, one or more agents to alleviate toxicity, or reduce the risk or likely risk of toxicity, can be administered to the individual. . Exemplary means to alleviate toxicity are known and are described, for example, in section IV.E.

[0407] In particular embodiments, the pharmacokinetic parameter is the number of recombinant receptor-expressing cells that is indicative of early or rapid cell proliferation in an individual. In some embodiments, such a detectable multiplication can be shown if at least or about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150 or 200 recombinant receptor-expressing cells per microliter are detected in a blood or serum sample collected within four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen, or more than fourteen days after the start of treatment. cell therapy. In some embodiments, the pharmacokinetic parameter is the number of recombinant receptor-expressing cells of at least, or about, 2 recombinant receptor-expressing cells per microliter in a blood or serum sample collected within four days of initiation of administration. In specific embodiments, the pharmacokinetic parameter is the number of recombinant receptor-expressing cells of at least, or about, 5 recombinant receptor-expressing cells per microliter in a blood or serum sample collected within five or six days of initiation of administration. In some embodiments, the pharmacokinetic parameter is the number of recombinant receptor-expressing cells of at least, or about, 10 recombinant receptor-expressing cells per microliter in a blood or serum sample collected within five or six days of initiation of administration. In specific embodiments, the pharmacokinetic parameter is the number of recombinant receptor-expressing cells of at least 15 recombinant receptor-expressing cells per microliter in a blood or serum sample collected within seven days of initiation of administration.

[0408] In specific embodiments, the present invention provides methods for designing a study for the administration of a T cell therapeutic composition. In some embodiments, the methods include allocating an individual to a treatment regimen based on the presence or absence of one or more patient-specific and/or clinical risk factors, such as any of those described. In some embodiments, the individual does not have or is not identified as having, or is not considered to have, a patient-specific and/or clinical factor and is dispensed for administration of a dose of a T cell therapeutic composition. In specific embodiments, the individual has, or is identified as having, or is considered to have, a patient-specific and/or clinical factor and is scheduled to receive a reduced and/or lower dose of cells. In specific embodiments, the reduced and/or lower dose is or is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60 %, 70%, 80%, 90%, or 95% less than the dose administered to an individual without a patient-specific and/or clinical factor. In specific embodiments, there is no significant difference in the incidence of toxicity, e.g., severe adverse side effects, CRS, and/or neurotoxicity, between individuals without a patient-specific and/or clinical factor dosed with cells and those with a patient-specific and/or a clinical factor administered at a lower dose of cells.

[0409] In specific embodiments, an individual is allocated to receive a low or reduced dose if the individual has one or more patient-specific and/or clinical factors, which may include, but are not limited to, (i) individuals subjected to fewer previous cycles. therapy, optionally less than two previous cycles of therapy, prior to the start of administration of the T cell therapeutic composition, (ii) young individuals, optionally less than 30 years of age, (iii) individuals whose CD4:CD8 ratio in the post-apheresis sample is from individual below a certain threshold, optionally below 1:1 or below 1.5:1, or below 0.5:1, or below, optionally, where the dose administered is based on the total number of T cells or the total number of T cells expressing the recombinant receptor; (iv) individuals having a weight greater than the average weight among the group of treated individuals; (v) individuals with a platelet count of less than or about less than 120,000; (vi) individuals having B-cell leukemia, optionally acute lymphocytic leukemia (ALL); (vii) individuals having a high disease burden prior to, e.g. immediately prior to or within one month prior to initiation of administration of the T cell therapeutic composition, optionally as determined based on a percentage of bone marrow blast cells greater than or equal to 5%, the sum of the products of the diameters (SPD), or lactate dehydrogenase levels; (ix) individuals undergoing transitional chemotherapy prior to initiation of administration of the T cell therapeutic composition; (x) individuals pre-treated with anti-lymphocyte therapy, optionally including administration of fludarabine and/or cyclophosphamide, prior to initiation of administration of the T cell therapeutic composition; (xi) individuals whose level, amount, or concentration of interleukin-15 (IL-15) in the blood sample prior to administration of the T cell therapeutic composition is greater than or equal to a threshold value, optionally, where the threshold value is 30 pg/mL plasma, ( xii) rapid in vivo expansion of cells expressing the recombinant receptor, (xiii) individuals lacking the Philadelphia chromosome (Ph+) molecular subtype and/or the Ph-like (Ph-like) molecular subtype of acute lymphoblastic leukemia (ALL).

C. Response Outcome

[0410] In some embodiments, the outcome of an individual's response after administration of a T cell composition can be monitored or assessed. In some embodiments, the outcome of the response is no response. In some embodiments, the outcome of the response is a partial response. In some embodiments, the response outcome is a complete response (CR). In some embodiments, the response outcome is assessed by monitoring the burden of disease in an individual. In some embodiments, no response, the presence of a partial response, or clinical or complete response can be assessed.

[0411] In some embodiments, a partial response or complete response is a response in which the drug reduces or prevents the increase or burden of a disease or condition in an individual. For example, when the disease or condition is a tumor, a reduction in disease burden exists or is present if there is a reduction in tumor size, core volume, metastasis, percentage of blast cells in the bone marrow, or molecularly detectable cancer, and/or improvement in prognosis or survival, or other symptom associated with tumor burden compared to before drug treatment (eg, CAR-T cells).

[0412] In some embodiments, the disease or condition is a tumor and the reduction in disease burden is a reduction in tumor size. In some embodiments, a reduction in disease burden is indicated by a reduction in one or more factors such as the burden or number of diseased cells in an individual or in their body fluid or organ or tissue, tumor mass or volume, or the number or extent of metastases. In some embodiments, disease burden, such as tumor burden, can be assessed or monitored by the extent of pathologically confirmed disease and/or minimal residual disease.

[0413] In some embodiments, the burden of a disease or condition in an individual is detected, assessed, or measured. Disease burden can be detected, in some aspects, by detecting the total number of disease-affected or disease-associated cells, such as tumor cells, in an individual, or in an organ, tissue, or body fluid of an individual, such as blood or serum. In some embodiments, disease burden, such as tumor burden, is assessed by measuring the mass of a solid tumor and/or the number or extent of metastases. In some aspects, the survival of the individual, survival within a certain period of time, the degree of survival, the presence or duration of the presence of survival in the absence of an adverse event or symptom, or recurrence-free survival are evaluated. In some embodiments, any symptom of a disease or condition is evaluated. In some embodiments, a disease or condition load score is indicated.

[0414] In some embodiments, the disease burden may include the total number of diseased cells in an individual or in an organ, tissue, or body fluid of an individual, such as an organ or tissue at a tumor site or other site, for example, which may be 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. Disease burden may include, in some embodiments, tumor weight, the number or extent of metastases, and/or the percentage of blast cells present in the bone marrow.

[0415] In some embodiments, the individual has leukemia. The degree of disease burden can be determined by assessing residual leukemia in the blood or bone marrow.

[0416] In some embodiments, a response outcome exists if there is a decrease in the percentage of blast cells in the bone marrow compared to the percentage of blast cells in the bone marrow prior to treatment with the drug. In some embodiments, a reduction in disease burden exists if there is a reduction or reduction of at least, or at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95 % or more of the number or percentage of blast cells in the bone marrow, compared to the number or percentage of blast cells in the bone marrow before treatment.

[0417] In some embodiments, a response is indicated for an individual if the individual does not show a pathologically confirmed disease (a pathologically confirmed disease) or does not show a significant pathologically confirmed disease. In some embodiments, an individual is shown to have morphologically confirmed disease if greater than or equal to 5% blast cells are present in the bone marrow, eg, as detected by light microscopy. In some embodiments, an individual is in complete or clinical remission if less than 5% of blast cells are present in the bone marrow.

[0418] In some embodiments, an individual is shown to have a reduction or reduction in disease burden if they show a pathologically confirmed disease prior to treatment and are in complete remission (e.g., less than 5% of blast cells in the bone marrow) in the presence or absence of a detectable at the molecular level of the disease (eg, minimal residual disease (MRD) that is detectable at the molecular level, eg, as detected by flow cytometry or quantitative PCR) after treatment. In some embodiments, an individual is shown to have a reduction or reduction in disease burden if it shows a molecularly detectable disease before treatment and does not show a molecularly detectable disease after treatment.

[0419] In some embodiments, the individual may show complete remission, but there is a small proportion of non-morphologically detectable (by light microscopy) residual leukemic cells. An individual is considered to have minimal residual disease (MRD) if the individual shows less than 5% blast cells 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 methods that allow sensitive detection of a small number of cells. In some aspects, such methods include PCR assays that can detect unique Ig/T cell receptor gene rearrangements or fusion transcripts produced by chromosomal translocations. In some embodiments, flow cytometry can be used to identify cancer cells based on leukemia-specific immunophenotypes. In some embodiments, cancer molecular detection can detect as little as 1 leukemic or blast cell out of 100,000 normal cells, or 1 leukemic or blast cell out of 10,000 normal cells. In some embodiments, an individual is shown to have an MRD that is detectable at the molecular level if at least or more than 1 leukemic cell is detected in 100,000 cells, for example, by PCR or flow cytometry.

[0420] In some embodiments, the individual's disease burden is not molecularly detectable, or MRD- ^, so that, in some cases, it is not possible to detect leukemic cells in an individual using PCR or flow cytometry methods.

[0421] In some embodiments, the response outcome is the absence of CR or the presence of a complete response, in which the individual achieves or exhibits minimal residual disease or molecularly detectable disease status. In some embodiments, the outcome of the response is the presence of a CR with a molecularly detectable disease or the presence of a CR with no molecularly detectable disease. In some embodiments, disease burden is assessed in individuals using methods as described herein, such as methods assessing blast cells in the bone marrow or molecularly detectable disease by flow cytometry or qPCR methods.

[0422] After the introduction of cells, the biological activity of the modified populations of cells in some embodiments, the implementation is measured, for example, by any of a number of known methods. Parameters for evaluation include specific binding of the modified or native T cell or other immunocyte to the antigen, in vivo , eg, by imaging, or ex vivo , eg, by ELISA or flow cytometry. In specific embodiments, the ability of modified cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays, as described, for example, in Kochenderfer et al., J. Immunotherapy, 32(7): 689 –702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25–40 (2004). In specific embodiments, the biological activity of cells is measured by analyzing the expression and/or secretion of one or more cytokines such as CD 107a, IFNγ, IL-2, and TNF. In some aspects, biological activity is measured by assessing a clinical outcome, such as a reduction in tumor burden or mass.

D. Agent for alleviating or treating toxicity

[0423] In some embodiments, the methods provided herein relate to monitoring individuals for risk or probable risk of toxicity, and/or identifying individuals at risk or probable risk for toxicity, e.g., severe toxicity such as severe CRS or severe neurotoxicity following cellular therapy involving cells expressing a recombinant receptor (eg, CAR). In some embodiments, the methods include administering one or more agents or therapies that treat cell therapy toxicity, such as CRS or neurotoxicity, eg, severe CRS or severe neurotoxicity). In some embodiments, the agent is administered at the time at which the individual is determined to be at risk for toxicity, such as severe toxicity, and/or at the time at which one or more signs or symptoms of toxicity occur.

[0424] In some embodiments, the agent is a steroid. In some embodiments, the agent is an antagonist or inhibitor of a cytokine receptor, such as IL-6 receptor, CD122 receptor (IL-2R-beta receptor), or CCR2, or is 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 TGFβ. In some embodiments, the agent, such as an agonist, antagonist, or inhibitor, is an antibody or antigen-binding fragment, a small molecule, a protein or peptide, or a nucleic acid.

[0425] In some embodiments, a liquid bolus can be used as an intervention, for example, to treat hypotension associated with CRS. In some embodiments, the target hematocrit is >24%. In some embodiments, the intervention includes the use of absorbent resin technology along with blood or plasma filtration. In some cases, the intervention includes dialysis, plasmapheresis, or similar technologies. In some embodiments, vasoconstrictors or acetaminophen may be used.

1. Steroid

[0426] In some embodiments, the agent that treats and/or prevents, delays, or reduces the development or risk of cell therapy toxicity, such as CRS or grade 2 or higher neurotoxicity, or severe CRS or neurotoxicity, is a steroid, for example, corticosteroid. Corticosteroids typically include glucocorticoids and mineralocorticoids.

[0427] Any corticosteroid, such as a glucocorticoid, can be used in the methods described herein. In some embodiments, glucocorticoids include synthetic and non-synthetic glucocorticoids. Illustrative glucocorticoids include, but are not limited to: aclomethasone, algestone, beclomethasone (eg, beclomethasone dipropionate), betamethasone (eg, betamethasone 17-valerate, betamethasone sodium acetate, betamethasone sodium phosphate, betamethasone valerate), budesonides, clobetasoles (eg, clobetasol propionate ), clobetasones, clocortolones (eg, clocortolone pivalate), cloprednols, corticosterones, cortisones and hydrocortisones (eg, cortisone hydroacetate), cortivasols, deflazacorts, desonides, deoxymethasones, dexamethasone (eg, dexamethasone-21 phosphate, dexamethasone acetate, dexamethasone sodium phosphate) , diflorazones (eg, diflorazone diacetate), difluorocortolones, difluprednates, enoxolones, fluazacorts, flucloronides, fludrocortisones (eg, cortisone fludroacetate), flumethasones (eg, flumethasone pivalate), flunisolides, fluocinolones (eg, fluocinolone acetonide), fluocionides, fluocortins, fluocortolones , fluorometholones (e.g. fluorometholone acetate), flu perolones (eg, fluperolone acetate), fluprednidenes, fluprednisolones, flurandrenolides, flucasones (eg, flucazone propionate), formocortals, halcinonides, halobetasols, halomethasones, halopredones, hydrocortamates, hydrocortisones (eg, hydrocortisone-21 butyrate, hydrocortisone aceponate, buteprate hydroacetate, cortisone hydroacetate гидрокортизона, бутират гидрокортизона, ципионата гидрокортизона, гемисукцинат гидрокортизона, пробутат гидрокортизона, фосфат гидрокортизона натрия, сукцинат гидрокортизона натрия, валерат гидрокортизона), этабонат лотепреднола, мазипредоны, медризоны, мепреднизоны, метилпреднизолоны (ацепонат метилпреднизолона, ацетат метилпреднизолона, гемисукцинат метилпреднизолона, сукцинат метилпреднизолона натрия) , mometasone (eg, mometasone furoate), paramethasones (eg, paramethasone acetate), prednisolones, prednisolones (eg, prednisolone-25 diethylaminoacetate, sodium prednisolone 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 (eg, 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).

[0428] In some examples, the glucocorticoid is selected from cortisones, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, and prednisone. In a specific example, the glucocorticoid is dexamethasone.

[0429] 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 cell therapy toxicity, such as CRS or neurotoxicity. In some embodiments, indicators of improvement or treatment success include determining the absence of an appropriate measure on a toxicity grading scale (e.g., CRS or neurotoxicity grading scale), such as a score of less than 3, or a change in grade or severity on a grading scale, as discussed in the present description, for example, a change from a score of 4 to a score of 3.

[0430] In some aspects, the corticosteroid is provided in a therapeutically effective dose. A therapeutically effective concentration can be determined empirically by testing in known in vitro or in vivo systems (eg, an animal model). For example, the amount of a selected corticosteroid to be administered to alleviate the symptoms or adverse effects of cell therapy toxicity, 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 exact dose that can be empirically determined may depend on the particular therapeutic agent, dosing regimen and schedule, route of administration, and severity of disease.

[0431] The corticosteroid can be administered in any amount that is effective in alleviating one or more symptoms associated with toxicity, such as CRS or neurotoxicity. A corticosteroid, such as a glucocorticoid, may be administered, for example, in an amount between or about 0.1 and 100 mg, per dose, 0.1 and 80 mg, 0.1 and 60 mg, 0.1 and 40 mg, 0. 1 and 30 mg, 0.1 and 20 mg, 0.1 and 15 mg, 0.1 and 10 mg, 0.1 and 5 mg, 0.2 and 40 mg, 0.2 and 30 mg, 0.2 and 20 mg, 0.2 and 15 mg, 0.2 and 10 mg, 0.2 and 5 mg, 0.4 and 40 mg, 0.4 and 30 mg, 0.4 and 20 mg, 0.4 and 15 mg, 0.4 and 10 mg, 0.4 and 5 mg, 0.4 and 4 mg, 1 and 20 mg, 1 and 15 mg, or 1 and 10 mg each, for a 70 kg adult human . Typically, a corticosteroid, such as a glucocorticoid, is administered in an amount between or about 0.4 and 20 mg, such as at 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, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg or 20 mg per dose, for the average adult human subject.

[0432] In some embodiments, the corticosteroid can be administered, for example, by dosing at or about 0.001 mg/kg (individual body weight), 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 human individual, typically weighing approximately 70 kg - 75 kg.

[0433] A corticosteroid or glucocorticoid, such as dexamethasone, can be administered orally (tablets, liquid, or liquid concentrate), PO, intravenously (IV), intramuscularly, or by any other known method or method described herein (for example, in relation to pharmaceutical formulations). In some aspects, the corticosteroid is administered in the form of a bolus, and in other aspects, it can be administered over a period of time.

[0434] In some aspects, the glucocorticoid can be administered over a period of more than one day, such as more than two days, for 3 days, or for 4 or more days. In some embodiments, the corticosteroid can be administered once a day, twice a day, or three times or more a day. For example, a corticosteroid, such as dexamethasone, may, in some instances, be administered at 10 mg (or equivalent) IV twice daily for three days.

[0435] In some embodiments, the dosing of a corticosteroid, such as a glucocorticoid, is administered in successively decreasing doses per treatment cycle. Thus, in some of these regimens, the corticosteroid dose is reduced. For example, a corticosteroid can be administered at an initial dose (or equivalent dose, for example, in the case of dexamethasone) of 4 mg, and with each subsequent administration, the dose can 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 introduction

[0436] As a rule, 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 drugs vary in potency and that doses can thus be varied to obtain equivalent effects. Table 4 shows the equivalence in terms of potency for different glucocorticoids and routes of administration. Equivalent activity for clinical dosing is well known. Information regarding equivalent dosing of steroids (not on a chronotherapeutic basis) can be found in the British National Formulary (BNF), 37 March 1999.

TABLE 4: Administration of glucocorticoids Glucocorticoid (method) Activity equivalence Hydrocortisone (IV or PO) twenty Prednisone 5 Prednisolone (IV or PO) 5 Sodium methylprednisolone succinate (IV) four Dexamethasone (IV or PO) 0.5–0.75

[0437] Thus, in some embodiments, the steroid is administered in an amount equivalent to a dosage 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 10 mg dexamethasone daily, 2.0 mg to 8 mg dexamethasone daily, or 2.0 mg to 6.0 mg dexamethasone daily, each included. In some cases, the steroid is administered at a dose equivalent to or approximately equivalent to 4 mg, or equivalent to or approximately equivalent to 8 mg of dexamethasone per day.

[0438] In some embodiments, the steroid is administered if fever persists after treatment with tocilizumab. For example, in some embodiments, dexamethasone is administered orally or intravenously at a dose of 5-10 mg up to every 6-12 hours during ongoing fever. In some embodiments, tocilizumab is administered simultaneously or after supplemental oxygen.

2. Other means

[0439] In some embodiments, the agent that treats or ameliorates the symptoms of cell therapy toxicity, e.g., CRS or neurotoxicity, is an agent that targets a cytokine, e.g., is an antagonist or inhibitor of a cytokine, 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 gamma (IFN-gamma), or monocytic chemotactic protein- 1 (MCP–1). In some embodiments, the agent that treats or ameliorates the symptoms of cell therapy toxicity, e.g., CRS or neurotoxicity, is an agent that targets (e.g., an inhibitor or antagonist) of a cytokine receptor, such as the IL-6 receptor (IL-6R), IL-2 receptor (IL-2R/CD25), MCP-1 receptor (CCL2) (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).

[0440] The amount of the selected agent that treats or alleviates the symptoms of cell therapy toxicity, e.g., CRS or neurotoxicity to be administered to alleviate the symptoms or adverse effects of cell therapy toxicity, e.g., CRS or neurotoxicity, can be determined by standard clinical methods. Illustrative 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, seizures, and/or tremor.

[0441] In some embodiments, the agent is administered at a dose level 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, 100mg to 1000mg, 100mg to 500mg, 100mg to 200mg, 200mg to 1000mg, 200mg to 500mg, or 500mg to 1000mg.

[0442] In some embodiments, the agent is administered at or about 0.5 mg/kg to 100 mg/kg, e.g., from or about 1 mg/kg to 50 mg/kg, 1 mg/kg to 25 mg /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 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 up to 50 mg/kg up to 50 mg/kg up to 100 mg/kg. In some embodiments, the agent is administered at a dose level 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 to 4 mg/kg or 6 mg/kg up to 8 mg/kg each included. In some aspects, the agent is administered at a dose level 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.

[0443] In some embodiments, the agent is administered by injection, e.g., intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, vitreous injection, septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjunctival injection , sub-Tenon injection, retrobulbar injection, peribulbar injection, or posterior parascleral delivery. In some embodiments, it is administered via parenteral, intrapulmonary, and intranasal, and if desired for topical treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal or subcutaneous administration.

[0444] In some embodiments, this amount of agent is administered about or about twice a day, once a day, every second day, three times a week, once a week, once every two weeks, or once a month .

[0445] 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 containing the agent is administered as described below. In other aspects, the agent is administered separately, and it can be administered via any known suitable route of administration or via the method described herein, for example, in relation to compositions and pharmaceutical formulations.

[0446] In some embodiments, the agent that treats or ameliorates the symptoms of 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.

[0447] 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 contains tocilizumab (atlizumab) or sarilumab, an anti-IL-6R antibody. In some embodiments, the agent is an anti-IL-6R antibody described in US Patent No: 8,562,991. In some instances, the agent targeting IL-6 is an anti-IL-6 antibody such as siltuximab, elsilimoab, 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 interaction. The usefulness of this general type of method has been demonstrated using a natural interleukin-1 receptor antagonist. Cm. Harmurn, C. H. et al., Nature (1990) 343:336–340. In some aspects, the antagonist or inhibitor of IL-6/IL-6R is an IL-6 mutein, such as described in US Patent No. 5,591,827. In some embodiments, the IL-6/IL-6R antagonist or inhibitor agent is a small molecule, protein or peptide, or nucleic acid.

[0448] In some embodiments, the agent is tocilizumab. In some embodiments, tocilizumab is administered as early intervention according to the presented dosing methods from or about 1 mg/kg to 12 mg/kg, e.g., at or about 4 mg/kg, 8 mg/kg, or 10 mg /kg. In some embodiments, tocilizumab is administered by intravenous infusion. In some embodiments, tocilizumab is administered for persistent fever greater than 39°C lasting 10 hours that does not respond to acetaminophen. In some embodiments, a second administration of tocilizumab is given if symptoms recur 48 hours after the initial dose.

[0449] In some embodiments, the agent is a TGF-β or TGF-β receptor agonist or stimulator (eg, TGF-β receptor I, II, or III). In some aspects, the agent is an antibody that increases the activity of TGF-β, for example, an antibody or antigen-binding fragment that binds to TGF-β or one of its receptors. In some embodiments, the agent that is an agonist or stimulant of TGFβ and/or its receptor is a small molecule, protein or peptide, or nucleic acid.

[0450] 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 any of those 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, the 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.

[0451] 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 the antibodies 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, the IFN-γ/IFNGR antagonist or inhibitor agent is a small molecule, protein or peptide, or nucleic acid.

[0452] 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 the antibodies 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, the IL-10/IL-10R antagonist or inhibitor agent is a small molecule, protein or peptide, or nucleic acid.

V. MODIFIED CELLS

[0453] The present invention relates to modified cells that express one or more recombinant antigen receptors. In some embodiments, the cells may include cells genetically modified with a recombinant receptor, such as a chimeric antigen receptor.

A. Recombinant antigen receptors

[0454] The present invention relates to modified cells, such as T cells, that express a recombinant receptor, including chimeric receptors containing ligand-binding domains or binding fragments thereof, such as functional non-TCR antigen receptors, such as chimeric antigen receptors ( CAR), as well as including T cell receptors (TCRs), such as transgenic TCRs, and their components. A chimeric receptor, such as a CAR, typically includes an extracellular antigen (or ligand) binding domain associated with one or more intracellular signaling components, in some aspects via linkers and/or a transmembrane domain(s).

1. Chimeric antigen receptors

[0455] Exemplary antigen receptors, including CARs, and methods for constructing and introducing such receptors into cells include those described, for example, in International Patent Application Publication Nos. , WO2013/123061, в публикациях Патентных заявок США номер US2002131960, US2013287748, US20130149337, в Патентах США No.: 6451995, 7446190, 8252592, 8339645, 8398282, 7446179, 6410319, 7070995, 7265209, 7354762, 7446191, 8324353 и 8479118, и in European patent application number EP2537416, and/or the methods described in Sadelain et al.,Cancer Discs., 3(4): 388–398 (2013); Davila et al.PLOS ONE 8(4): e61338 (2013); Turtle et al.,Curr. Opin. Immunol., 24(5): 633–39 (2012); Wu et al.,cancer, 18(2): 160–75 (2012). In some aspects, antigen receptors include CAR as described in US Patent No.: 7446190 and receptors described in International Patent Application Publication No.: WO/2014055668 A1. Examples of CARs include CARs as described in any of the above publications such as WO2014031687, US 8339645, US 7446179, US 2013/0149337, US Patent No.: 7446190, US Patent No.: 8389282, Kochenderfer et al.,Nature Reviews Clinical Oncology, 10, 267–276 (2013); Wang et al.J. Immunother. 35(9): 689–701 (2012); and Brentjens et al.,Sci Transl Med. 5(177) (2013). See also WO2014031687, US 8339645, US 7446179, US 2013/0149337, US Patent No.: 7446190, and US Patent No.: 8389282. Chimeric receptors such as CAR typically include an extracellular antigen binding domain, such as part of a antibodies, typically the heavy (VH) chain variable region and/or the light (VL) chain variable region of the antibody, for example,scFv antibody fragment.

[0456] In some embodiments, the recombinant receptor, e.g., an antigen receptor, comprises an extracellular antigen or ligand binding domain that binds, e.g., specifically binds, to an antigen, ligand, and/or marker. Among antigen receptors, there are functional non-TCR antigen receptors such as chimeric antigen receptors (CARs). In some embodiments, the antigen receptor is a CAR containing an extracellular antigen recognition domain that specifically binds to an antigen. In some embodiments, the CAR is designed with specificity for a particular antigen, marker, or ligand, such as an antigen expressed in a particular cell type to be targeted by adoptive therapy, for example, a cancer marker, and/or an antigen intended to induce an attenuated response, for example, an antigen expressed in normal or disease-free cells. Thus, a CAR typically includes, in its extracellular portion, one or more ligand (e.g., antigen)-binding molecules, such as one or more antigen-binding fragments, domains, or moieties, or one or more antibody variable domains, and/or antibody molecules. In some embodiments, the CAR comprises an antigen-binding portion or portions of an antibody molecule, such as a single chain antibody fragment (scFv) derived from the variable heavy (V _H ) and variable light (V _L ) chains of a monoclonal antibody (mAb), or a single domain antibody (sdAb ), such as sdFv, nanobody, V _H H and V _NAR. In some embodiments, the antigen binding fragment comprises antibody variable regions connected by a flexible linker.

[0457] In some embodiments, among the antigens targeted by the chimeric receptors are antigens expressed in the context of the disease, condition, or cell type to be targeted by adoptive cell therapy. Among the diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, including malignancies and tumors, including hematologic malignancies, malignancies of the immune system, such as lymphomas, leukemias, and/or myelomas, such as B–, T–, and myeloid leukemias. , lymphomas, and multiple myelomas. In some embodiments, the antigen targeted by the receptor is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on cells affected by the disease or condition, such as tumor cells or pathogenic cells, compared to normal or untargeted cells or tissues, such as healthy cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or is expressed on modified cells.

[0458] The antigens targeted by the receptors, in some embodiments, include the orphan tyrosine kinase receptor ROR1, tEGFR, Her2, Ll-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, 0EPHa2, ErbB2, 3 or 4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL –13R-alpha2, kdr, kappa light chain, Lewis Y, cell adhesion molecules L1, MAGE–A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligands, NY–ESO–1, MART–1, gp100, oncofetal antigen, ROR1 , TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate-specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, c-Met, GD-2, and MAGE A3, CE7, Wilms tumor antigen 1 (WT-1), a cyclin such as cyclin A1 (CCNA1) and/or biotinylated molecules and/or molecules expressed by HIV, HCV, HBV or other pathogens.

[0459] In some embodiments, the antigen targeted by the receptors includes the αvβ6 integrin (avb6 integrin), B-cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), cancer-testicular antigen, cancer-testicular antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, chemokine ligand 1 with C-C motif ( CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD138, CD171, epidermal growth factor protein (EGFR), truncated epidermal growth factor protein (tEGFR ), epidermal growth factor receptor type III mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin A2 receptor (EPHa2), estrogen receptor, Fc receptor-like protein 5 (FCRL5; also known as Fc receptor homologue 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), folate-binding protein (FBP), folate receptor alpha, fetal acetylcholine receptor, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), G protein-coupled receptor 5D (GPCR5D), Her2/neu (erb– receptor tyrosine kinase B2), Her3 (erb–B3), Her4 (erb–B4), erbB dimers, melanoma-associated high molecular weight human antigen (HMW–MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA–AIA1), human leukocyte A2 antigen (HLA-A2), IL-22 alpha receptor (IL-22Ra), IL-13 alpha 2 receptor (IL-13Ra2), kinase insert domain-containing receptor (kdr), kappa light chain, L1 cell adhesion molecule ( L1-CAM), CE7 epitope from L1-CAM, member A of family 8 containing leucine-rich repeats (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, mesothelin, c- Met, mouse cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D ligands (NKG2D), melan A (MART-1), adhesion molecule neuronal cell antigen (NCAM), oncofetal antigen, preferably expressed melanoma antigen (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), orphan receptor tyrosine kinase-like receptor 1 (ROR1), survivin , trophoblastic cell glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor antigen 1 (WT-1), specific for pathogen antigen, or antigen associated with a universal label, and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV or other pathogens. The antigens targeted by the receptors, in some embodiments, include antigens associated with B-cell cancer, such as any of a number of known B-cell markers. In some embodiments, the antigen targeted by the receptor is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Ig-kappa, Ig-lambda, CD79a, CD79b, or CD30.

[0460] In some embodiments, the CAR binds a pathogen-specific antigen. In some embodiments, the CAR is specific for viral antigens (such as a viral antigen from HIV, HCV, HBV, etc.), bacterial antigens, and/or parasitic antigens.

[0461] In some embodiments, the antibody or antigen-binding fragment (eg , scFv or V _H domain) specifically recognizes an antigen, such as CD19. In some embodiments, the antibody or antigen-binding fragment is derived from or is an antibody variant or antigen-binding fragment that specifically binds to CD19.

[0462] In some embodiments, the scFv and/or V _H domains are derived from FMC63. FMC63 refers generally to a mouse IgG1 monoclonal antibody raised against Nalm-1 and -16 cells expressing human CD19 (Ling, NR, et al. (1987). Leucocyte typing III. 302). The FMC63 antibody contains the CDR H1 shown in SEQ ID NO: 38; CDR H2 specified in SEQ ID NO:39; CDR H3 specified in SEQ ID NO: 40 or 54; and CDR L1 specified in SEQ ID NO: 35; CDR L2 specified in SEQ ID NO:36 or 55; and CDR L3 as set forth in SEQ ID NO:37 or 56. The FMC63 antibody comprises a heavy chain variable region (V _H ) containing the amino acid sequence from SEQ ID NO: 41 and a light chain variable region (V _L ) containing the amino acid sequence from SEQ ID NO: 42. In some embodiments, the scFv comprises a variable light chain comprising the L1 CDR sequence of SEQ ID NO:35, the L2 CDR sequence of SEQ ID NO:36, and the L3 CDR sequence of SEQ ID NO:37, and/ or a variable heavy chain comprising the H1 CDR sequence of SEQ ID NO:38, the H2 CDR sequence of SEQ ID NO:39, and the H3 CDR sequence of SEQ ID NO:40, 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 them. In some embodiments, the scFv comprises the heavy chain variable region from FMC63 as set forth in SEQ ID NO:41 and the light chain variable region from FMC63 as set forth in SEQ ID NO:42, 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 them. In some embodiments, the variable heavy and variable light chains are connected via a linker. In some embodiments, the linker is listed in SEQ ID NO:58. In some embodiments, the scFv contains, in the following order, V _H , a linker, and V _L . In some embodiments, the scFv contains, in the following order, V _L , a linker, and V _H . In some embodiments, the scFv is encoded by the nucleotide sequence shown in SEQ ID NO:57 or by a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:57. In some embodiments, the scFv contains the amino acid sequence shown in SEQ ID NO:43 or a sequence having 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.

[0463] In some embodiments, the scFv and/or V _H domain is derived from SJ25C1. SJ25C1 is a mouse IgG1 monoclonal antibody raised against Nalm-1 and -16 cells expressing human CD19 (Ling, NR, et al. (1987). Leucocyte typing III. 302). The SJ25C1 antibody contains the H1, H2 and H3 CDRs shown in SEQ ID NOs: 47-49, respectively, and the L1, L2 and L3 CDRs of the sequence shown in SEQ ID NOs: 44-46, respectively. The SJ25C1 antibody comprises a heavy chain variable region (V _H ) comprising the amino acid sequence from SEQ ID NO: 50 and a light chain variable region (V _L ) containing the amino acid sequence from SEQ ID NO: 51. In some embodiments, the svFv comprises a variable light chain containing the CDR L1 sequence specified in SEQ ID NO:44; CDR L2 specified in SEQ ID NO: 45; and CDR L3 specified in SEQ ID NO:46; and/or a variable heavy chain comprising an H1 CDR as set forth in SEQ ID NO:47, an H2 CDR as set forth in SEQ ID NO:48, and an H3 CDR as set forth in SEQ ID NO:49, 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 identical sequences with them. In some embodiments, the scFv comprises the heavy chain variable region from SJ25C1 as set forth in SEQ ID NO:50 and the heavy chain variable region from SJ25C1 as set forth in SEQ ID NO:51, 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 them. In some embodiments, the variable heavy and variable light chains are connected via a linker. In some embodiments, the linker is listed in SEQ ID NO:52. In some embodiments, the scFv contains, in the following order, V _H , a linker, and V _L . In some embodiments, the scFv contains, in the following order, V _L , a linker, and V _H . In some embodiments, the scFv contains the amino acid sequence shown in SEQ ID NO:53 or a sequence having 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.

[0464] In some aspects, a CAR contains a ligand-binding (eg, antigen) domain that binds or recognizes, eg, specifically binds, a universal tag, or a universal epitope. In some aspects, a binding domain may bind a molecule, tag, polypeptide, and/or epitope, which may be linked to another binding molecule (eg, an antibody or antigen-binding fragment) that recognizes an antigen associated with a disease or disorder. An exemplary label or epitope includes a dye (eg, fluorescein isothiocyanate) or biotin. In some aspects, the binding molecule (e.g., an antibody or antigen-binding fragment) is coupled to a label that recognizes an antigen associated with a disease or disorder, such as a tumor antigen, together with a modified cell expressing a CAR specific for the label to cause cytotoxicity or other effector functions of the modified cell. In some aspects, the specificity of a CAR for an antigen associated with a disease or disorder is provided by a labeled binding molecule (eg, an antibody), and different labeled binding molecules can be used to target different antigens. Exemplary CARs specific for a universal tag or universal epitope include those described in, for example, U.S. 9233125, WO 2016/030414, Urbanska et al., (2012) Cancer Res 72: 1844-1852, and Tamada et al., (2012). Clin Cancer Res 18:6436–6445.

[0465] In some embodiments, the CAR comprises a TCR-like antibody, such as an antibody or antigen-binding fragment (e.g. , scFv), that specifically recognizes an intracellular antigen, such as a tumor-associated antigen, presented on the cell surface in the form of a major histocompatibility complex (MHC )-peptide. In some embodiments, an antibody or antigen-binding portion thereof that recognizes the MHC-peptide complex can be expressed on cells as part of a recombinant receptor, such as an antigen receptor. Among antigen receptors, there are functional non-T cell receptor (TCR) antigen receptors such as chimeric antigen receptors (CARs). In some embodiments, a CAR containing an antibody or antigen-binding fragment having a TCR-like specificity against peptide-MHC complexes may also be referred to as a TCR-like CAR. In some embodiments, the CAR is a TCR-like CAR and the antigen is a truncated peptide antigen, such as a peptide antigen from an intracellular protein that, like TCR, performs cell surface recognition in the context of an MHC molecule. In some embodiments, an extracellular antigen-binding domain specific for an MHC-peptide complex from a TCR-like CAR is linked to one or more intracellular signaling components, in some aspects via linkers and/or a transmembrane domain(s). In some embodiments, such molecules can typically mimic or approximate a signal through a natural antigen receptor such as a TCR, and optionally a signal through such a receptor in combination with a co-stimulatory receptor.

[0466] Reference to "major histocompatibility complex" (MHC) refers to a protein, typically a glycoprotein, that contains a polymorphic peptide binding site or binding groove that can, in some cases, complex with peptide antigens from polypeptides, including peptide antigens , processed by the cellular apparatus. In some cases, MHC molecules may be displayed or expressed on the cell surface, including in the form of a complex with a peptide, ie. an MHC-peptide complex to present the antigen in a conformation recognized by an antigen receptor on T-cells, such as a TCR or TCR-like antibody. Typically, class I MHC molecules are heterodimers having a transmembrane α-chain, in some cases with three α-domains, and a non-covalently bound β2-microglobulin. As a rule, MHC class II molecules consist of two transmembrane glycoproteins, α and β, both of which tend to cross the membrane. The MHC molecule may include an effective MHC region, which contains the antigen-binding site or sites for peptide binding, and the sequences necessary for recognition by the corresponding antigen receptor. In some embodiments, MHC class I molecules deliver peptides formed in the cytosol to the cell surface, where the MHC-peptide complex is recognized by T cells, typically such as CD8 ⁺ T cells, but, in some cases, CD4 ⁺ T -cells. In some embodiments, class II MHC molecules deliver peptides generated in the vesicular system to the cell surface, where they are typically recognized by CD4 ⁺ T cells. Typically, MHC molecules are encoded by a group of linked loci collectively referred to as H-2 in the mouse and human leukocyte antigens (HLA) in humans. Thus, in general, human MHC may also be referred to as human leukocyte antigen (HLA).

[0467] The term "MHC-peptide complex" or "peptide-MHC complex", or variants thereof, refers to the complex or association of a peptide antigen and an MHC molecule, for example, typically through non-covalent interactions of the peptide in the binding groove or cleft of the MHC molecule. In some embodiments, the MHC-peptide complex is present or exposed on the cell surface. In some embodiments, the MHC-peptide complex can be specifically recognized by an antigen receptor, such as a TCR, TCR-like CAR, or antigen-binding fragments thereof.

[0468] In some embodiments, a peptide, such as a peptide antigen or an epitope from a polypeptide, can bind to an MHC molecule, for example, to be recognized by the antigen receptor. Typically, a peptide is derived from or derived from a fragment of a longer biological molecule, such as a polypeptide or protein. In some embodiments, the peptide is typically about 8 to about 24 amino acids in length. In some embodiments, the peptide is from or about 9 to 22 amino acids in length for recognition in complex with MHC class II. In some embodiments, a peptide is from or about 8 to 13 amino acids in length for recognition in a complex with MHC class I. In some embodiments, after recognition of the peptide in the context of an MHC molecule, e.g. such as a TCR or TCR-like CAR, provides or triggers an activation signal to a T cell that induces a T cell response, such as T cell proliferation, cytokine production, cytotoxic T cell response, or other response.

[0469] In some embodiments, the TCR-like antibody or antigen-binding fragment is known or can be obtained by known methods (see, for example, US Patent Application Publication No. US 2002/0150914; US 2003/0223994; US 2004/0191260; US 2006/0034850; US 2007/00992530; US20090226474; US20090304679; and International Patent Application Publication No. WO 03/068201).

[0470] In some embodiments, an antibody or antigen-binding portion thereof that specifically binds to the MHC-peptide complex can be obtained by immunizing the host with an effective amount of an immunogen containing the specific MHC-peptide complex. In some instances, the peptide from the MHC-peptide complex is an epitope of an antigen capable of binding to MHC, such as a tumor antigen, eg universal tumor antigen, myeloma antigen, or other antigen, as described below. In some embodiments, an effective amount of the immunogen is then administered to the host to elicit an immune response, wherein the immunogen retains its three-dimensional shape for a period of time sufficient to elicit an immune response against the three-dimensional representation of the peptide in the binding groove of the MHC molecule. Serum collected from the host is then analyzed to determine if the desired antibodies have been produced that recognize the three-dimensional representation of the peptide in the binding groove of the MHC molecule. In some embodiments, antibodies produced can be assessed to confirm that the antibody can distinguish between an MHC-peptide complex from a single MHC molecule, a single peptide of interest, and a complex of MHC and an irrelevant peptide. The desired antibodies can then be isolated.

[0471] In some embodiments, an antibody, or antigen-binding portion thereof, that specifically binds to the MHC-peptide complex can be generated using antibody library display methods, such as antibody phage libraries. In some embodiments, phage display libraries for mutated Fab, scFv, or other antibody forms can be generated, for example, in which library members are mutated at one or more CDR residues or multiple CDRs. See, for example, US Patent Application Publication No. US20020150914, US20140294841; and Cohen CJ. et al . (2003) J Mol. Recogniz . 16:324-332.

[0472] The term "antibody" is used in the present description in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) fragments of antibodies, including fragments of antigen-binding (Fab) fragments, F(ab') ₂ fragments, fragments Fab', Fv fragments, recombinant IgG (rIgG) fragments, heavy chain variable regions (V _H ) capable of specifically binding antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody , V _H H or V _NAR ) or fragments. The term includes genetically modified and/or otherwise modified forms of immunoglobulins such as intraantibodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugated antibodies, multispecific e.g. -scFv, tandem tri-scFv. Unless otherwise indicated, the term "antibody" should be understood to include functional antibody fragments thereof. The term also includes intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA and IgD. In some aspects, a CAR is a bispecific CAR, eg, containing two antigen-binding domains with different specificities.

[0473] In some embodiments, antigen-binding proteins, antibodies, and antigen-binding fragments thereof specifically recognize the antigen of a full-length antibody. In some embodiments, the heavy and light chains of the antibody may be full length or may be an antigen binding fragment (a Fab, F(ab')2, Fv or single chain Fv fragment (scFv)). In other embodiments, the antibody heavy chain constant region is selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, specifically selected from, for example, IgG1, IgG2, IgG3, and IgG4, more specifically, IgG1 (eg, human IgG1). In another embodiment, the antibody light chain constant region is selected from, for example, kappa or lambda, in particular kappa.

[0474] Among the presented antibodies are fragments of antibodies. "Antibody fragment" refers to a molecule, other than an intact antibody, that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; diabody; linear antibodies; heavy chain variable regions (V _H ), single chain antibody molecules such as scFv and single domain V _H antibodies; and multispecific antibodies formed from antibody fragments. In specific embodiments, the antibodies are single chain antibody fragments containing a heavy chain variable region and/or a light chain variable region, such as scFv.

[0475] The term "variable region" or "variable domain" refers to the heavy or light chain domain of an antibody involved in the binding of an antibody to an antigen. The heavy chain and light chain variable domains (V _H and V _L , respectively) of a native antibody typically have similar structures, with each domain containing four conserved framework regions (FRs) and three CDRs. (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007). A single V _H or V _L domain may be sufficient to provide antigen binding specificity. binding a particular antigen can be isolated using the V _H or V _L domain from an antibody that binds to the antigen to screen for a library of complementary V _L or V _H domains, respectively See, for example, Portolano et al., J. Immunol.150 :880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0476] Single domain antibodies (sdAb) are antibody fragments containing all or part of the heavy chain variable domain, or all or part of the light chain variable domain of an antibody. In specific embodiments, the single domain antibody is a human single domain antibody. In some embodiments, the CAR comprises an antibody heavy chain domain that specifically binds to an antigen, such as a cancer marker or cell surface antigen for the cell or disease to be targeted, e.g., a tumor cell or cancer cell, such as any of the antigens targets described in the present description or known. Exemplary single domain antibodies include sdFv, nanobody, V _H H or V _NAR .

[0477] Antibody fragments can be obtained by various methods, including, but not limited to, proteolytic cleavage of intact antibodies, as well as production by recombinant host cells. In some embodiments, the antibodies are recombinantly produced fragments, such as fragments containing arrangements not naturally occurring, such as fragments with two or more antibody regions or chains connected via synthetic linkers, e.g., peptide linkers, and/or which cannot be obtained by enzymatic cleavage of natural intact antibodies. In some embodiments, the antibody fragments are scFv.

[0478] A "humanized" antibody is an antibody in which all or substantially all of the CDR amino acid residues are derived from non-human CDRs and all or substantially all of the FR amino acid residues are derived from human FRs. The humanized antibody may optionally include at least a portion of the antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody refers to a variant of a non-human antibody that has been humanized, typically to reduce immunogenicity in humans, while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (eg, the antibody from which the CDR residues are derived), for example, to maintain or improve the specificity or affinity of the antibody.

[0479] Thus, in some embodiments, the chimeric antigen receptor, including TCR-like CARs, includes an extracellular fragment containing an antibody or antibody fragment. In some embodiments, the antibody or fragment comprises an scFv. In some aspects, an antibody or antigen-binding fragment can be obtained by screening a plurality, eg a library, antigen-binding fragments or molecules, eg by screening an scFv library for binding to a specific antigen or ligand.

[0480] In some aspects, a recombinant receptor, e.g., a chimeric antigen receptor, includes an extracellular fragment containing one or more ligand (e.g., antigen) binding domains, such as an antibody or fragment thereof, and one or more intracellular signaling regions or domains. (also referred to interchangeably as cytoplasmic signaling domains or regions). In some aspects, the recombinant receptor, eg CAR, further includes a spacer and/or a transmembrane domain or fragment. In some aspects, a spacer and/or a transmembrane domain may connect an extracellular fragment containing a ligand (eg, antigen) binding domain and intracellular signaling region(s) or domain(s).

[0481] 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 CH1/CL, and/or an Fc region. In some embodiments, the constant region or fragment is 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 an increased ability of the cell to respond after antigen binding, compared to no spacer. Exemplary spacers, such as hinge regions, include those described in International Patent Application Publication Number WO2014031687. In some examples, the spacer is either about 12 amino acids long, or no more than 12 amino acids long. Exemplary spacers include spacers having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids , about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and includes any integer between the end points of any of the listed ranges. In some embodiments, the spacer region is about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. In some embodiments, the spacer is less than 250 amino acids in length, less than 200 amino acids in length, less than 150 amino acids in length, less than 100 amino acids in length, less than 75 amino acids in length, less than 50 amino acids in length, less than less than 25 amino acids in length, less than 20 amino acids in length, less than 15 amino acids in length, less than 12 amino acids in length, or less than 10 amino acids in length. In some embodiments, the spacer is from or about 10 to 250 amino acids in length, 10 to 150 amino acids in length, 10 to 100 amino acids in length, 10 to 50 amino acids in length, 10 to 25 amino acids in length, 10 to 15 amino acids in length, 15 to 250 amino acids in length, 15 to 150 amino acids in length, 15 to 100 amino acids in length, 15 to 50 amino acids in length, 15 to 25 amino acids in length, 25 to 250 amino acids in length, 25 to 100 amino acids in length, 25 to 50 amino acids in length, 50 to 250 amino acids in length, 50 to 150 amino acids in length, 50 to 100 amino acids in length, 100 to 250 amino acids in length length, 100 to 150 amino acids in length, or 150 to 250 amino acids in length. Exemplary spacers include an IgG4 hinge alone, an IgG4 hinge associated with the CH2 and CH3 domains, or an IgG4 hinge associated with the CH3 domain. Illustrative spacers include, but are not limited to, those described in Hudecek et al. Clin. Cancer Res ., 19:3153 (2013), International Patent Publication No. WO2014031687, U.S. Patent No. 8822647 or patent application publication no. US2014/0271635.

[0482] In some embodiments, the constant region or fragment is from human IgG, such as IgG4 or IgG1. In some aspects, the spacer is a polypeptide spacer, such as one or more selected from one that: (a) contains or consists of all or part of an immunoglobulin hinge or a modified variant thereof, or contains about 15 amino acids or less, and does not contain an extracellular region of CD28 or an extracellular region of CD8, (b) contains or consists of all or part of an immunoglobulin hinge, optionally an IgG4 hinge or a modified variant thereof, and/or contains about 15 amino acids or less, and does not contain an extracellular region of CD28 or an extracellular region of CD8 or (c) is or is about 12 amino acids in length and/or contains or consists of all or part of an immunoglobulin hinge, optionally IgG4, or a modified variant thereof; or (d) contains or consists of the formula X ₁ PPX ₂ P (SEQ ID NO: 31), where X ₁ is glycine, cysteine or arginine, and X ₂ is cysteine or threonine. In some embodiments, the spacer has the sequence ESKYGPPCPPCP (as shown in SEQ ID NO: 1) and is encoded by the sequence as shown in SEQ ID NO: 2. In some embodiments, the spacer has the sequence as shown in SEQ ID NO: 3. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 4. In some embodiments, the constant region or fragment is 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 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 1, 3, 4 or 5 or 27-34.

[0483] This antigen recognition domain is typically associated with one or more components for intracellular signaling, such as signaling components mimicking activation through an antigen receptor complex, such as the TCR complex, in the case of CAR, and/or signaling via another cell surface receptor. Thus, in some embodiments, an antigen-binding component (e.g., an antibody) is associated with one or more transmembrane and intracellular signaling domains. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain naturally associated with one of the domains in the receptor, such as CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0484] The transmembrane domain, in some embodiments, is derived from either a natural or synthetic source. When the source is natural, the domain, in some aspects, is derived from any membrane-bound or transmembrane protein. Transmembrane regions include regions derived from (i.e., containing at least the transmembrane region(s) from) the T cell receptor alpha, beta, or zeta chain, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9 , CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137(4-1BB), CD 154. Alternatively, the transmembrane domain, in some embodiments, is synthetic. In some aspects, the synthetic transmembrane domain contains predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine can be found at each end of the synthetic transmembrane domain. In some embodiments, binding is via linkers, spacers, and/or transmembrane domain(s). In some aspects, the transmembrane domain contains a transmembrane fragment from CD28 or a variant thereof. The extracellular and transmembrane domains can be linked directly or indirectly. In some embodiments, the extracellular and transmembrane domain are linked via a spacer, such as any described herein.

[0485] Among the intracellular signaling domains are domains that mimic or approximate 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, such as a linker between 2 and 10 amino acids in length, such as a linker containing glycine and serine residues, such as a glycine-serine doublet, is present and forms a link between the transmembrane domain and the cytoplasmic transmitter signals by the CAR domain.

[0486] A receptor, such as a CAR, typically includes at least one intracellular signaling component or components. In some embodiments, the receptor comprises an intracellular component of the TCR complex, such as the CD3 TCR chain mediating T-cell activation and cytotoxicity, such as the CD3-zeta chain. Thus, in some aspects, the antigen binding fragment is associated with one or more signaling modules. In some embodiments, signaling modules include a CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In some embodiments, the receptor, eg, CAR, further comprises a portion of one or more additional molecules, such as an Fcγ receptor, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR or other chimeric receptor includes a chimeric molecule between the CD3-zeta (CD3-ζ) or Fcγ receptor and CD8, CD4, CD25, or CD16.

[0487] In some embodiments, upon binding to 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 responses of an immunocyte, such as a T cell modified to express CAR. For example, in some contexts, CAR induces T-cell function, such as cytolytic activity or T-helper cell activity, such as cytokine secretion or other factors. In some embodiments, a truncated fragment of the intracellular signaling domain of an antigen receptor component or co-stimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it signals an effector function. In some embodiments, the intracellular domain or signaling domains include cytoplasmic T-cell receptor (TCR) sequences, and in some aspects, also cytoplasmic co-receptor sequences that naturally act in conjunction with such receptors to initiate signaling upon recruitment of an antigen receptor. .

[0488] In the context of a natural TCR, full activation typically requires not only signaling through the TCR, but also a costimulatory signal.

[0489] Thus, in some embodiments, to stimulate full activation, a component for generating a secondary or costimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, the additional CAR is expressed on the same cell and provides a component for generating a secondary or co-stimulatory signal.

[0490] T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: sequences that initiate antigen-dependent primary activation via TCRs (primary cytoplasmic signaling sequences), and sequences that act in an antigen-independent manner to generate secondary or costimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components.

[0491] In some aspects, CAR includes a primary cytoplasmic signaling sequence that regulates the primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling known as immunoreceptor tyrosine activating motifs or ITAMs. Examples of ITAMs containing primary cytoplasmic signaling sequences include ITAMs derived from TCR-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-delta, CD3-epsilon, CD8, CD22, CD79a, CD79b, and CD66d. 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.

[0492] In some embodiments, the CAR includes a signaling domain and/or a transmembrane fragment of a costimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some aspects, the same CAR includes both an activating and a costimulatory component.

[0493] In some embodiments, the activating domain is included in one CAR while the co-stimulatory component is provided to other CARs recognizing a different antigen. In some embodiments, CARs include activating or stimulatory CARs, costimulatory CARs, both expressed on the same cell (see WO2014/055668). In some aspects, the cells include one or more stimulatory or activating CARs and/or a co-stimulatory CARs. In some embodiments, the cells further comprise inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine , 5(215) (2013), such as CARs recognizing an antigen other than the antigen associated with and/or specific for a disease or condition, whereby the activating signal delivered through the disease-targeted CAR is reduced or inhibited by binding the inhibitory CAR to its ligand, for example, to reduce non-specific effects.

[0494] In specific embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain associated with an intracellular CD3 domain (eg, CD3-zeta). In some embodiments, the intracellular signaling domain comprises chimeric CD28 and CD137 (4-1BB, TNFRSF9) co-stimulatory domains associated with the CD3-zeta intracellular domain.

[0495] In some embodiments, the CAR includes one or more, eg, two or more, costimulatory domains and an activating domain, eg, a primary activating domain, in the cytoplasmic portion. Exemplary CARs include the intracellular components CD3-zeta, CD28, and 4-1BB.

[0496] In some embodiments, the CAR or other antigen receptor further comprises a marker, such as a cell surface marker, that can be used to confirm transduction or modification of the cell to express the receptor, such as a truncated cell surface receptor variant, such as truncated EGFR (tEGFR ). In some aspects, the marker includes all or part (eg, a truncated form) of CD34, NGFR, or epidermal growth factor receptor (eg, tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding a linker sequence, such as a cleavable linker sequence, eg, T2A. For example, the marker, and optionally, the linker sequence, can be any of those as described in Published Patent Application No. WO2014031687. For example, the marker may be a truncated EGFR (tEGFR), optionally linked to a linker sequence, such as a cleavable T2A linker sequence. An exemplary polypeptide for a truncated EGFR (e.g., tEGFR) comprises an amino acid sequence as set forth in SEQ ID NO: 7 or 23, or an amino acid sequence having 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 23. An exemplary T2A linker sequence contains the amino acid sequence specified in SEQ ID NO: 6 or an amino acid sequence having 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.

[0497] In some embodiments, the marker is a molecule, such as a cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a fragment thereof. In some embodiments, the molecule is a non-self molecule, eg, a non-self protein, ie, a protein not recognized as "self" by the immune system of the host to which the cells will be adoptively transferred.

[0498] In some embodiments, the marker does not perform a therapeutic function and/or has no effect other than being used as a marker for genetic modification, for example, to select successfully modified cells. In other embodiments, the marker may be a therapeutic molecule or a molecule otherwise having some desired effect, such as a ligand for a cell that may be encountered in vivo , such as a co-stimulatory molecule or an immune checkpoint molecule to enhance and/or attenuate cell responses. after adoptive transfer and encounter with the ligand.

[0499] In some cases, CARs are referred to as first, second, third, or fourth generation CARs. In some aspects, a first generation CAR is a CAR that provides the sole primary stimulatory or activating signal, eg via a CD3-chain induced signal after antigen binding; in some aspects, a second generation CAR is a CAR providing such a signal and a costimulatory signal such as a CAR comprising intracellular signaling region(s) or domain(s) from one or more costimulatory receptors such as CD28, CD137 (4- 1BB), OX40 (CD134), CD27, DAP10, DAP12, NKG2D, ICOS and/or other costimulatory receptors; in some aspects, a third generation CAR is a CAR comprising multiple costimulatory domains from various costimulatory receptors, e.g., selected from CD28, CD137 (4-1BB), OX40 (CD134), CD27, DAP10, DAP12, NKG2D, ICOS and/or other costimulatory receptors; in some aspects, a fourth generation CAR is a CAR comprising three or more costimulatory domains from different costimulatory receptors, e.g., selected from CD28, CD137 (4-1BB), OX40 (CD134), CD27, DAP10, DAP12, NKG2D, ICOS, and /or other costimulatory receptors.

[0500] In some embodiments, the chimeric antigen receptor includes an extracellular fragment containing an antibody or antibody fragment. In some aspects, the chimeric antigen receptor includes an extracellular fragment containing an antibody or fragment, and an intracellular signaling domain. In some embodiments, the antibody or fragment comprises scFv and the intracellular domain contains ITAM. In some aspects, the intracellular signaling domain includes the zeta chain signaling domain of the CD3 zeta chain (CD3ζ). In some embodiments, the chimeric antigen receptor includes a transmembrane domain that binds an extracellular domain and an intracellular signaling domain. In some aspects, the transmembrane domain contains a CD28 transmembrane fragment. In some embodiments, the chimeric antigen receptor contains an intracellular domain from a costimulatory T cell molecule. The antigen-binding fragment of the extracellular domain and the transmembrane domain can be linked directly or indirectly. In some embodiments, the antigen-binding fragment of the extracellular domain and the transmembrane domain are linked via a spacer, such as any of those described herein. In some embodiments, the receptor comprises an extracellular fragment of the molecule from which the transmembrane domain is derived, such as an extracellular fragment of CD28. In some embodiments, the chimeric antigen receptor comprises an intracellular domain derived from a costimulatory T cell molecule, or a functional variant thereof, e.g., chimeric between a transmembrane domain and an ITAM-containing, e.g., CD3-zeta-derived, signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 41BB.

[0501] For example, in some embodiments, the CAR comprises an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains a CD28 transmembrane fragment or a functional variant thereof, and an intracellular signaling domain containing a CD28 signaling fragment or a functional variant thereof. and a signaling fragment of 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 fragment or a functional variant thereof, and an intracellular signaling domain containing a 4-1BB signaling fragment or a functional variant thereof, and signals fragment CD3-zeta or its functional variant. In some such embodiments, the receptor further comprises a spacer comprising a fragment of an Ig molecule, eg a human Ig molecule, such as an Ig hinge or variant thereof, eg an IgG4 hinge or variant thereof, eg a hinge-only spacer.

[0502] In some embodiments, the recombinant receptor transmembrane domain, e.g., CAR, is or includes a human CD28 transmembrane domain (e.g., Accession No. P10747.1) or a variant thereof, such as a transmembrane domain containing the amino acid sequence specified in SEQ ID NO: 8, or an amino acid sequence having 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 recombinant receptor fragment contains 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.

[0503] In some embodiments, the intracellular signaling component(s) of the recombinant receptor, e.g., CAR, comprises an intracellular costimulatory signaling domain from human CD28, or a functional variant or fragment thereof, such as a domain with an LL to GG substitution at positions 186 –187 native protein CD28. For example, an intracellular signaling domain may comprise an amino acid sequence as set forth in SEQ ID NO: 10 or 11, or an amino acid sequence having 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: 10 or 11. In some embodiments, the intracellular domain comprises an intracellular costimulatory signaling domain of 4 –1BB (e.g. (Access No. Q07011.1) or a functional variant or fragment thereof, such as the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence having 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: 12.

[0504] In some embodiments, the intracellular signaling domain of the recombinant receptor, e.g., CAR, comprises a human CD3-zeta stimulatory signaling domain or a functional variant thereof, such as the 112 AA cytoplasmic domain from human CD3ζ isoform 3 (Access No: P20963 .2), or the signaling domain of CD3-zeta, as described in US Patent No.: 7446190 or US Patent No. 8911993. 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 having 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.

[0505] In some aspects, the spacer comprises only an IgG hinge, e.g., only a hinge of IgG4 or IgG1, such as the hinge-only spacer indicated in SEQ ID NO: 1. In other embodiments, the spacer is or contains an Ig hinge , for example, an IgG4-derived hinge, optionally associated with CH2 and/or CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge associated with CH2 and CH3 domains, such as indicated in SEQ ID NO: 4. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, associated only with the CH3 domain, such as indicated in SEQ ID NO: 3. In some embodiments, the spacer is or contains a glycine-serine rich sequence or other flexible linker, such as known flexible linkers.

[0506] For example, in some embodiments, a CAR includes an antibody, e.g., an antibody fragment, including scFv, a spacer, such as a spacer containing an immunoglobulin molecule fragment, such as a hinge region and/or one or more constant regions from a heavy chain molecule, for example, an Ig hinge-containing spacer, a transmembrane domain containing all or part of the CD28-derived transmembrane domain, the CD28-derived intracellular signaling domain, and the 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 hinge-containing Ig spacers, a CD28-derived transmembrane domain, a 4-1BB-derived intracellular signaling domain, and a CD3-zeta-derived signaling domain. .

[0507] In some embodiments, the nucleic acid molecules encoding such CAR constructs further comprise a T2A ribosome slippage element encoding sequence and/or a tEGFR sequence, for example, downstream of the CAR encoding sequence. In some embodiments, the sequence encodes a T2A ribosome slip element as set forth in SEQ ID NO: 6, or an amino acid sequence having 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. In some embodiments, T cells expressing an antigen receptor (e.g., CAR) , can also be generated to express a truncated EGFR (EGFRt) as a non-immunogenic epitope for selection (eg, by introducing a construct encoding CAR and EGFRt separated by a T2A ribosome switch to express two proteins from the same construct), which can then be use as a marker for the detection of such cells (see, for example, US Patent No. 8802374). In some embodiments, the sequence encodes a tEGFR sequence as set forth in SEQ ID NO: 7 or 23, or an amino acid sequence having 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 23.

[0508] Recombinant receptors, such as CARs, expressed by cells administered to an individual will typically recognize or specifically bind a molecule that is expressed in, associated with, and/or specific for the disease or condition being treated, or associated cells. Upon specific binding to a molecule, such as an antigen, the receptor typically delivers an immunostimulatory signal, such as an ITAM-transmitted signal, to the cell, thereby stimulating an immune response that targets the disease or condition. For example, in some embodiments, the cells express a CAR that specifically binds to an antigen expressed by a cell or tissue affected by the disease or condition, or associated with the disease or condition.

B. TCR

[0509] In some embodiments, the invention relates to modified cells, such as T cells, that express a T cell receptor (TCR) or antigen-binding fragment thereof, that recognize a peptide epitope or T cell epitope of a target polypeptide, such as tumor antigen, viral or autoimmune protein.

[0510] In some embodiments, a "T-cell receptor" or "TCR" is a molecule that contains the α and β variable chains (also known as TCRα and TCRβ, respectively) or the γ and δ variable chains (also known as TCRα and TCRβ, respectively), or antigen-binding fragments thereof, and which is capable of specifically binding a peptide associated with an MHC molecule. In some embodiments, the TCR is in the αβ form. In general, TCRs that exist in the αβ and γδ forms tend to be structurally similar, but T cells expressing them may have distinct anatomical locations or functions. TCR can be detected on the cell surface or in soluble form. Typically, TCR is found on the surface of T cells (or T lymphocytes), where it is typically responsible for recognizing antigens associated with major histocompatibility complex (MHC) molecules.

[0511] Unless otherwise indicated, the term "TCR" should be understood to include full length TCRs as well as their antigen-binding portions or antigen-binding fragments. In some embodiments, the TCR is an intact or full length TCR, including TCR in the αβ form or in the γδ form. In some embodiments, the TCR is an antigen-binding fragment that is smaller than the full-length TCR, but that binds to a specific peptide linked to an MHC molecule, eg, binds to an MHC-peptide complex. In some cases, an antigen-binding portion or fragment of a TCR may contain only a portion of the structural domains of the full-length or intact TCR, but still be able to bind a peptide epitope, such as the MHC-peptide complex, to which the full-length TCR binds. In some cases, the antigen-binding fragment contains TCR variable domains, such as the TCR variable α chain and the 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.

[0512] In some embodiments, the TCR variable domains contain hypervariable loops, or complementarity determining regions (CDRs), which typically make a primary contribution to antigen recognition and binding capacity and specificity. In some embodiments, the CDR from a TCR, or a combination thereof, forms all or substantially all of the antigen-binding site of a given TCR molecule. The various CDRs in the TCR variable region are generally separated by framework regions (FRs), which tend to have less variability among TCR molecules compared to CDRs (see, e.g., Jores et al., Proc. Nat'l Acad. Sci U.S.A. 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 main CDR responsible for antigen binding or specificity, or is the most important of the three CDRs in 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 of the beta chain may interact with the C-terminus of the peptide. In some contexts, CDR2 has the strongest influence or is the primary CDR responsible for interacting with a portion of the MHC or recognizing a portion of the MHC from the MHC-peptide complex. In some embodiments, the β-chain variable region may contain an additional hypervariable region (CDR4 or HVR4) that is generally involved in superantigen binding rather than antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426 ).

[0513] In some embodiments, the TCR may also contain a constant domain, a transmembrane domain, and/or a short cytoplasmic tail (see, e.g., 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 from the CD3 complex involved in mediating signal transduction.

[0514] In some embodiments, the TCR chain contains one or more constant domains. For example, an extracellular fragment 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 Vβ; typically amino acids 1-116, based on the numbering of Kabat, 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, usually , chain positions 117–259 based on Kabat numbering, or a β chain constant domain, or Cβ, typically chain positions 117–295 based on Kabat) adjacent to the cell membrane. For example, in some cases, an extracellular TCR fragment formed by two chains contains two constant domains closest to the membrane and two variable domains distant from the membrane, where each of the variable domains contains a CDR. The TCR constant domain may contain short binding sequences in which the cysteine residue forms a disulfide bond, thus linking two TCR chains. In some embodiments, the TCR may have an additional cysteine residue on each of the α and β chains such that the TCR contains two disulfide bonds in the constant domains.

[0515] In some embodiments, the TCR chains contain a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain contains a cytoplasmic tail. In some cases, the structure allows the TCR to associate with other molecules like CD3 and its subunits. For example, a TCR containing constant domains with a transmembrane region can anchor a protein in the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex. The intracellular tails of the CD3 signaling subunits (eg, CD3γ, CD3δ, CD3ε, and CD3ζ chains) contain one or more immunoreceptor tyrosine activating motifs, or ITAMs, which are involved in the signaling ability of the TCR complex.

[0516] 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 single chain TCR 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, via a disulfide bond or disulfide bonds.

[0517] In some embodiments, TCRs can be derived 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 cellular sources are well known. In some embodiments, TCR-encoding nucleic acids can be obtained from a variety of sources, for example, by polymerase chain reaction (PCR) amplification of TCR-encoding nucleic acids within or after isolation from a given cell or cells, or by synthesizing a publicly available TCR DNA sequence.

[0518] In some embodiments, the TCR is derived from a biological source, such as cells, such as a T cell (eg, cytotoxic T cell), T cell hybridoma, or other publicly available source. In some embodiments, T cells can be obtained from in vivo isolated cells. In some embodiments, the TCR is a thymus-selected TCR. In some embodiments, the TCR is a neoepitope-restricted TCR. In some embodiments, the T cells may be a cultured hybridoma or T cell clone. In some embodiments, the TCR, or an antigen-binding portion thereof, can be generated synthetically from knowledge of the sequence of the TCR.

[0519] In some embodiments, the TCR is derived from a TCR identified or selected by screening a candidate TCR library against a target polypeptide antigen, or a target T-cell epitope from it. TCR libraries can be obtained by amplifying the Vα and Vβ repertoire from T cells isolated from an individual, including cells present in the PBMC, spleen, or other lymphoid organ. In some cases, T cells can be amplified from tumor infiltrating lymphocytes (TILs). In some embodiments, TCR libraries can be obtained from CD4+ or CD8+ cells. In some embodiments, the TCR may be amplified from a T cell source from a normal or healthy individual, i. normal TCR libraries. In some embodiments, the TCR may be amplified from a T cell source from a diseased individual, i.e. disease-associated TCR libraries. In some embodiments, degenerate primers are used to amplify the Vα and Vβ gene repertoire, for example by RT-PCR, in samples such as human T cells. In some embodiments, scTv libraries can be assembled from naïve Vα and Vβ libraries in which amplified products are cloned or harvested, separated by a linker. Depending on the source of the individual and the cells, the libraries may be specific for the HLA allele. Alternatively, in some embodiments, TCR libraries can be generated by mutagenesis or diversification of the parent or framework TCR molecule. In some aspects, TCR is subjected to directed evolution, for example, through mutagenesis, for example, chain α or β. In some aspects, specific residues within the CDR of the TCR are changed. In some embodiments, the 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, eg, those present on antigen-specific T cells, may be selected, eg, by binding activity, eg, specific affinity or avidity for the antigen.

[0520] In some embodiments, the TCR or antigen-binding portion thereof is modified or engineered. In some embodiments, guided evolution methods are used to generate TCRs with altered properties, such as higher affinity for a specific MHC-peptide complex. In some embodiments, directed evolution is performed 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 USA, 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, the display methods include constructing or modifying a known, original, or reference TCR. For example, in some cases, wild-type TCRs can be used as a template to generate mutated TCRs in which one or more CDR residues are mutated and mutants with a desired altered property, such as higher affinity for the desired target antigen, are selected.

[0521] In some embodiments, the target polypeptide peptides for use in the production or production of TCRs of interest are known or can be easily identified by one of skill in the art. In some embodiments, peptides suitable for use in generating TCRs or antigen-binding fragments can be determined based on the presence of an HLA-restricted motif in a target polypeptide of interest, such as the target polypeptide described below. In some embodiments, peptides are identified using computer predictive models known to those skilled in the art. In some embodiments, 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 thus is a suitable choice of MHC antigen to be used to generate a TCR or other MHC-peptide binding molecule.

[0522] HLA-A0201 binding motifs and cleavage sites for proteasomes and immunoproteasomes using computer predictive models are known to those skilled in the art. For prediction of MHC class I binding sites, 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)

[0523] In some embodiments, the TCR, or an antigen-binding fragment thereof, may be a recombinantly produced natural protein or a mutant form thereof in which one or more properties, such as binding characteristics, are altered. In some embodiments, the TCR may be from one of a variety of animal species such as a human, mouse, rat, or other mammal. The TCR may be in cell-bound or soluble form. In some embodiments, for the purposes of the presented methods, the TCR is in cell-bound form, expressed on the cell surface.

[0524] In some embodiments, the TCR is a full length TCR. In some embodiments, the TCR is an antigen binding fragment. 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.

[0525] In some embodiments, the TCR contains a sequence corresponding to a transmembrane sequence. In some embodiments, the TCR contains a sequence corresponding to a cytoplasmic sequence. 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 signaling domains resulting in the formation of an active TCR on the surface of the T cell. In some embodiments, the TCR is expressed on the cell surface.

[0526] In some embodiments, the dTCR comprises a first polypeptide, wherein the sequence corresponding to the TCR α-chain variable region sequence is fused to the N-terminus of the sequence corresponding to the TCR α-chain extracellular constant region sequence, and a second polypeptide, wherein the sequence corresponding to the sequence variable region of the TCR β chain, fused to the N-terminus of a sequence corresponding to the extracellular sequence of the TCR β chain constant region, where the first and second polypeptides are linked via a disulfide bond. In some embodiments, the bond may correspond to a natural inter-chain disulfide bond present in naturally occurring αβ dimeric TCRs. In some embodiments, inter-chain disulfide bonds are not present in native TCR. For example, in some embodiments, one or more cysteine residues can be included in the extracellular constant region sequences of a pair of dTCR polypeptides. In some cases, both natural and non-natural disulfide bonds may be desirable. In some embodiments, the TCR contains a transmembrane sequence for anchoring in the membrane.

[0527] In some embodiments, the dTCR comprises a TCR α-chain containing an α variable domain, an α constant domain and a first dimerization motif attached to the C-terminus of an α constant domain, and a TCR β-chain containing a β variable domain, a β constant domain and a first dimerization motif attached to the C-terminus of the β constant domain, where the first and second dimerization motifs readily interact to form a covalent bond between the amino acid in the first dimerization motif and the amino acid in the second dimerization motif, linking together α– TCR chain and TCR β-chain.

[0528] In some embodiments, the TCR is an scTCR. Typically, scTCRs can be generated using methods known to those of skill in the art. See, for example, Soo Hoo, WF 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); International PCT Publications No. WO 96/13593, WO 96/18105, WO99/60120, WO99/18129, WO 03/020763, WO2011/044186; and Schlueter, CJ et al. J. Mol. Biol. 256, 859 (1996). In some embodiments, the scTCR contains introduced non-natural interchain disulfide bonds to facilitate association of the TCR chains (see, for example, PCT International Publication No. WO 03/020763). In some embodiments, the scTCR is a non-disulfide truncated TCR in which heterologous leucine zippers fused at its C-terminus facilitate chain association (see, for example, PCT International Publication No. WO99/60120). In some embodiments, the scTCR comprises a TCRα variable domain covalently linked to a TCRβ variable domain via a peptide linker (see, for example, PCT International Publication No. WO99/18129).

[0529] In some embodiments, the scTCR comprises a first fragment consisting of an amino acid sequence corresponding to the TCR α chain variable region, a second fragment consisting of an amino acid sequence corresponding to the TCR β chain variable region sequence, fused to the N-terminus of the amino acid sequence , corresponding to the extracellular sequence of the constant domain of the TCR β-chain, and a linker sequence connecting the C-terminus of the first fragment with the N-terminus of the second fragment.

[0530] In some embodiments, the scTCR comprises a first fragment consisting of an α-chain variable region sequence fused to the N-terminus of an extracellular α-chain constant domain sequence and a second fragment consisting of a β-chain variable region sequence fused to The N-terminus of an extracellular constant and transmembrane β-chain sequence, and optionally a linker sequence linking the C-terminus of the first fragment to the N-terminus of the second fragment.

[0531] In some embodiments, the scTCR comprises a first fragment consisting of the TCR β-chain variable region sequence fused to the N-terminus of the extracellular β-chain constant domain sequence, and a second fragment consisting of the α-chain variable region sequence fused with the N-terminus of the sequence of the extracellular constant and transmembrane α-chain sequences, and, optionally, a linker sequence linking the C-terminus of the first fragment to the N-terminus of the second fragment.

[0532] In some embodiments, the scTCR linker binding the first and second TCR fragments can be any linker capable of forming a single polypeptide chain while retaining TCR binding specificity. In some embodiments, the linker sequence may, for example, have the formula -P-AA-P-, where P is a proline and AA is an amino acid sequence, where the amino acids are glycine and serine. In some embodiments, the first and second fragments are paired such that their variable region sequences are oriented for such binding. Thus, in some cases, the linker is long enough to span the distance between the C-terminus of the first fragment and the N-terminus of the second fragment, or vice versa, but not too long to block or reduce scTCR binding to its target ligand. In some embodiments, the linker may contain from or about 10 to 45 amino acids, such as 10 to 30 amino acids, or 26 to 41 amino acid residues, such as 29, 30, 31, or 32 amino acids. In some embodiments, the linker has the formula -PGGG-(SGGGG)5-P-, where P is proline, G is glycine, and S is serine (SEQ ID NO: 16). In some embodiments, the linker has the sequence GSADDAKKDAAKKDGKS (SEQ ID NO: 17)

[0533] In some embodiments, the scTCR contains a covalent disulfide bond linking a residue from the immunoglobulin region of the α chain constant domain to a residue from the immunoglobulin region of the β chain constant domain. In some embodiments, no interchain disulfide bond is present in natural TCR. For example, in some embodiments, one or more cysteine residues can be included in the extracellular constant region sequences of the first and second scTCR polypeptide fragments. In some cases, both natural and non-natural disulfide bonds may be desirable.

[0534] In some embodiments, the implementation of dTCR or scTCR, containing introduced interchain disulfide bonds, natural disulfide bonds are not present. In some embodiments, one or more of the naturally occurring cysteine residues forming natural interchain disulfide bonds are replaced with another residue, such as serine or alanine. In some embodiments, the introduced disulfide bond may be formed by mutagenesis of non-cysteine residues in the first and second fragments to cysteine. Exemplary unnatural TCR disulfide bonds are described in International PCT Publication No. WO2006/000830.

[0535] In some embodiments, the TCR or antigen-binding fragment thereof has an affinity with an equilibrium binding constant for the target antigen between or between about 10 ^-5 and 10 ^-12 M, and all of its individual values and ranges. In some embodiments, the target antigen is an MHC-peptide or ligand complex.

[0536] In some embodiments, the TCR-encoding nucleic acid or nucleic acids, such as the α and β chains, can be amplified by PCR, cloning, or other suitable methods 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 vectors designed for propagation and propagation, or for expression, or both, such as plasmids and viruses.

[0537] In some embodiments, the vector may be a vector from the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden) or the pEX series (Clontech, Palo Alto, Calif.). In some cases, bacteriophage vectors such as λG10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 can 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.

[0538] In some embodiments, recombinant expression vectors can be generated using standard recombinant DNA techniques. In some embodiments, the vectors may contain regulatory sequences, such as transcription and translation initiation and termination codons, that are specific for the type of host (e.g., bacteria, fungus, plant, or animal) to which the vector is intended to be administered, where appropriate and taking whether the vector is based on DNA or RNA. In some embodiments, the vector may contain a non-natural promoter operably linked to a nucleotide sequence encoding a TCR or an antigen-binding fragment (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 mouse stem cell virus. Other promoters known to the person skilled in the art are also contemplated.

[0539] In some embodiments, to obtain a vector encoding a TCR, the α and β chains are PCR amplified with 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 resulting α and β chains are inserted into a retroviral, eg, lentiviral, vector.

C. Multiple targeting

[0540] In some embodiments, cells and methods are incorporated into multiple targeting strategies, such as expressing two or more genetically modified receptors on a cell, where each recognizes the same or different antigen, and typically each includes a different intracellular signaling component. Such multiple targeting methods are described, for example, in International Patent Application Publication No.: WO 2014055668 A1 (describing combinations of activating and costimulatory CARs, e.g. targeting two different antigens, present individually on non-targeted, e.g. on cells affected by the disease or condition being treated) and Fedorov et al., Sci. Transl. Medicine, 5(215) (2013) (describes cells expressing both an activating and an inhibitory CAR, such as CARs, in which the activating CAR binds to a single antigen expressed on both normal or disease-free cells and diseased or diseased cells). the condition to be treated and the inhibitory CAR binds to another antigen expressed only on normal cells or cells that are undesirable to be treated).

[0541] For example, in some embodiments, the cells include a receptor that expresses a first genetically modified antigen receptor (e.g., CAR or TCR) that is capable of inducing an activating signal for 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 genetically modified antigen receptor (e.g., CAR or TCR), e.g., a chimeric costimulatory receptor, that is capable of inducing a costimulatory signal for an immunocyte, typically upon 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.

[0542] In some embodiments, the first and/or second genetically modified antigen receptor (eg, CAR or TCR) is capable of inducing an activating signal for the cell. In some embodiments, the receptor includes an intracellular signaling component containing ITAM or ITAM-like motifs. In some embodiments, activation induced by the first receptor comprises signal transduction or a change in protein expression in the cell resulting in the initiation of an immune response such as ITAM phosphorylation and/or initiation of an ITAM-mediated signaling cascade, formation of an immunological synapse and/or clustering of a molecule around an 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.

[0543] In some embodiments, the first and/or second receptor includes the intracellular signaling domains of costimulatory receptors such as CD28, CD137 (4-1 BB), OX40, CD27, DAP10, DAP12, NKG2D, ICOS, and/or other costimulatory receptors. In some embodiments, the first and second receptors comprise intracellular signaling domains of costimulatory receptors that are different. In one embodiment, the first receptor contains the costimulatory signaling region of CD28 and the second receptor contains the costimulatory signaling region 4-1BB, or vice versa.

[0544] 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.

[0545] In some embodiments, the first receptor comprises an intracellular signaling domain containing ITAM or ITAM-like motifs, and the second receptor comprises an intracellular signaling domain of a costimulatory receptor. A costimulatory signal in combination with an activating signal induced in the same cell is what leads to an immune response, such as a reliable and long lasting immune response such as increased gene expression, cytokine secretion and other factors, and mediated T- cell effector functions, such as killing cells.

[0546] In some embodiments, neither binding of the first receptor alone nor binding of the second receptor alone induces a reliable immune response. In some aspects, if only one receptor is bound, the cell becomes tolerant or non-responsive to the antigen, or inhibited and/or not induced to proliferate or secrete factors or perform effector functions. In some such embodiments, however, when a plurality of receptors are associated, for example, upon encountering a cell expressing the first and second antigens, the desired response is achieved, such as complete immune activation or stimulation, for example, as shown by the secretion of one or more cytokines, proliferation, persistence and/or performance of an immunoeffector function such as cytotoxic killing of the target cell.

[0547] In some embodiments, two receptors induce, respectively, an activating and inhibitory signal for a cell such that binding of one receptor to its antigen activates the cell or induces a response, however, binding of the second inhibitory receptor to its antigen induces a signal that suppresses or attenuates such a response. Examples are combinations of activating CARs and inhibitory CARs, or iCARs. For example, one can use such a method in which an activating CAR binds to an antigen that is expressed in a disease or condition, but which is also expressed on normal cells, and an inhibitory receptor binds to a separate antigen, which is expressed on normal cells, but not on diseased cells. or state of the cells.

[0548] In some embodiments, a multi-targeting method is used when an antigen associated with a particular disease or condition is expressed on a disease-free cell and/or is expressed on a self-modified cell, either transiently (e.g., upon stimulation in combination with genetic modification), or permanently. In such cases, specificity, selectivity and/or efficacy can be improved by requiring two separate and individually specific antigen receptors to bind.

[0549] In some embodiments, a plurality of antigens, eg, the first and second antigens, are expressed on the cell, tissue, or disease or condition being targeted, eg, on 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 that is undesirable to be targeted by cell therapy, such as a normal or disease-free cell or tissue, and/or self-modified cells. In such embodiments, through the need to bind multiple receptors to achieve a cell response, specificity and/or efficacy is achieved.

D. Cells and preparation of cells for modification

[0550] the Present invention also relates to cells, such as cells containing a modified recombinant receptor, such as described in the present description. The present invention also relates to populations of such cells, compositions containing such cells and/or enriched in such cells, for example, those in which cells expressing a recombinant receptor, for example, a chimeric receptor, are at least 50, 60, 70, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the total number of cells in the composition or of a specific cell type such as T cells or CD8+ or CD4+ cells. Among the compositions are pharmaceutical compositions and formulations for administration, for example, for adoptive cell therapy. The present invention also relates to therapeutic methods for administering cells and compositions to individuals, for example, patients, for example, in accordance with the presented methods.

[0551] Thus, the present invention also relates to genetically modified cells expressing recombinant receptors such as CAR. 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, are cells of the immune system, such as cells of innate or adaptive immunity, for example, myeloid or lymphoid cells, including lymphocytes, typically T cells and/or or NK cells. Other exemplary cells include stem cells such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells are typically primary cells, such as cells isolated directly from the individual and/or isolated from the individual and frozen. In some embodiments, the cells include one or more subgroups of T cells or other cell types such as complete populations of T cells, CD4+ cells, CD8+ cells, and subpopulations thereof, such as subpopulations defined by function, activation state, maturity, potential differentiation, multiplication, recycling, localization and/or persistence ability, antigenic specificity, type of antigen receptor, presence in a particular organ or compartment, cytokine secretion marker or profile, and/or degree of differentiation. In relation to the individual to be treated, the cells may be allogeneic and/or autologous. Among the methods included are methods of using the product ready for immediate use. In some aspects, such as for immediate use technologies, the cells are pluripotent and/or multipotent, eg stem cells such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from an individual, preparing, processing, culturing and/or modifying them as described herein, and reintroducing them to the same patient, before or after cryopreservation.

[0552] Among the subtypes and subpopulations of T cells and/or CD4+ and/or CD8+ T cells, there are naïve T cells (T _N ), effector T cells (T _EFF ), memory T cells and their subtypes, such as memory T cell stem (T _SCM ), memory T cell (T _CM ), effector memory T cell (T _EM ), or terminally differentiated effector memory T cell, tumor infiltrating lymphocyte (TIL), immature T cell , mature T-cells, helper T-cells, cytotoxic T-cells, mucosal-associated invariant T-cells (MAIT) cells, natural and adaptive regulatory T-cells (T-reg) cells, T-helper cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.

[0553] 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.

[0554] In some embodiments, the cells include one or more nucleic acids introduced through genetic modification and thus express recombinant or genetically modified 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 the cell being modified, and /or in the organism from which such a cell originates. In some embodiments, the nucleic acids are non-natural, such as a nucleic acid not found in nature, including a nucleic acid containing chimeric combinations of nucleic acids encoding different domains from many different cell types.

[0555] In some embodiments, the manufacture, formation, or preparation of a cell therapy agent, for example, cell therapeutic compositions containing cells expressing the recombinant receptor, can be carried out in a manner that includes one or more additional processing steps, such as steps for activation or stimulating, transducing, culturing, expanding, washing, suspending, diluting, concentrating and/or formulating cells. In some embodiments, methods for generating or obtaining a cell therapy agent include isolating cells from an individual, preparing, processing, culturing under one or more stimulating conditions. In some embodiments, the method includes processing steps in the order that: cells, eg, primary cells, are first isolated, eg, harvested or separated, from a biological sample; the selected cells are incubated with the viral vector particles for transduction, optionally after a step of stimulating the isolated cells in the presence of a stimulating reagent; culturing the transduced cells, for example for cell expansion; formulating the transduced cells into a composition; and introducing the composition into the provided biomedical vessel. In some embodiments, the resulting modified cells are reintroduced to the same individual, before or after cryopreservation.

[0556] In some embodiments, one or more processing steps may include one or more of (a) washing the biological sample containing cells (e.g., whole blood sample, buffy coat sample, peripheral blood mononuclear cell (PBMC) sample, unfractionated T cells, lymphocyte sample, leukocyte sample, apheresis product, or leukapheresis product), (b) isolating, e.g., selecting, a desired subgroup or population of cells (e.g., CD4+ and/or CD8+ T cells) from the sample, e.g., by incubation cells with a selective or immunoaffinity reagent for separation based on immunoaffinity; (c) activating and/or stimulating cells by exposing the cells to stimulating conditions and/or stimulating reagents, which can be performed before, during and/or after incubation of cells with viral vector particles, (d) incubation of isolated, for example, selected cells, with viral vector particles, e.g. cell transduction, (e) culturing, culturing or expanding the cells, e.g. using the methods as described, and (f) preparing the composition of the transduced cells, e.g. in a pharmaceutically acceptable buffer, cryopreservant or other suitable medium . In some embodiments, cells from a PBMC sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product are cryopreserved and then thawed prior to any steps for isolation, selection, incubation, transduction, transfection, culture, propagating and/or obtaining cell composition.

[0557] In some embodiments, one or more additional washing steps or suspension steps, for example, to dilute, concentrate, and/or exchange cell buffer, can also be performed before or after any of the above steps. In some aspects, the resulting modified cell composition is introduced into one or more of the provided biomedical culture vessels.

[0558] In some embodiments, one, more or all of the steps in preparing cells for clinical use, such as in adoptive cell therapy, are performed without exposing the cells to non-sterile conditions and without the need for a sterile room or cabinet. In some embodiments of such a method, cells are isolated, separated or selected, transduced, washed, optionally activated or stimulated, and constituted, all within a closed system. In some aspects of such a method, cells are removed from a closed system and introduced into one or more biological material vessels. In some embodiments, the methods are carried out in automatic mode. In some embodiments, one or more of the steps is carried out outside of a closed system or device.

[0559] In some embodiments, a closed system is used to conduct one or more of the other processing steps of a method to make, form, or obtain a cell therapy agent. In some embodiments, one or more or all of the processing steps, e.g., isolation, selection and/or enrichment, processing, incubation in combination with transduction and modification steps, and formulation steps are carried out using a system, device, or apparatus in an integrated or stand-alone system, and/or in automated or programmed mode. In some aspects, the system or apparatus includes a computer and/or a computer program in communication with the system or apparatus, allowing the user to program, control, evaluate the outcome and/or correct various aspects of the stages of processing, isolation, modification and formulation. In one example, the system is as described in International Patent Application Publication Number WO2009/072003, or US 20110003380 A1. In one example, the system is the system as described in International Publication Number WO2016/073602.

[0560] In some embodiments, obtaining modified cells includes one or more stages of culturing and/or obtaining. Cells for administration of a recombinant receptor, eg, CAR, can be isolated from a sample, such as a biological sample, eg, a sample derived from or derived from an individual. In some embodiments, the individual from which the cell is isolated is an individual who has a disease or condition, or who is in need of cell therapy, or who will be subjected to cell therapy. The subject, 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 modified.

[0561] Accordingly, the cells, in some embodiments, are primary cells, eg, primary human cells. Samples include tissue, fluid, and other samples taken directly from the individual, as well as samples obtained from one or more processing steps such as separation, centrifugation, genetic modification (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 that has been processed. Biological samples include, but are not limited to, samples of body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissues and organs, including processed samples derived from them.

[0562] In some aspects, the sample from which cells are obtained or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Illustrative specimens include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosal-associated lymphoid tissue, spleen, other lymphoid tissue, 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.

[0563] In some embodiments, the 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, or pig.

[0564] In some embodiments, cell isolation includes one or more steps of cell generation and/or non-affinity-based cell separation. 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 desired components, lyse, or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more properties such as density, adherence properties, size, sensitivity, and/or resistance to particular components.

[0565] In some examples, cells from the circulating blood of an individual are obtained, 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.

[0566] In some embodiments, blood cells collected from an individual are washed, for example, to remove a plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all of the divalent cations. In some aspects, the washing step is performed using a semi-automated "flow-through" centrifuge (eg, Cobe 2991 Cell Processor, Baxter), according to the manufacturer's instructions. In some aspects, the washing step is carried out in a centrifuge chamber, for example, manufactured and sold by Biosafe SA, including chambers for use with the Sepax® and Sepax® 2 system, including A-200/F and A-200 centrifuge chambers, according to with manufacturer's instructions. In some aspects, the washing step is carried out by flow filtration along the flow (TFF) in accordance with the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as Ca++/Mg++ free PBS. In specific embodiments, components of the blood cell sample are removed and the cells directly resuspended in culture medium.

[0567] In some embodiments, the methods include methods for separating cells based on density, such as obtaining leukocytes from peripheral blood by lysing erythrocytes and centrifuging in a Percoll or Ficoll gradient.

[0568] 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 can be used. In some embodiments, the separation is a separation based on affinity or immunoaffinity. For example, isolation, in some aspects, includes separation of cells and cell populations based on the expression or level of expression in the cells of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, which are typically followed by washing and separating cells that have bound to the antibody or binding partner from cells that have not bound to the antibody or binding partner.

[0569] Such separation steps can be based on positive selection, in which cells that have bound to the reagents are retained for later use, and/or negative selection, in which cells that have not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for later use. In some aspects, negative selection can be particularly useful when no antibody is available that specifically identifies a cell type in a heterogeneous population, so that separation is best done based on markers expressed by cells other than the desired population.

[0570] Separation need not result in 100% enrichment or removal of a particular population of cells or cells expressing a particular marker. For example, positive selection or enrichment of cells of a particular type, such as cells 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 the marker. Similarly, negative selection, removal or depletion of cells of a particular type, such as cells expressing a marker, refers to a decrease in the number or percentage of such cells, but should not result in the complete removal of all such cells.

[0571] In some examples, multiple cycles of separation steps are performed, where a positively or negatively selected fraction from one stage is 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, such as by incubating the cells with multiple antibodies or binding patterns, each specific for the marker targeted for negative selection. Similarly, multiple cell types can be simultaneously positively selected by incubating cells with multiple antibodies or binding patterns expressed on different cell types.

[0572] For example, in some aspects, specific T cell subsets, such as cells positive for or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and /or CD45RO+ T cells are isolated by positive or negative selection methods.

[0573] For example, CD3+, CD28+ T cells can be positively selected using CD3/CD28 conjugated magnetic beads (eg, DYNABEADS® M-450 CD3/CD28 T Cell Expander).

[0574] In some embodiments, isolation is carried out by enriching a particular cell population through positive selection, or depleting a particular cell population through negative selection. In some embodiments, positive or negative selection is performed by incubating cells with one or more antibodies or other binders that specifically bind to one or more surface markers expressed or expressed (marker+) at a relatively higher level (marker ^high ) at positively or negatively selected cells, respectively.

[0575] In some embodiments, T cells are separated from the PBMC sample by negative selection for markers expressed on non-T cells, such as B cells, monocytes, or other leukocytes such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ T helper cells from CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or relatively highly expressed on one or more subpopulations of naive T cells, memory T cells, and/or effector T cells.

[0576] In some embodiments, CD8+ cells are further enriched or depleted for naïve cells, central memory cells, effector memory cells, and/or central memory stem cells, e.g., through positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, central memory TCM (TCM) enrichment is performed to increase efficacy, e.g., to improve long-term survival, expansion, and/or engraftment after administration, which, in some aspects, is particularly potent in such subpopulations. See Terakuraet 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 improves efficacy.

[0577] In some embodiments, memory T cells are present in both CD62L+ and CD62L– subgroups of CD8+ peripheral blood lymphocytes. PBMC can be enriched or depleted in CD62L-CD8+ and/or CD62L+CD8+ fractions, for example using anti-CD8 and anti-CD62L antibodies.

[0578] In some embodiments, central memory TCM (TCM) enrichment is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8+ population enriched in TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment of cells expressing CD62L. In one aspect, enrichment for central memory T cells (TCM) is performed starting with a negative cell fraction selected based on CD4 expression, which is negatively selected based on CD14 and CD45RA expression, and positively selected based on CD62L. Such selections, in some aspects, are carried out simultaneously, and in other aspects, they are carried out sequentially, in any order. In some aspects, the same selection step based on CD4 expression is used to obtain a population or subset of CD8+ cells, is also used to obtain a population or subset of CD4+ cells, such that both positive and negative fractions after separation based on CD4 are retained and used. in subsequent steps of the methods, optionally after one or more additional positive or negative selection steps.

[0579] In a specific example, a PBMC sample or a sample of other white blood cells is subjected to CD4+ cell screening, in which both negative and positive fractions are retained. The negative fraction is then subjected to negative selection based on CD14 and CD45RA or ROR1 expression, and positive selection based on a central memory T cell marker such as CD62L or CCR7, where positive and negative selections are made in any order.

[0580] CD4+ T helper cells are sorted into naive cells, central memory cells, and effector cells by identifying cell populations having cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, the naïve CD4+ T lymphocytes are CD45RO–, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, the central CD4+ memory cells are CD62L+ and CD45RO+. In some embodiments, the CD4+ effector cells are CD62L- and CD45RO-.

[0581] In one example, to enrich CD4+ cells through negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is associated with a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow cell separation for positive and/or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation methods (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In vitro and In vivo , p 17–25 Edited by: SA Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ).

[0582] In some aspects, two or more selection steps can be performed sequentially. For example, the cell sample or composition to be separated is subjected to CD8+ cell selection, in which both negative and positive fractions are retained. The CD8 negative fraction can then be subjected to a selection of CD4+ cells. In some aspects, the cell sample or composition to be separated is subjected to CD4+ cell selection, wherein both the negative and positive fractions are retained, and the CD4 negative fraction can be subjected to CD8+ cell selection. Exemplary cell selection methods are described in International Patent Publication Numbers WO2015157384 and/or WO 2015/164675, the entire contents of which are incorporated by reference, and all or part of them can be used in combination with the methods described herein.

[0583] In some aspects, the cell sample or composition to be separated is incubated with a small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as beads Dynalbeads or MACS). A magnetically responsive material, such as a particle, is typically 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 cell population that is desired to be isolated. , for example, which it is desirable to select negatively or positively.

[0584] In some embodiments, the magnetic particle or bead comprises a magnetically responsive material associated with a specific binding member, such as an antibody or other binding partner. There are many well known magnetically responsive materials used in magnetic separation processes. Suitable magnetic particles include those described in Molday, US Patent No. 4452773, and in the description of the European patent application EP 452342 B, the contents of which are thus given as a reference. Other examples are colloidal sized particles such as those described in Owen, U.S. Patent No. 4795698, and Liberti et al., US Patent No. 5200084.

[0585] Incubation is generally carried out under conditions in which antibodies or binding patterns, or molecules such as secondary antibodies or other reagents that specifically bind to such antibodies or binding patterns that are attached to the magnetic particle or bead, specifically bind with cell surface molecules, if they are present on the cells in the sample.

[0586] In some aspects, the sample is placed in a magnetic field, and cells having associated magnetically responsive or magnetizable particles can be associated with a magnet and separated from unlabeled cells. For positive selection, cells that are associated with the magnet are retained; for negative selection, cells that are not bound (unlabeled cells) are kept. In some aspects, the combination of positive and negative selection is carried out during the same selection stage, where the positive and negative fractions are stored and further processed or subjected to further separation steps.

[0587] In specific embodiments, magnetically responsive particles are coated with primary antibodies or other binding patterns, secondary antibodies, lectins, enzymes, or streptavidin. In specific embodiments, the magnetic particles are attached to cells by coating with primary antibodies specific for one or more markers. In specific embodiments, cells, rather than beads, 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 beads are added. In specific embodiments, streptavidin-coated magnetic particles are used in combination with biotinylated primary or secondary antibodies.

[0588] In some embodiments, the magnetically responsive particles remain attached to cells that are subject to subsequent incubation, culture, and/or modification; in some aspects, the particles remain attached to the cells for administration to the patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of competitive unlabeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, and the like. In some embodiments, the magnetizable particles are biodegradable.

[0589] In some embodiments, selection based on affinity is performed by activated cell magnetic sorting (MACS) (Miltenyi Biotec, Auburn, CA). Activated Cell Magnetic Sorting (MACS) systems are capable of high frequency selection of cells having magnetized particles attached to them. In specific embodiments, the MACS operates in a mode where non-target and target molecules are sequentially eluted following the application of an external magnetic field. That is, cells attached to magnetized particles are held in place while non-attached molecules are eluted. Then, upon completion of the first elution step, the molecules trapped in the magnetic field and protected from elution are released in some way so that they can be eluted and isolated. In specific embodiments, non-target cells are labeled and depleted from a heterogeneous population of cells.

[0590] In specific embodiments, the isolation or separation is carried out using a system, device, or apparatus that performs one or more of the steps of the isolation, cell production, separation, processing, incubation, culture, and/or formulation methods. In some aspects, the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user manipulation, and/or contamination. In one example, the system is the system as described in International Patent Application, publication number WO2009/072003, or US 20110003380 A1. In some aspects, the apheresis or leukapheresis product, or a sample derived therefrom, is processed and/or isolated or selected using a system, device, apparatus and/or method as described in International Patent Application Publication number WO2016/073602 or US 2016/ 0122782, the full content of which is given as a reference. In some embodiments, the isolation or separation is carried out according to the methods described in International Patent Application Publication No. WO 2015/164675, the entire contents of which are incorporated by reference.

[0591] In some embodiments, the system or apparatus performs one or more, for example, all, of the steps of isolation, processing, modification, and formulation in an integrated or stand-alone 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 the user to program, control, evaluate the outcome and/or correct various aspects of the stages of processing, isolation, modification and formulation.

[0592] In some aspects, the separation and/or other steps are carried out using the CliniMACS system (Miltenyi Biotic), for example, for automated separation of cells at the clinical scale level in a closed and sterile system. Components may include an integrated microcomputer, magnetic separator, peristaltic pump, and various check valves. The integrated computer in some aspects controls all the components of the device and controls the system to carry out repeated procedures in a standardized sequence. The magnetic separator in some aspects includes a movable permanent magnet and a holder for a sampling column. The peristaltic pump controls the flow rate through the tubing and, together with check valves, provides a controlled flow of buffer through the system and a continuous cell suspension.

[0593] The CliniMACS system, in some aspects, uses antibody-bound, magnetizable particles that are supplied in a sterile, pyrogen-free solution. In some embodiments, after cell labeling with magnetic particles, the cells are washed to remove excess particles. The cell preparation bag is then connected to the tubing set, which in turn is connected to the buffer containing bag and the cell collection bag. The tubing set consists of pre-assembled sterile tubing including guard column and separation column and is intended for single use only. After starting the separation program, the system automatically applies the cell sample to the separation column. Labeled cells remain on the column while unlabeled cells are removed through a series of washing steps. In some embodiments, cell populations for use in the methods described herein are not labeled and do not remain on the column. In some embodiments, cell populations for use in the methods described herein are labeled and remain on the column. In some embodiments, cell populations for use in the methods described herein are eluted from the column after removal of the magnetic field and collected in a cell collection bag.

[0594] In specific embodiments, separation and/or other steps are performed using a CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy System is in some aspects equipped with a cell processing unit allowing automated cell washing and fractionation by centrifugation. The CliniMACS Prodigy system may also include an integrated camera and image recognition software that determines the optimal cell fractionation endpoint by distinguishing macroscopic layers of the cell product source. For example, peripheral blood can be automatically separated into layers of erythrocytes, leukocytes and plasma. The CliniMACS Prodigy system may also include an integrated cell culture chamber performing cell culture methods such as cell differentiation and expansion, antigen loading, and long term cell culture, for example. Inlets may allow sterile removal and replenishment of media, and cells may be monitored using an integrated microscope. See, for example, Klebanoff et al. (2012) J Immunother. 35(9): 651–660, Terakura et al. (2012) Blood.1:72–82, and Wang et al. (2012) J Immunother. 35(9):689–701.

[0595] In some embodiments, the cell population described herein is harvested and enriched (or depleted) by flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluid stream. In some embodiments, the cell population described herein is harvested and enriched (or depleted) by preparative scale sorting (FACS). In specific 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, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10, 1567–1573 and Godin et al (2008) J Biophoton 1(5):355–376 In both cases, cells can be labeled using a variety of markers allowing the isolation of well-defined T– high purity cells.

[0596] In some embodiments, the antibodies or binding patterns are labeled with one or more detectable markers to facilitate separation for positive and/or negative selection. For example, separation may be based on the binding of fluorescently labeled antibodies. In some examples, cell separation based on antibody binding or other binding patterns specific to one or more cell surface markers is carried out in a fluid stream, for example, by fluorescence-activated cell sorting (FACS), including preparative scale sorting (FACS) and/or microelectromechanical systems (MEMS) chips, for example, in combination with a flow cytometric detection system. Such methods allow positive and negative selection based on multiple markers simultaneously.

[0597] In some embodiments, methods of preparation include the steps of freezing, eg, cryopreserving, cells, either before or after isolation, incubation, and/or modification. 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 freezing solution, for example, after a washing step to remove plasma and platelets. In some aspects, any of a variety of known freezing solutions and parameters may be used. One example involves the use of PBS containing 20% DMSO and 8% human serum albumin (HSA) or other suitable cell freezing media. This is then diluted 1:1 with medium so that the final concentrations of DMSO and HSA are 10% and 4%, respectively. In some embodiments, cells are frozen by any method and/or using any of the reagents, containers, freezing solutions, and/or cryoprotectants described herein. In some embodiments, the cells are then frozen to −80° C. at or at a rate of approximately 1° per minute and stored in the vapor phase in a liquid nitrogen storage vessel.

[0598] In some embodiments, the methods provided include the steps of culture, incubation, cultivation, and/or genetic modification. For example, in some embodiments, the present invention relates to methods for incubating and/or modifying depleted cell populations and compositions for starting culture.

[0599] Thus, in some embodiments, cell populations are incubated in the composition to start culture. The incubation and/or modification can be carried out in a culture vessel such as a unit, chamber, well, column, tube, tubing, valve, vial, culture dish, bag, or other cell culture or culture container.

[0600] In some embodiments, cells are incubated and/or cultured prior to or in combination with genetic modification. Incubation steps may include culturing, cultivating, stimulating, activating and/or propagating. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulating agent. Such conditions include conditions designed to induce proliferation, expansion, activation and/or survival of cells in a population, to mimic antigen exposure, and/or to prime cells for genetic modification, for example, to introduce a recombinant antigen receptor.

[0601] Conditions may include one or more of specific environments, temperature, oxygen content, carbon dioxide content, time, means, such as nutrients, amino acids, antibiotics, ions, and/or stimulatory factors such as cytokines, chemokines, antigens, binding patterns, fusion proteins, recombinant soluble receptors, and any other means designed to activate cells. In some aspects, cells are incubated in the presence of one or more cytokines, and in some embodiments, a cytokine cocktail can be used, for example, as described in International Patent Application Publication number WO2015157384. In some embodiments, the cells are incubated with one or more cytokines and/or a cytokine cocktail before, during, or after transduction.

[0602] In some embodiments, the stimulatory conditions or agents include one or more agents, for example, a ligand capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the agent starts or initiates an intracellular TCR/CD3 signaling cascade in a T cell. Such agents may include antibodies, such as antibodies specific for TCR, for example, an anti-CD3 antibody. In some embodiments, the stimulatory conditions include one or more agents, such as a ligand capable of stimulating a costimulatory receptor, such as an anti-CD28 antibody. In some embodiments, such agents and/or ligands may be associated with a solid support, such as a bead, and/or with one or more cytokines. Illustrative stimulatory reagents are described in the present description, for example, in section V-D-1. Optionally, the propagation method may further comprise the step of adding the anti-CD3 antibody and/or the 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. In some embodiments, the stimulants include IL-2, IL-7 and/or IL-15, e.g., at concentrations of at least or at concentrations of about 10 units/mL, 20 units/mL, 50 units/mL , 100 units/ml, 250 units/ml, 500 units/ml, 600 units/ml, 700 units/ml, 800 units/ml, 900 units/ml or 1000 units/ml.

[0603] In some aspects, the incubation is carried out in accordance with such methods as described in US Patent No. 6.040.1 77 from 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. In some aspects, incubation is carried out using a system, device, apparatus and/or method as described in International Patent Application Publication No. WO2016/073602 or US 2016/0122782, the entire contents of which are incorporated by reference. In some embodiments, the incubation and/or culture is carried out in accordance with the methods described in International Patent Application Publication No. WO 2015/164675, the entire contents of which are incorporated by reference. In some embodiments, at least a portion of the incubation conducted in the centrifuge chamber includes mixing with a reagent or reagents to induce stimulation and/or activation. In some embodiments, cells, such as selected cells, are combined with a stimulus condition or stimulus in a centrifuge chamber. In some aspects of such methods, cell volume is combined with a level of one or more stimulus conditions, or with an amount of agents much less than is normally used when conducting similar stimulations in a culture plate or other system.

[0604] In some embodiments, the stimulant is added to the cells in the chamber cavity in an amount significantly less than (e.g., no more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% of the amount), compared to the amount of stimulant that is typically used or needed to be used to achieve approximately the same or similar selection efficiency for the same number of cells or the same cell volume when the selection is carried out without mixing in the cell for centrifugation, for example, in a test tube or bag with periodic shaking or rocking. In some embodiments, the incubation is carried out with the addition of incubation buffer to the cells and a stimulant to achieve the target reagent incubation volume, e.g., 10 ml to 200 ml, e.g., at least or about at least, or about, or 10 ml, 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, 100 ml, 150 ml or 200 ml. In some embodiments, the incubation buffer and stimulant are premixed before being added to the cells. In some embodiments, the incubation buffer and the stimulant are separately added to the cells. In some embodiments, the stimulation incubation is carried out under conditions of occasional gentle agitation, which can promote the stimulation of energetically beneficial interactions and thus allow the use of less total stimulant while achieving cell stimulation and activation.

[0605] In some embodiments, the incubation is typically carried out under agitated conditions, e.g., in the presence of rotation, typically at a relatively low acceleration or speed, such as a speed lower than that used to pellet the cells, e.g., from or approximately 600 rpm to 1700 rpm (e.g., at or about or at least 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm) e.g. at RCF for sample or chamber wall or other container from or about 80 g to 100 g (e.g., at or about or at least 80 g, 85 g, 90 g, 95 g, or 100 g) . In some embodiments, rotation is performed using repeated intervals of rotation at this low speed followed by a period of rest, such as rotation and/or rest for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, for example, rotation for about 1 or 2 seconds followed by rest for about 5, 6, 7, or 8 seconds.

[0606] In some embodiments, the total duration of incubation, for example, with a stimulant, is between or between about 1 hour and 96 hours, 1 hour and 72 hours, 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hours, or 12 hours and 24 hours, such as at least or about at least 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 72 hours, each included. In some embodiments, the additional incubation is carried out for a time between or approximately between 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hours, or 12 hours and 24 hours, each included.

[0607] In some embodiments, the processing steps include introducing a nucleic acid molecule encoding a recombinant protein. Among such recombinant proteins are recombinant receptors, such as any of those described in section V. Introduction of nucleic acid molecules encoding a recombinant protein, such as a recombinant receptor, into a cell can be accomplished using any of a number of known vectors. Such vectors include viral and non-viral systems, including lentiviral and gammaretroviral systems, as well as transposon based systems such as PiggyBac or Sleeping Beauty based gene transfer systems. Exemplary methods include methods for transferring receptor-encoding nucleic acids, including methods via viral, eg, retroviral or lentiviral transduction, transposons, and electroporation.

[0608] In some embodiments, gene transfer is accomplished by first stimulating the cell, e.g., by combining it with a stimulus that induces a response, such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or an activation marker, followed by transduction of activated cells, and propagation in culture to quantities sufficient for clinical applications.

[0609] 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), and human immunodeficiency virus ( HIV). In some embodiments, the recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors such as gamma retroviral vectors.

[0610] Methods for viral transduction, such as lentiviral transduction, are known. Illustrative methods are described, for example, in Wang et al. (2012) J. Immunother. 35(9): 689–701; Cooper et al. (2003) Blood. 101:1637–1644; Verhoeyen et al. (2009) Methods Mol Biol. 506:97–114; and Cavalieri et al. (2003) Blood. 102(2): 497–505.

[0611] In some embodiments, the introduction is carried out by bringing one or more cells from the composition into contact with a nucleic acid molecule encoding a recombinant protein, for example, a recombinant receptor. In some embodiments, the implementation, bringing into contact can be performed using centrifugation, such as spinoculation (eg, centrifugal inoculation). Such methods include any of the methods as described in International Publication Number WO2016/073602. Illustrative centrifuge chambers include those manufactured and sold by Biosafe SA, including chambers for use with the Sepax® and Sepax® 2 systems, including A-200/F and A-200 centrifuge chambers, and various kits for use with such systems. Exemplary chambers, systems, and processing equipment and cabinets are described, for example, in US Patent No. 6123655, US Patent No. 6,733,433 and U.S. Patent Application Publication No.: US 2008/0171951 and International Patent Application Publication No. WO 00/38762, the entire content of each of which is given in the present description by reference. Illustrative kits for use with such systems include, but are not limited to, disposable kits sold by BioSafe SA under the product names CS-430.1, CS-490.1, CS-600.1, or CS-900.2.

[0612] In some embodiments, the system includes and / or the system is placed in interaction with other equipment, including equipment for performing, automating, controlling and / or monitoring aspects of the transduction step and one or more various other processing steps performed in the system, for example, one or more processing steps that can be carried out with or in combination with a centrifuge chamber system as described herein or in International Publication Number WO2016/073602. This equipment, in some embodiments, is contained within a cabinet. In some embodiments, the equipment includes a cabinet including a housing containing control circuits, a centrifuge, a lid, motors, pumps, sensors, displays, and a user interface. Exemplary devices are described in US Patent No. 6123655, US Patent No. 6733433 and US 2008/0171951.

[0613] In some embodiments, the system comprises a series of containers, such as bags, tubes, electronic circuits, clips, connectors, and a centrifuge chamber. In some embodiments, containers, such as pouches, include one or more containers, such as pouches, containing cells to be transduced and viral vector particles in the same container or in separate containers, such as the same pouch. or individual packages. In some embodiments, the system further includes one or more containers, such as pouches, containing a medium such as a diluent and/or wash solution that are included with the chamber and/or other components for dilution, resuspension, and/or washing of the components and /or compositions during the methods. The containers can be attached at one or more positions in the system, for example, at a position corresponding to an inlet line, a dilution line, a flush line, a waste line, and/or an exit line.

[0614] In some embodiments, the chamber cooperates with a centrifuge capable of rotating the chamber, for example, about its axis of rotation. Rotation may occur before, during and/or after incubation in combination with cell transduction, and/or at one or more of the other processing steps. Thus, in some embodiments, one or more of the various stages of processing is carried out with rotation, for example, with a certain acceleration. The chamber is typically capable of vertical or substantially vertical rotation such that the chamber is vertical during centrifugation and the side wall and axis are vertical or generally vertical where the end wall(s) are horizontal or generally horizontal .

[0615] In some embodiments, a composition containing cells, viral particles, and a reagent can be subjected to rotation, typically at a relatively low acceleration or speed, such as a speed lower than that used to precipitate the cells, for example, from or about 600 rpm up to 1700 rpm (for example, from or about from, or at least 600 rpm 1000 rpm, or 1500 rpm, or 1700 rpm). In some embodiments, the rotation is performed at acceleration, for example, relative centrifugal acceleration, from or about 100 g to 3200 g (for example, at or about at, or at least at or about 100 g, 200 g, 300 g, 400 g, 500 g, 1000 g, 1500 g, 2000 g, 2500 g, 3000 g or 3200 g) as measured, for example, against the inner or outer wall of the chamber or cavity. The term "relative centrifugal acceleration" or RCF is generally understood as the effective acceleration imparted to an object or substance (such as a cell, sample or sediment, and/or a point in a chamber or other container subjected to rotation) relative to the gravitational force of the Earth, in a specific point in space, compared to the axis of rotation. This value can be determined using well-known formulas, taking into account the gravitational force, the speed of rotation and the radius of rotation (distance from the axis of rotation and the object, substance or particle for which the RCF is measured).

[0616] In some embodiments, during at least a portion of the genetic modification, e.g., transduction, and/or after the genetic modification, the cells are transferred to a container, such as a genetically modified cell culture bag, e.g., for culturing or expanding cells, as described above. In some embodiments, the cell culture or expansion container is a bioreactor bag, such as a perfusion bag.

[0617] In some embodiments, the present methods include one or more steps of culturing the modified cells, for example, culturing the cells under conditions that promote proliferation and/or multiplication. In some embodiments, the modified cells are cultured under conditions that promote proliferation and/or reproduction after a genetic modification step, such as introducing a recombinant polypeptide into the cells via transduction or transfection. In specific embodiments, the cells are cultured after the cells have been incubated under stimulating conditions and transduced or transfected with a recombinant polynucleotide, eg, a polynucleotide encoding a recombinant receptor. In some embodiments, the culture results in one or more cultured compositions of enriched T cells.

[0618] In some aspects, the culture medium is an adapted culture medium that supports the growth, cultivation, expansion, or proliferation of cells, such as T cells. In some aspects, the medium may be a liquid containing a mixture of salts, amino acids, vitamins, sugars, or any combination thereof. In some embodiments, the culture medium further comprises one or more stimulating conditions or agents, for example, to stimulate cell culture, expansion, or proliferation during incubation. In some embodiments, the stimulatory condition is or includes one or more cytokines selected from IL-2, IL-7, or IL-15. In some embodiments, the cytokine is a recombinant cytokine. In some embodiments, the concentration of one or more cytokines in the culture medium during culture or incubation is independently from or about 1 IU/mL to 1500 IU/mL, e.g., from or about 1 IU/mL to 100 IU/mL , 2 IU/mL to 50 IU/mL, 5 IU/mL to 10 IU/mL, 10 IU/mL to 500 IU/mL, 50 IU/mL to 250 IU/mL or 100 IU/mL up to 200 IU/ml, from 50 IU/ml to 1500 IU/ml, from 100 IU/ml to 1000 IU/ml or from 200 IU/ml to 600 IU/ml. In some embodiments, the concentration of one or more cytokines is independently at least or at least about 1 IU/mL, 5 IU/mL, 10 IU/mL, 50 IU/mL, 100 IU/mL, 200 IU/mL , 500 IU/ml, 1000 IU/ml or 1500 IU/ml.

[0619] In some aspects, the cells are incubated for at least a portion of the time following the transfer of the modified cells and culture medium. In some embodiments, stimulating conditions typically include a temperature suitable for growth of primary immunocytes such as human T-lymphocytes, e.g., at least about 25 degrees Celsius, typically at least about 30 degrees, and typically , at or approximately at 37 degrees Celsius. In some embodiments, cells are incubated at 25 to 38 degrees Celsius, eg 30 to 37 degrees Celsius, eg at or about 37 degrees Celsius±2 degrees Celsius. In some embodiments, the incubation is carried out for a period of time until culture, such as culture or propagation, results in the desired or threshold cell density, number, or dose. In some embodiments, the incubation is more than or more than about, or is about, or is 24 hours, 48 hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, or more.

[0620] In some embodiments, the cells are incubated under conditions to maintain the target amount of carbon dioxide in the cell culture. In some aspects, this provides optimal cultivation, expansion and proliferation of cells during growth. In some aspects, the amount of carbon dioxide (CO ₂ ) is between 10% and 0% (v/v) of said gas, such as between 8% and 2% (v/v) of said gas, such as the amount equal to or approximately equal to 5% (v/v) CO ₂ .

[0621] In some embodiments, cells are incubated using containers, such as bags, that are used in conjunction with a bioreactor. In some cases, the bioreactor may be subjected to movement or rocking, which, in some aspects, may increase oxygen transfer. Movement of the bioreactor may include, but is not limited to, rotation about a horizontal axis, rotation about a vertical axis, rocking about an inclined or angled horizontal axis of the bioreactor, or any combination thereof. In some embodiments, at least part of the incubation is carried out with shaking. The swing speed and swing angle can be adjusted to achieve the desired shaking. In some embodiments, the rocking angle is or is approximately 20°, 19°, 18°, 17°, 16°, 15°, 14°, 13°, 12°, 11°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2° or 1°. In particular embodiments, the rocking angle is between 6-16°, inclusive. In other embodiments, the rocking angle is between 7-16°. In other embodiments, the rocking angle is between 8-12°, inclusive. In some embodiments, the swing speed is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 1 12, 13, 14 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 rpm In some embodiments, the swing speed is between 4 and 12 rpm, such as between 4 and 6 rpm, inclusive. At least part of the expansion of the cell culture is carried out with a rocking motion, for example, with an angle between 5° and 10° inclusive, such as 6°, at a constant rocking speed, such as a speed between 5 and 15 rpm, inclusive, for example , 6 RMP or 10 RPM The CD4+ and CD8+ cells are each separately expanded until they each reach a threshold cell number or density.

[0622] In some embodiments, at least a portion of the incubation is carried out under static conditions. In some embodiments, at least a portion of the incubation is perfused to perfuse spent media and perfuse fresh media during culture. In some embodiments, the method includes the step of perfusing fresh culture medium into the cell culture, for example, through a feed port. In some embodiments, the culture medium added during the perfusion contains one or more stimulants, for example, one or more recombinant cytokines such as IL-2, IL-7 and/or IL-15. In some embodiments, the culture medium added during the perfusion is the same culture medium used during the static incubation.

[0623] In some embodiments, after incubation, the container, e.g., a pouch, is reattached to the system for one or more other processing steps to manufacture, form, or obtain a cell therapy agent, e.g., reattached to the system containing the chamber for centrifugation. In some aspects, the cultured cells are transferred from the bag to the interior of the chamber to obtain a cultured cell composition.

[0624] In some embodiments, T cells are expanded by adding feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs), to the composition to start culture (e.g., such that the resulting cell population contains at least about 5 , 10, 20, or 40 or more PBMC feeder cells for each T-lymphocyte in the initial population to be expanded); and culture incubation (eg, for a period of time sufficient for these numbers of T cells to expand). In some aspects, non-dividing feeder cells may contain gamma irradiated PBMC feeder cells. In some embodiments, the PBMC is irradiated using gamma radiation in the range of about 3000 to 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 populations.

[0625] In some embodiments, the stimulating conditions include a temperature suitable for human T-lymphocyte 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 lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated using gamma radiation in the range of approximately 6000 to 10000 rad. LCL feeder cells, in some aspects, are provided in any suitable amount, for example, in a ratio of LCL feeder cells to original T-lymphocytes of at least about 10:1.

[0626] In some embodiments, antigen-specific T cells, such as antigen-specific CD4+ and/or CD8+ T cells, are obtained by antigen stimulation of naive or antigen-specific T lymphocytes. For example, antigen-specific T cell lines or clones can be generated against cytomegalovirus antigens by isolating T cells from infected individuals and stimulating the cells in vitro with the same antigen.

[0627] In some embodiments, the modified cells are accumulated, eg harvested, to obtain a composition after the cells have been transduced, transfected, cultured and/or expanded. In some embodiments, the cells are harvested within a certain amount of time, for example, within or within 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after the start and/or initiation, or the end and/or completion of a processing step such as isolation, selection, incubation, stimulation, activation, transduction, transfection, cultivation and/or propagation steps. In specific embodiments, the modified cells are harvested after culture, for example, for or within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days of culture and/ or after the modified cells and/or cell compositions comprising the modified cells have expanded to a threshold cell density, cell number, and/or expansion level. In some embodiments, the harvested cells are made up by any of the methods described herein, such as, for example, as described in Section VI.

1. Stimulating reagents

[0628] In some embodiments, cells can be incubated and/or contacted with a stimulatory reagent capable of activating and/or expanding T cells. In specific embodiments, the stimulatory reagent comprises a particle, eg a bead, that is conjugated or linked to one or more agents, eg biomolecules, that are capable of activating and/or proliferating cells, eg T cells. In some embodiments, one or more agents are associated with the bead. In some embodiments, the bead is biocompatible, i.e., consists of a material suitable for biological use. In some embodiments, the beads are non-toxic to cultured cells, such as cultured T cells. In some embodiments, the beads may be any particles capable of attaching agents in a manner that allows interaction between the agent and the cell.

[0629] In some embodiments, the stimulatory reagent contains one or more agents that are capable of activating and/or expanding cells, e.g., T cells, that are associated with or otherwise attached to the bead, e.g., the surface of the bead . In specific embodiments, the bead is a non-cellular particle. In specific embodiments, the bead may include a colloidal particle, a microsphere, a nanoparticle, a magnetic bead, or the like. In some embodiments, the beads are agarose beads. In specific embodiments, the beads are Sepharose beads.

[0630] In specific embodiments, the implementation, stimulating reagent contains beads that are monodisperse. In specific embodiments, beads that are monodisperse have a size dispersion with a diameter standard deviation of less than 5% from each other.

[0631] In some embodiments, the bead contains one or more agents, such as an agent, connected, conjugated, or associated (directly or indirectly) with the surface of the bead. In some embodiments, an agent as provided herein may include, but is not limited to, RNA, DNA, proteins (e.g., enzymes), antigens, polyclonal antibodies, monoclonal antibodies, antibody fragments, carbohydrates, lipids, lectins, or any other biomolecule. with affinity for the desired target. In some embodiments, the desired target is a T cell receptor and/or a T cell receptor component. In specific embodiments, the desired target is CD3. In a specific embodiment, the desired target is a costimulatory molecule, eg CD28. One or more means can be attached directly or indirectly to the bead through a variety of methods known and available in this field. The connection may be covalent, non-covalent, electrostatic, or hydrophobic, and may be carried out by a variety of attachment methods, including, for example, chemical methods, mechanical methods, or enzymatic methods. In some embodiments, a biomolecule (eg, a biotinylated anti-CD3 antibody) can be attached indirectly to the bead via another biomolecule (eg, an anti-biotin antibody) directly attached to the bead.

[0632] In some embodiments, one or more of the agents attached to the bead is an antibody. An antibody may include a polyclonal antibody, a monoclonal antibody (including full length antibodies having an immunoglobulin Fc region), antibody compositions with polyepitope specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single chain molecules, as well as antibody fragments (e.g., Fab, F( ab')2 and Fv).In some embodiments, the stimulatory reagent is an antibody fragment (including an antigen binding fragment), e.g. any isotype can be used for the antibodies provided herein, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be derived from any human or animal species (e.g., mouse species).In some embodiments, implementation, an agent is an antibody that binds and/or recognizes one or more components of the T-cell receptor. x embodiments, the agent is an anti-CD3 antibody. In specific embodiments, the agent is an antibody that binds and/or recognizes a co-receptor. In some embodiments, the stimulatory reagent contains an anti-CD28 antibody.

[0633] In some embodiments, the bead has a diameter greater than about 0.001 microns, greater than about 0.01 microns, greater than about 0.1 microns, greater than about 1.0 microns, greater than about 10 microns, greater than about 50 microns, greater than about 100 microns, or greater than about 1000 microns and not greater than about 1500 microns. In some embodiments, the bead has a diameter of from about 1.0 µm to about 500 µm, from about 1.0 µm to about 150 µm, from about 1.0 µm to about 30 µm, from about 1.0 µm to about 10 µm, from about 1.0 µm to about 5.0 µm, from about 2.0 µm to about 5.0 µm, or from about 3.0 µm to about 5.0 µm. In some embodiments, the bead has a diameter of from about 3 µm to about 5 µm. In some embodiments, the bead has a diameter of at least or at least about, or about 0.001 µm, 0.01 µm, 0.1 µm, 0.5 µm, 1.0 µm, 1.5 µm, 2.0 µm, 2.5 µm, 3.0 µm, 3.5 µm, 4.0 µm, 4.5 µm, 5.0 µm, 5.5 µm, 6.0 µm, 6.5 µm, 7.0 µm, 7.5 µm, 8.0 µm, 8.5 µm, 9.0 µm, 9.5 µm, 10 µm, 12 µm, 14 µm, 16 µm, 18 µm or 20 µm. In specific embodiments, the bead has or has a diameter of approximately 4.5 microns. In particular embodiments, the bead has or has a diameter of approximately 2.8 microns.

[0634] In some embodiments, the beads have a density greater than 0.001 g/cm ³ , greater than 0.01 g/cm ³ , greater than 0.05 g/cm ³ , greater than 0.1 g/cm ³ , greater than more than 0.5 g/cm ³ , more than 0.6 g/cm ³ , more than 0.7 g/cm ³ , more than 0.8 g/cm ³ , more than 0.9 g/cm ³ , more more than 1 g/cm ³ , more than 1.1 g/cm ³ , more than 1.2 g/cm ³ , more than 1.3 g/cm ³ , more than 1.4 g/cm ³ , more than 1 .5 g/cm ³ , more than 2 g/cm ³ , more than 3 g/cm ³ , more than 4 g/cm ³ or more than 5 g/cm ³ . In some embodiments, the beads have a density between about 0.001 g/cm ³ and about 100 g/cm ³ , about 0.01 g/cm ³ and about 50 g/cm ³ , about 0.1 g/cm ³ and about 10 g/cm ³ , about 0.1 g/cm ³ and about 0.5 g/cm ³ , about 0.5 g/cm ³ and about 1 g/cm ³ , about 0.5 g/cm ³ and about 1 .5 g/cm ³ , about 1 g/cm ³ and about 1.5 g/cm ³ , about 1 g/cm ³ and about 2 g/cm ³ , or about 1 g/cm ³ and about 5 g/cm ³ , including each. In some embodiments, the beads have a density of about 0.5 g/cm ³ , about 0.5 g/cm ³ , about 0.6 g/cm ³ , about 0.7 g/cm ³ , about 0.8 g/cm 3 cm ³ , approximately 0.9 g/cm ³ , approximately 1.0 g/cm ³ , approximately 1.1 g/cm ³ , approximately 1.2 g/cm ³ , approximately 1.3 g/cm ³ , approximately 1 .4 g/cm ³ , approximately 1.5 g/cm ³ , approximately 1.6 g/cm ³ , approximately 1.7 g/cm ³ , approximately 1.8 g/cm ³ , approximately 1.9 g/cm ³ , or approximately 2.0 g/cm ³ . In specific embodiments, the beads have a density of approximately 1.6 g/cm ³ . In particular embodiments, the beads or particles have a density of about 1.5 g/cm ³ . In specific embodiments, the particles have a density of approximately 1.3 g/cm ³ .

[0635] In specific embodiments, the plurality of beads have a uniform density. In specific embodiments, the uniform density has a density standard deviation of less than 10%, less than 5%, or less than 1% of the average bead density.

[0636] In some embodiments, the beads have a surface area between about 0.001 m ² for each gram of particles (m ² /g) and about 1000 m ² /g, about 0.010 m ² /g and about 100 m ² /g, approximately 0.1 m ² /g and about 10 m ² /g, about 0.1 m ² /g and about 1 m ² /g, about 1 m ² /g and about 10 m ² /g, about 10 m ² / g and approximately 100 m ² /g, approximately 0.5 m ² /g and approximately 20 m ² /g, approximately 0.5 m ² /g and approximately 5 m ² /g, or approximately 1 m ² /g and approximately 4 m ² /g, including each. In some embodiments, the particles or beads have a surface area of from about 1 m ² /g to about 4 m ² /g.

[0637] In some embodiments, the bead contains at least one material on or near the surface of the bead that can be combined with, associated with, or conjugated with an agent. In some embodiments, the bead has a functionalized surface, i. contains functional groups that are capable of forming a covalent bond with a binding molecule, such as a polynucleotide or polypeptide. In specific embodiments, the bead contains surface-exposed carboxyl, amino, hydroxyl, tosyl, epoxy, and/or chloromethyl groups. In specific embodiments, the beads contain surface-exposed agarose and/or sepharose. In specific embodiments, the surface of the bead contains attached stimulatory reagents that can bind or attach binding molecules. In specific embodiments, the biomolecules are polypeptides. In some embodiments, the beads contain surface-exposed protein A, protein G, or biotin.

[0638] In some embodiments, the bead reacts in a magnetic field. In some embodiments, the bead is a magnetic bead. In some embodiments, the magnetic bead is paramagnetic. In particular embodiments, the magnetic bead is superparamagnetic. In specific embodiments, the beads do not exhibit any magnetic properties until they are exposed to a magnetic field.

[0639] In specific embodiments, the bead comprises a magnetic core, a paramagnetic core, or a superparamagnetic core. In some embodiments, the magnetic core contains a metal. In some embodiments, the metal may be, but is not limited to, iron, nickel, copper, cobalt, halolinium, manganese, tantalum, zinc, zirconium, or any combination thereof. In specific embodiments, the magnetic core comprises metal oxides (eg, iron oxides), ferrites (eg, manganese ferrites, cobalt ferrites, nickel ferrites, etc.), hematite, and metal alloys (eg, CoTaZn). In some embodiments, the magnetic core comprises one or more of ferrite, metal, metal alloy, iron oxide, or chromium dioxide. In some embodiments, the magnetic core contains elemental iron or a compound thereof. In some embodiments, the magnetic core comprises one or more of magnetite (Fe3O4), maghemite (γFe2O3), or greigite (Fe3S4). In some embodiments, the inner core contains iron oxide (eg, Fe ₃ O ₄ ).

[0640] In specific embodiments, the bead comprises a magnetic, paramagnetic, and/or superparamagnetic core coated with a shell or coating with a functionalized surface. In some embodiments, the shell may contain a material that may include, but is not limited to, a polymer, polysaccharide, silicon dioxide, fatty acid, protein, carbon, agarose, sepharose, or a combination thereof. In some embodiments, the polymer may be polyethylene glycol, (lactic-glycolic acid) copolymer, polyglutaraldehyde, polyurethane, polystyrene, or polyvinyl alcohol. In specific embodiments, the outer shell or cover comprises polystyrene. In specific embodiments, the surface of the outer coating is functionalized.

[0641] In some embodiments, the stimulating reagent comprises a bead containing a metal oxide core (e.g., an iron oxide core) and a shell, where the metal oxide core contains at least one polysaccharide (e.g., dextran), and where the shell contains at least one polysaccharide (eg aminodextran), at least one polymer (eg polyurethane) and silicon dioxide. In some embodiments, the metal oxide core is a colloidal iron oxide core. In specific embodiments, one or more agents comprise an antibody, or an antigen-binding fragment thereof. In specific embodiments, the one or more agents comprise an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the stimulatory reagent comprises an anti-CD3 antibody, an anti-CD28 antibody, and an anti-biotin antibody. In some embodiments, the stimulatory reagent contains an anti-biotin antibody. In some embodiments, the bead has a diameter of about 3 microns to about 10 microns. In some embodiments, the bead has a diameter of from about 3 µm to about 5 µm. In specific embodiments, the bead has a diameter of approximately 3.5 microns.

[0642] In some embodiments, the stimulating reagent comprises one or more agents that are attached to a bead containing a metal oxide core (e.g., an iron oxide inner core) and a shell (e.g., a containment shell) where the shell contains polystyrene. In specific embodiments, the beads are monodisperse, superparamagnetic beads comprising a superparamagnetic iron core, such as a core containing magnetite (Fe ₃ O ₄ ) and/or maghemite (γFe ₂ O ₃ ), and a polystyrene sheath or coating. In some embodiments, the bead is non-porous. In some embodiments, the beads comprise a functionalized surface to which one or more agents are attached. In specific embodiments, one or more agents are covalently bonded to the beads on the surface. In some embodiments, one or more agents comprise an antibody, or an antigen-binding fragment thereof. In some embodiments, the one or more agents comprise an anti-CD3 antibody and an anti-CD28 antibody. In particular embodiments, the beads have a density of about 1.5 g/cm ³ and a surface area of about 1 m ² /g to about 4 m ² /g. In specific embodiments; the beads are monodisperse superparamagnetic beads having a diameter of approximately 4.5 μm and a density of approximately 1.5 g/cm ³ . In some embodiments, the beads are monodisperse superparamagnetic beads having an average diameter of about 2.8 microns and a density of about 1.3 g/cm ³ .

E. Vectors and methods for genetic engineering

[0643] Polynucleotides (nucleic acid molecules) encoding recombinant receptors can be included in vectors for genetically modifying cells to express such receptors. In some embodiments, the vectors or constructs contain one or more promoters operably linked to a nucleotide encoding a polypeptide or receptor to direct its expression. In some embodiments, the promoter is operably linked to one or more nucleic acid molecules.

[0644] In some cases, the vector is a viral vector, such as a retroviral vector, such as a lentiviral vector or a gammaretroviral vector. In some embodiments, a polynucleotide, such as a vector encoding a recombinant receptor, is introduced into a composition containing cultured cells, eg, via retroviral transduction, transfection, or transformation.

[0645] Various methods of introducing genetically modified components, for example, recombinant receptors, such as CAR or TCR, are well known and can be used with the presented methods and compositions. Exemplary methods include methods for transferring nucleic acids encoding polypeptides or receptors, e.g. via viral vectors, e.g. retroviral or lentiviral, non-viral vectors or transposons, e.g. transposonsleeping beauty. Gene transfer methods may include transduction, electroporation, or other method that transfers genes into a cell.

[0646] In some embodiments, gene transfer occurs first by stimulating a cell, such as by combining it with a stimulus that induces a response, such as proliferation, survival, and/or activation, such as as measured by expression of a cytokine or activation marker, with subsequent transduction of activated cells, and propagation in culture to quantities sufficient for clinical applications.

[0647] In some contexts, it may be desirable to guard against the possibility that overexpression of a stimulatory factor (e.g., a lymphokine or cytokine) could potentially lead to an undesirable outcome or lower efficacy in an individual, e.g., a factor associated with toxicity in an individual. Thus, in some contexts, modified cells include gene fragments that render cells susceptible to negative selection in vivo , such as when administered in adoptive immunotherapy. For example, in some aspects, the cells are modified in such a way that they can be destroyed by changing the in vivo conditions for the patient to whom they are administered. A negatively selectable phenotype may result from the insertion of a gene conferring sensitivity to an administered agent, such as a compound. Negatively selectable genes include the herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell II:223, I977), conferring sensitivity to ganciclovir; cellular hypoxanthine-phosphoribosyltransferase (HPRT) gene, cellular adenine-phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase gene, (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

[0648] 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, the recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors such as gamma retroviral vectors (see, for example, Koste et al. (2014) Gene Therapy 2014 Apr 3. doi: 10.1038 /gt.2014, 25; Carlens et al. (2000) Exp Hematol 28(10): 1137–46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November 29(11): 550–557.

[0649] In some embodiments, the polynucleotide encoding the recombinant receptor and/or additional polypeptide is contained in a vector or can be cloned into one or more vector(s). In some embodiments, one or more vector(s) can be used to transform or transfect a host cell, such as a cell for modification. Illustrative vectors include vectors designed for introduction, propagation and propagation, or expression, or both, such as plasmids and viral vectors. In some aspects, the vector is an expression vector, such as a recombinant expression vector. In some embodiments, recombinant expression vectors can be generated using standard recombinant DNA techniques.

[0650] In some embodiments, the vector may be a vector from the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), or the pEX series (Clontech, Palo Alto, Calif.). In some cases, bacteriophage vectors such as λG10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 can 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).

[0651] In some embodiments, the vector is a viral vector, such as a retroviral vector. In some embodiments, the polynucleotide encoding the recombinant receptor and/or additional polypeptide(s) is introduced into the cell via retroviral or lentiviral vectors, or via transposons (see, for example, Baum et al. (2006) Molecular Therapy: The Journal of the American Society of Gene Therapy 13:1050-1063; Frecha et al. (2010) Molecular Therapy 18:1748-1757; and Hackett et al. (2010) Molecular Therapy 18:674-683).

[0652] In some embodiments, the transcription units may be modified in the form of a bicistronic unit containing an IRES that allows co-expression of gene products (eg , encoding a recombinant receptor and an additional polypeptide) via messenger RNA from a single promoter. Alternatively, in some cases, a single promoter may direct the expression of an RNA containing, in a single open reading frame (ORF), two or three genes (eg, a coding marker and coding for a recombinant receptor) separated from each other by sequences encoding a self-cleaving peptide (eg , sequences 2A) or a protease (eg, furin) recognition site. The ORF thus encodes a single polypeptide which, either during (in the case of 2A) or after translation, is processed into individual proteins. In some cases, a peptide such as T2A can cause a ribosome to skip (ribosome slip) the synthesis of a peptide bond at the C-terminus of element 2A, resulting in a separation between the end of the 2A sequence and the next peptide below (see, e.g., de Felipe, Genetic Vaccines and Ther 2:13 (2004) and de Felipe et al Traffic 5:616–626 (2004)). Various 2A elements are known. Examples of 2A sequences that can be used in the methods and system described herein include, but are not limited to, 2A sequences from foot and mouth disease virus (F2A, e.g. SEQ ID NO: 21), equine rhinitis virus A (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.

[0653] In some embodiments, the recombinant receptor, such as CAR, is under the control of a promoter. In some embodiments, the vector includes a promoter. In some embodiments, the promoter is or contains a constitutive promoter. Exemplary constitutive promoters include, for example, the simian virus 40 (SV40) early promoter, the cytomegalovirus immediate early (CMV) promoter, the human ubiquitin C (UBC) promoter, the human elongation factor 1α (EF1α) promoter, the mouse phosphoglycerate kinase 1 (PGK) promoter, and the chicken β-actin in combination with the CMV early enhancer (CAGG). In some embodiments, the constitutive promoter is a synthetic or modified promoter. In some embodiments, the promoter is or contains an MND promoter, a synthetic promoter containing a U3 region from a modified MoMuLV LTR with a myeloproliferative sarcoma virus enhancer (see Challita et al. (1995) J. Virol. 69(2):748-755) . In some embodiments, the promoter is a tissue-specific promoter. In another embodiment, the promoter is a viral promoter. In another embodiment, the promoter is a non-viral promoter. In some embodiments, exemplary promoters may include, but are not limited to, the human elongation factor 1 alpha (EF1α) promoter or a modified form thereof, or the MND promoter.

[0654] In some embodiments, the promoter is a regulated promoter (eg, an inducible promoter). In some embodiments, the promoter is an inducible promoter or a repressible promoter. In some embodiments, the promoter comprises a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence, or a doxycilin operator sequence, or an analogue thereof, or is a binding or recognition capable Lac repressor or a tetracycline repressor, or an analogue thereof. In some embodiments, the polynucleotide does not include a regulatory element, such as a promoter.

[0655] In some cases, the nucleic acid sequence encoding the recombinant receptor contains a signal sequence encoding a signal peptide. In some aspects, the signal sequence may encode a signal peptide derived from a native polypeptide. In other aspects, the signal sequence may encode a heterologous or non-natural signal peptide, such as the exemplary GMCSFR alpha chain signal peptide shown in SEQ ID NO:25 and encoded by the nucleotide sequence shown in SEQ ID NO:24. In some cases, the nucleic acid sequence encoding a recombinant receptor, for example, a chimeric antigen receptor (CAR), contains a signal sequence encoding a signal peptide. Non-limiting exemplary signal peptides include, for example, the GMCSFR alpha chain signal peptide set forth in SEQ ID NO: 25 and encoded by the nucleotide sequence set forth in SEQ ID NO: 24, or the CD8 alpha signal peptide set forth in SEQ ID NO: 26.

[0656] In some embodiments, the polynucleotide contains a nucleic acid sequence encoding one or more additional polypeptides, for example, one or more marker(s) and/or one or more effector molecules. In some embodiments, the one or more marker(s) includes a transduction marker, a surrogate marker, and/or a selection marker. Among the additional nucleic acid sequences introduced, for example, encoding one or more additional polypeptide(s), included are nucleic acid sequences that can improve the efficacy of therapy, for example, by promoting the viability and/or function of the transferred cells; a nucleic acid sequence to provide a genetic marker for selection and/or evaluation of cells, for example, to assess survival or localization in vivo ; nucleic acid sequences to improve safety, for example, by making the cell susceptible to in vivo negative selection as described in Lupton SD et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319–338 (1992); see also WO 1992008796 and WO 1994028143 describing the use of bifunctional selectable fusion genes resulting from the fusion of a dominant positive selectable marker with a negative selectable marker, and US Patent No. 6040177.

[0657] In some embodiments, a single promoter may direct the expression of an RNA containing, in one open reading frame (ORF), two or three genes (e.g., encoding a molecule involved in metabolic pathway modulation and encoding a recombinant receptor) separated by each other from each other by sequences encoding a self-cleaving peptide (eg 2A sequences) or a protease recognition site (eg furin). The ORF thus encodes a single polypeptide which, either during (in the case of 2A) or after translation, is processed into individual proteins. In some cases, a peptide such as T2A can cause a ribosome to skip (ribosome slip) the synthesis of a peptide bond at the C-terminus of element 2A, resulting in a separation between the end of the 2A sequence and the next peptide below (see , e.g., de Felipe. Genetic Vaccines and Ther 2:13 (2004) and deFelipe et al Traffic 5:616–626 (2004)). The set of elements 2A is known. Examples of 2A sequences that can be used in the methods and nucleic acids described herein include, without limitation, 2A sequences from foot-and-mouth disease virus (F2A, e.g. SEQ ID NO: 22), equine rhinitis virus A (E2A, e.g. SEQ ID NO: 21), Thosea asigna virus (T2A, eg, SEQ ID NO: 6 or 18), and porcine teshovirus-1 (P2A, eg, SEQ ID NO: 19 or 20), as described in US Patent Publication No. 20070116690.

[0658] In some embodiments, the vector contains a nucleic acid sequence encoding one or more marker(s). In some embodiments, the one or more marker(s) is a transduction marker, a surrogate marker, and/or a selection marker.

[0659] In some embodiments, the marker is a transduction marker or a surrogate marker. A transduction marker or a surrogate marker can be used to detect cells into which a polynucleotide has been introduced, for example, a polynucleotide encoding a recombinant receptor. In some embodiments, the transduction marker may indicate or confirm cell modification. In some embodiments, the surrogate marker is a protein that is co-expressed on the cell surface with a recombinant receptor, such as CAR. In specific embodiments, such a surrogate marker is a surface protein modified to have little or no activity. In specific embodiments, the surrogate marker is encoded by the same polynucleotide that encodes the recombinant receptor. In some embodiments, a nucleic acid sequence encoding a recombinant receptor is operably linked to a nucleic acid sequence encoding a marker, optionally separated by an internal ribosome binding site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome slippage, such as a 2A sequence, such as T2A, P2A, E2A, or F2A. External marker genes can, in some cases, be used in combination with a modified cell to allow detection or selection of cells and, in some cases, also stimulate cell suicide.

[0660] Exemplary surrogate markers can include truncated forms of cell surface polypeptides, such as truncated forms that are non-functional and do not or are unable to transmit a signal, or the signal normally transmitted by the full-length form of the cell surface polypeptide, and/or that are not exposed or are unable to be internalized. Exemplary truncated cell surface polypeptides include truncated forms of growth factors or other receptors, such as truncated human epidermal growth factor receptor 2 (tHER2), truncated epidermal growth factor receptor (tEGFR, exemplary tEGFR sequence shown in SEQ ID NO:7 or 23), or prostate-specific membrane antigen (PSMA) or a modified form thereof. tEGFR may contain an epitope recognized by a cetuximab (Erbitux®) or other therapeutic anti-EGFR antibody or binding molecule that can be used to identify or select cells modified with a tEGFR construct and an encoded exogenous protein and/or to exclude or isolate cells, expressing the encoded exogenous protein. See US Patent No. 8802374 and Liu et al., Nature Biotech. April 2016; 34(4): 430–434). In some aspects, the marker, e.g. , a surrogate marker, includes all or part (e.g., a truncated form) of CD34, NGFR, CD19, or a truncated CD19, e.g., truncated non-human CD19, or an epidermal growth factor receptor (e.g., tEGFR).

[0661] In some embodiments, the marker is or comprises a fluorescent protein such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP) such as superfolded GFP (sfGFP), red fluorescent protein (RFP) such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, blue fluorescent protein (CFP), blue-green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), and variants thereof, including species variants , monomeric variants and codon optimized and/or improved variants of fluorescent proteins. In some embodiments, the marker is or contains an enzyme, such as luciferase, E. coli lacZ gene, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT). Exemplary light emitting reporter genes include luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or variants thereof.

[0662] In some embodiments, the marker is a selection marker. In some embodiments, the selection marker is or contains a polypeptide conferring resistance to exogenous agents or drugs. In some embodiments, the selection marker is an antibiotic resistance gene. In some embodiments, the selection marker is an antibiotic resistance gene conferring antibiotic resistance to a mammalian cell. In some embodiments, the selection marker is or comprises a puromycin resistance gene, a hygromycin resistance gene, a blasticidin resistance gene, a neomycin resistance gene, a geneticin resistance gene, or a zeocin resistance gene, or a modified form thereof.

[0663] In some embodiments, the retroviral vector has a long terminal repeat (LTR) sequence, e.g., from a retroviral vector, derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV) , mouse stem cell virus (MSCV), splenic foci-forming virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include retroviruses derived from any source in avian or mammalian cells. Retroviruses are generally amphotropic, meaning that they are capable of infecting host cells from several species, including humans. In one embodiment, the genome to be expressed is replaced by the gag, pol and/or env retroviral sequences. A number of illustrative 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.

[0664] Methods for lentiviral transduction are known. Illustrative methods are described, for example, in Wang et al. (2012) J. Immunother. 35(9): 689–701; Cooper et al. (2003) Blood. 101:1637–1644; Verhoeyen et al. (2009) Methods Mol Biol. 506:97–114; and Cavalieri et al. (2003) Blood. 102(2): 497–505. In some embodiments, a polynucleotide encoding a recombinant receptor and/or one or more additional polypeptide(s) is introduced into a composition containing cultured cells, eg, via retroviral transduction, transfection, or transformation.

[0665] In some embodiments, recombinant nucleic acids are transferred to T cells via electroporation (see , for example, Chicaybam et al, (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, the recombinant nucleic acids are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74 and Huang et al (2009) Methods Mol Biol 506: 115–126). Other methods for introducing and expressing genetic material into immunocytes include calcium phosphate transfection (eg, as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. NY), protoplast fusion, cationic liposome-mediated transfection; microparticle bombardment using tungsten particles (Johnston, Nature, 346: 776-777 (1990)); and coprecipitation of strontium phosphate and DNA (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)). In some aspects, a wash step is carried out in a centrifuge chamber, such as those manufactured and sold by Biosafe SA, including chambers for use with the Sepax® and Sepax® 2 system, including A-200/F and A-200 centrifuge chambers in manufacturer's instructions.

[0666] Other methods and vectors for transferring nucleic acids encoding recombinant products are those described, for example, in International Patent Application Publication No.: WO2014055668, and US Patent No. 7446190.

[0667] In some embodiments, cells, eg, T cells, can be transfected either during or after expansion, eg, using a T cell receptor (TCR) or a chimeric antigen receptor (CAR). This transfection to introduce the desired polypeptide or receptor gene can be carried out, for example, using any suitable retroviral vector. The genetically modified cell population can then be released from the original stimulus (eg, CD3/CD28 stimulus) and then stimulated using a second type of stimulus (eg, using a newly introduced receptor). This second type of stimulus may include an antigenic stimulus in the form of an MHC peptide/molecule, a cognate (cross-linking) ligand for a genetically introduced receptor (e.g., a naturally occurring CAR ligand), or any ligand (such as an antibody) that binds directly within the framework of the novel receptor ( for example, recognizing constant regions in a 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).

[0668] Among the introduced additional nucleic acids, for example, genes for introduction, included are nucleic acids that can improve the effectiveness of therapy, for example, by stimulating the viability and/or function of the transferred cells; genes to provide a genetic marker for selection and/or evaluation of cells, for example, to assess survival or localization in vivo ; genes to improve safety, for example, by rendering the cell susceptible to in vivo negative selection, as described in 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 by Lupton et al. describing the use of bifunctional selectable fusion genes resulting from the fusion of a dominant positive selectable marker with a negative selectable marker. See , for example , Riddell et al., U.S. Patent No. 6040177, in columns 14–17.

[0669] In some embodiments, cells are incubated and/or cultured prior to or in combination with genetic modification. The incubation steps may include culturing, culturing, stimulation, activation and/or propagation. The incubation and/or modification can be carried out in a culture vessel such as a unit, chamber, well, column, tube, tubing, valve, vial, culture dish, bag, or other cell culture or culture container. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulating agent. Such conditions include conditions designed to induce proliferation, expansion, activation and/or survival of cells in a population, to mimic antigen exposure, and/or to prime cells for genetic modification, for example, to introduce a recombinant antigen receptor. In some embodiments, one or more of the incubation steps may be carried out using a rocking bioreactor, such as a WAVE™ bioreactor (GE Healthcare) or BIOSTAT® RM (Sartorius). In some embodiments, one or more of the incubation steps may be carried out using a static bioreactor or incubation chamber. In specific embodiments, a shear reducing agent, such as poloxamer, may be added to the composition if the oscillating bioreactor is used for one or more of the incubation steps.

[0670] In some cases, a vector may be used that does not require cells, such as T cells, to be activated. In some of these cases, cells can be selected and/or transduced prior to activation. Thus, cells can be modified before or after cell culture, and, in some cases, during or during at least part of the culture.

[0671] Conditions may include one or more of specific environments, temperature, oxygen content, carbon dioxide content, time, means, such as nutrients, amino acids, antibiotics, ions, and/or stimulatory factors such as cytokines, chemokines, antigens, binding patterns, fusion proteins, recombinant soluble receptors, and any other means designed to activate cells. In some aspects, the cells are incubated in the presence of one or more cytokines, and in some embodiments, a cytokine cocktail may be used, for example, as described in International Patent Application Publication number WO2015157384. In some embodiments, cells are incubated with one or more cytokines and/or a cytokine cocktail before, during, or after transduction.

[0672] In some embodiments, the stimulatory conditions or agents include one or more agents, for example, a ligand capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the agent starts or initiates an intracellular TCR/CD3 signaling cascade in a T cell. Such agents may include antibodies, such as antibodies specific for a TCR component, for example, an anti-CD3 antibody. In some embodiments, the stimulatory conditions include one or more agents, such as a ligand capable of stimulating a costimulatory receptor, such as an anti-CD28 antibody. In some embodiments, such agents and/or ligands may be associated with a solid support, such as a bead, and/or with one or more cytokines. Optionally, the propagation method may further comprise the step of adding the anti-CD3 antibody and/or the anti-CD28 antibody to the culture medium (eg, at a concentration of at least about 0.5 ng/mL). In some embodiments, the stimulants include IL-2 and/or IL-15, eg, at an IL-2 concentration of at least about 10 units/mL. In specific embodiments, the stimulation conditions include incubation with any of the stimulation reagents described herein, such as those described in Section V-D-1.

[0673] In some aspects, the incubation is carried out in accordance with such methods as described in US Patent No. 60401 77 from 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. In some aspects, transduction is carried out using a system, device, apparatus and/or method as described in International Patent Application Publication No. WO2016/073602 or US 2016/0122782, the entire contents of which are incorporated by reference. In some embodiments, transduction is carried out according to the methods described in International Patent Application Publication number WO 2015/164675, the contents of which are hereby incorporated by reference.

[0674] In some embodiments, T cells are expanded by adding feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs), to the composition to start culture (e.g., such that the resulting cell population contains at least about 5 , 10, 20, or 40 or more PBMC feeder cells for each T-lymphocyte in the initial population to be expanded); and culture incubation (eg, for a period of time sufficient for these numbers of T cells to expand). In some aspects, non-dividing feeder cells may contain gamma irradiated PBMC feeder cells. In some embodiments, the PBMC is irradiated using gamma radiation in the range of about 3000 to 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 populations.

[0675] In some embodiments, the stimulating conditions include a temperature suitable for growth of human T-lymphocytes, e.g., at least about 25 degrees Celsius, typically at least about 30 degrees, and typically at or about 37 degrees Celsius. Optionally, the incubation may further include the addition of non-dividing transformed EBV lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated using gamma radiation in the range of approximately 6000 to 10000 rad. LCL feeder cells, in some aspects, are provided in any suitable amount, for example, in a ratio of LCL feeder cells to original T-lymphocytes of at least about 10:1.

[0676] In some embodiments, methods of preparation include the steps of freezing, eg, cryopreserving, cells, either before or after isolation, incubation, and/or modification. 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 freezing solution, for example, after a washing step to remove plasma and platelets. In some aspects, any of a variety of known freezing solutions and parameters may be used. One example includes the use of PBS containing 20% DMSO and 8% human serum albumin (HSA) or other suitable cell freezing media. This is then diluted 1:1 with medium so that the final concentrations of DMSO and HSA are 10% and 4%, respectively. Cells are then typically frozen to −80° C. at a rate of 1° per minute and stored in the vapor phase in a liquid nitrogen storage vessel. In some embodiments, the composition is enclosed in a bag suitable for cryopreservation (eg, CryoMacs® freeze bags, Miltenyi Biotec). In some embodiments, the composition is enclosed in a vial suitable for cryopreservation (eg CellSeal® vials, Cook Regentec). In some embodiments, the freezing steps and/or reagents are or include any of the freezing steps and/or reagents that are described herein.

[0677] In specific embodiments, the cells are frozen, for example, after a washing step, for example, to remove plasma and platelets. In some embodiments, the cells are frozen before, after and/or during any of the steps associated with making and/or obtaining cells, eg CD4+ and/or CD8+ T cells, that express a recombinant receptor, eg CAR. In specific embodiments, such steps may include any steps associated with obtaining modified cells, including, but not limited to, selecting and/or isolating a subset of cells, such as CD4+ and/or CD8+ T cells, stimulating and/or expanding cells, for example, T cells or subgroups thereof, or transfection or transduction of cells. In some embodiments, the cells are cells from a post-apheresis sample taken from an individual, prior to cell selection and/or isolation, cell stimulation and/or expansion, or cell transfection or transduction. In specific embodiments, the cells are frozen after the completion of the modification process, for example, after a process involving one or more steps of isolation, selection, stimulation, activation, transduction, transfection, and/or expansion.

[0678] In some embodiments, the cells are suspended in a solution for freezing, for example, in a cryoprotectant and/or in a solution containing a cryoprotectant. In some aspects, any of a variety of known freezing solutions and parameters may be used, including freezing solutions containing media or cryopreservation or glazing solutions containing a cryoprotectant. Suitable cryoprotectants include, but are not limited to, dimethyl sulfoxide (DMSO), glycerol, glycol, propylene glycol, ethylene glycol, propanediol, polyethylene glycol (PEG), 1,2-propanediol (PROH), or a mixture thereof. In some examples, the cryopreservation solution may contain one or more non-cell-penetrating cryopreservatives, including, but not limited to, polyvinylpyrrolidone, hydroxyethyl starch, polysaccharide, monosaccharide, alginate, trehalose, raffinose, dextran, human serum albumin, ficoll, lipoproteins, polyvinylpyrrolidone, hydroxyethyl starch, autologous plasma, or a mixture thereof. In some embodiments, the cells are suspended in a freezing solution with a final concentration of cryoprotectant between about 1% and about 20%, between about 3% and about 9%, or between about 6% and about 9% by volume, each included. In specific embodiments, the final concentration of cryoprotectant in the freezing solution is about 3%, about 4%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5% , about 8%, about 8.5%, about 9%, about 9.5%, or about 10% by volume.

[0679] In some embodiments, the cells are, for example, frozen at a specific cell density, such as a known or controlled cell density. In specific embodiments, cell density during the freezing process may affect cell death and/or cell damage occurring during and/or as a result of the freezing process.

[0680] For example, in specific embodiments, cell density affects the equilibrium, eg, osmotic equilibrium, with the environment during the freezing process. In some embodiments, this equilibrium is, includes, and/or results in dehydration. In specific embodiments, dehydration is or includes cellular dehydration that occurs upon contact, association, and/or incubation with a freezing solution, such as DMSO and/or a DMSO-containing solution. In specific embodiments, dehydration is or includes dehydration resulting from nucleation and an increase in ice crystals in the extracellular space, for example, through a decrease in the effective concentration of liquid water that affects cells. In some embodiments, cells are frozen at a cell density resulting in slower and/or less rapid dehydration than cells that are frozen at a different, eg higher or lower, cell density. In some embodiments, cells are frozen at a cell density resulting in approximately, at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80 %, 90%, 100%, 125%, 150%, 175%, 200%, 1 time, 2 times, 3 times, 4 times, 5 times, 10 times, 50 times or 100 times slower dehydration than for cells frozen at a different cell density, eg higher or lower, under the same or similar conditions.

[0681] In some embodiments, the cryoprotectant is DMSO. In specific embodiments, cells are suspended in freezing solution with a final concentration of DMSO between about 1% and about 20%, between about 3% and about 9%, or between about 6% and about 9% by volume, each included. In specific embodiments, the final concentration of DMSO in the freezing solution is about 3%, about 4%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5% , about 8%, about 8.5%, about 9%, about 9.5%, or about 10% by volume.

[0682] In specific embodiments, the cells are suspended in the freezing solution at a density between or between about 0.1 x 10 ⁶ cells/ml and about 5,000 x 10 ⁶ cells/ml, between or between about 1 x 10 ⁶ cells/ml and about 500 x 10 ⁶ cells/ml, between or between about 5 x 10 ⁶ cells/ml and about 150 x 10 ⁶ cells/ml, between or between about 10 x 10 ⁶ cells/ml and about 70 x 10 ⁶ cells/ml ml, or between or between about 15 x 10 ⁶ cells/ml and about 60 x 10 ⁶ cells/ml, each included. In some embodiments, the cells are viable cells.

[0683] In specific embodiments, the cells are suspended in the freezing solution at a density between or between about 1 x 10 ⁶ cells/ml and about 1 x 10 ⁸ cells/ml, between about 1 x 10 ⁶ cells/ml and about 2 x 10 ⁷ cells/ml, between about 1 x 10 ⁷ cells/ml and about 5 x 10 ⁷ cells/ml, or between about 1 x 10 ⁷ cells/ml and 5 x 10 ⁷ cells/ml, each included. In specific embodiments, the cells are suspended in the freezing solution at a density of about 1 x 10 ⁶ cells/ml, about 2 x 10 ⁶ cells/ml, about 5 x 10 ⁶ cells/ml, about 1 x 10 ⁷ cells/ml, about 1.5 x 10 ⁷ cells/ml, approx. 2 x 10 ⁷ cells/ml, approx. 2.5 x 10 ⁷ cells/ml, approx. 2.5 x 10 ⁷ cells/ml, approx. 2.5 x 10 ⁷ cells/ml ml, approximately 3 × 10 ⁷ cells/ml, approximately 3.5 × 10 ⁷ cells/ml, approximately 4 × 10 ⁷ cells/ml, approximately 4.5 × 10 ⁷ cells/ml, or approximately 5 × 10 ⁷ cells/ml ml, including each. In specific embodiments, the cells are suspended in the freezing solution at a density between about 1.5 x 10 ⁷ cells/ml and about 6 x 10 ⁷ cells/ml, inclusive. In specific embodiments, the cells are suspended in the freezing solution at a density of at least about 1 x 10 ⁷ cells/mL. In specific embodiments, the cells are suspended in the freezing solution at a density of at least about 1.5 x 10 ⁷ cells/mL. In some embodiments, the cells are viable cells.

[0684] In some embodiments, the cells are frozen in a container. In specific embodiments, the container is a freezing container and/or a cryoprotection container. Containers suitable for cryofreezing include, but are not limited to, ampoules, pouches such as plastic bags, and tubes. In specific embodiments, cells, e.g., cells from the same cell composition, such as a cell composition containing CAR expressing cells, are frozen at 1, 2, 3, 4, 5, 6, 7, 8, 9 10 or more than 10 separate containers. For example, in some embodiments, the cells and/or cell composition are suspended in a volume, such as a volume of solution, freezing solution, and/or cryoprotectant, and it exceeds the volume suitable for the container, and thus, the volume is placed in two or more container. In some embodiments, the volume is, is about, or less than 100 ml, 50 ml, 25 ml, 20 ml, 15 ml, 10 ml, 5 ml, or less than 5 ml, and the cells are frozen in two, three, four, five, six, seven, eight, nine, ten or more than ten individual ampoules. In specific embodiments, the same volume of cells is placed in each ampoule. In some embodiments, the ampoules are identical ampoules, eg, ampoules from the same cycle, model, and/or production lot. In specific embodiments, the volume is, is about, or is greater than 10 ml, 15 ml, 20 ml, 25 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, 100 ml, 120 ml, 150 ml, 200 ml or more than 200 ml, and the cells are frozen in two, three, four, five, six, seven, eight, nine, ten or more than ten individual bags. In specific embodiments, the same volume of cells is placed in each bag. In some embodiments, the pouches are identical pouches, such as pouches from the same cycle, model, and/or production lot.

[0685] In some embodiments, the container is an ampoule. In specific embodiments, the container is an ampoule with a fill volume of, approximately, or at least 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 11 ml, 12 ml, 13 ml, 14 ml, 15 ml, 16 ml, 17 ml, 18 ml, 19 ml, 20 ml, 25 ml, 30 ml, 35 ml, 40 ml, 45 ml or 50 ml. In some embodiments, the vial has a fill volume between 1 ml and 120 ml, 1 ml and 20 ml, 1 ml and 5 ml, 1 ml and 10 ml, 1 ml and 40 ml, or 20 ml and 40 ml, each included. In some embodiments, the vial is a freezing vial, a cryoprotection vial, and/or a cryo vial. Suitable ampoules are known and include, but are not limited to, CellSeal® ampoules (Cook Regentec), and the ampoules described in US Patent Nos.

[0686] In specific embodiments, the container is a package. In specific embodiments, the container is a pouch with a fill volume of, about, or at least 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml , 9 ml, 10 ml, 11 ml, 12 ml, 13 ml, 14 ml, 15 ml, 16 ml, 17 ml, 18 ml, 19 ml, 20 ml, 25 ml, 30 ml, 35 ml, 40 ml, 45 ml or 50 ml. In some embodiments, the package has a fill volume between 1 ml and 120 ml, 1 ml and 20 ml, 1 ml and 5 ml, 1 ml and 40 ml, 20 ml and 40 ml, 1 ml and 70 ml, or 50 ml and 70 ml including each. In some embodiments, the bag is filled with a volume of, about, or less than 100 ml, 75 ml, 70 ml, 50 ml, 25 ml, 20 ml, or 10 ml. Suitable bags are known and include, but are not limited to, CryoMacs® freezer bags (Miltenyi Biotec). In particular embodiments, the volume is the volume at room temperature. In some embodiments, the volume is the volume at between 37°C and 4°C, 16°C and 27°C, inclusive, or at, at about, or at least 16°C, 17°C, 18° C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C or 37°C. In some embodiments, the volume is the volume at 25°C.

[0687] In some embodiments, cells in a volume of medium or solution, such as freezing solution, between 1 ml and 20 ml, are frozen in one or more ampoules, inclusive. In some embodiments, one or more ampoules have a fill volume between 1 ml and 5 ml, inclusive. In specific embodiments, cells in a volume of medium or solution, eg, freezing solution, between 20 ml and 120 ml, inclusive, are frozen in one or more bags. In specific embodiments, one or more of the bags have a fill volume between 20 ml and 40 ml, inclusive. In some embodiments, cells in a volume of medium or solution, such as freezing solution, 120 ml or more, are frozen in one or more bags. In specific embodiments, one or more bags have a fill volume between 50 ml and 70 ml, inclusive.

[0688] In specific embodiments, the cells are frozen in a solution, eg, freezing solution, placed in a container, eg, a bag or vial, at a certain ratio of surface area to volume. In particular embodiments, the surface area to volume ratio is from or about 0.1 cm ^-1 to 100 cm ^-1 ; from 1 cm– ¹ to 50 cm– ¹ , from 1 cm– ¹ to 20 cm– ¹ , from 1 cm– ¹ to 10 cm– ¹ , from 2 cm– ¹ to 10 cm– ¹ , from 3 cm– ¹ to 7 cm– ¹ or 3 cm– ¹ to 6 cm– ¹ , each included. In particular embodiments, the surface area to volume ratio is between or between about 3 cm ^-1 and 6 cm ^-1 . In some embodiments, the surface area to volume ratio is, is approximately, or is at least 3 cm ^-1 , 4 cm ^-1 , 5 cm ^-1 , 6 cm ^-1 , or 7 cm ^-1 .

[0689] In some embodiments, transfer to the cryopreservation medium is associated with one or more processing steps, which may include washing the sample, e.g., the modified cell composition, e.g., to remove the medium and/or replace the cell medium with an appropriate cryopreservation buffer, or medium for subsequent freezing. In specific embodiments, the transfer to the cryopreservation medium is fully automated at the clinical scale level in a closed and sterile system. In specific embodiments, transfer to cryopreservation medium is carried out using the CliniMACS system (Miltenyi Biotec).

[0690] In some embodiments, the cells are thawed. In specific embodiments, the cells are thawed rapidly, eg, as quickly as possible, without overheating the cells or exposing the cells to high temperatures, eg, above 37°C. In some embodiments, rapid thawing reduces and/or prevents cells from being exposed to high concentrations of cryoprotectant and/or DMSO. In particular embodiments, the rate at which thawing occurs can be influenced by the properties of the container, such as the ampoule and/or bag, in which the cells are frozen and thawed. In specific embodiments, cells are thawed at a temperature of, about, or less than 37°C, 35°C, 32°C, 30°C, 29°C, 28°C, 27°C, 26°C, 25°C, 24°C, 23°C, 22°C, 21°C, 20°C or 15°C, or between 15°C and 30°C, between 23°C and 28°C or between 24° C and 26°C, including each. In some embodiments, the cells are thawed in a thermal block or in a water bath. In specific embodiments, cells are not thawed in a thermoblock or water bath. In some embodiments, cells are thawed at room temperature. In some embodiments, the wall thickness of the container affects the rate at which cells are thawed, such that, for example, cells in thick-walled containers thaw at a slower rate than those in thinner-walled containers. In some embodiments, containers having a low surface area to volume ratio have a slow and/or uneven defrosting rate. In some embodiments, cryopreserved cells are rapidly thawed in a container having a surface area to volume ratio of, about, or at least 1 cm ^{-1 .2} cm ^{-1 .3} cm ^{-1 .4} cm ^{-1 .5} cm– ¹ , 6 cm– ¹ or 7 cm– ¹ , 8 cm– ¹ , 9 cm– ¹ , or 10 cm– ¹ . In specific embodiments, cells are thawed in, about, or less than 120 minutes, 90 minutes, 60 minutes, 45 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, or ten minutes. In some embodiments, cells are thawed for between 10 minutes and 60 minutes, 15 minutes and 45 minutes, or 15 minutes and 25 minutes each. In specific embodiments, cells are thawed in, about, or less than 20 minutes.

[0691] In specific embodiments, the thawed cells are provided with a dormant period, such as incubation or culture, prior to administration or prior to any subsequent modification and/or processing steps. In some embodiments, cells are provided with a dormant period at low and/or undetectable amounts of a cryoprotectant, or in the absence of a cryoprotectant, such as DMSO. In specific embodiments, the thawed cells are provided with a dormant period after or immediately after washing steps, for example, to remove cryoprotectant and/or DMSO. In some embodiments, the rest period is or includes culturing and/or incubation at or about 37°C. In some embodiments, the rest period is conducted in the absence of any reagents, eg, stimulatory reagents, bead reagents, or recombinant cytokines used for and/or associated with any processing or modification step. In some embodiments, cells are provided with a dormant period of approximately or at least 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours , 12 hours, 18 hours or 24 hours. In specific embodiments, the cells are provided with a dormant period of approximately, or for at least 2 hours.

[0692] In some embodiments, cells are frozen, eg, cryopreserved, before, during, or after said cell processing and/or modification methods. In some embodiments, the freezing and subsequent thawing step removes granulocytes and, to some extent, monocytes in the cell population. Cells are then typically frozen to −80° C. at a rate of 1° per minute and stored in the vapor phase in a liquid nitrogen storage vessel. In some embodiments, the cells are actively and/or efficiently cooled at a rate of or at about 1° per minute using a controlled rate freezer. In some embodiments, cells may be frozen using a controlled rate freezer. In some aspects, rate controlled freezing devices are used to freeze cells with programmable cooling profiles, such as multiple cooling and/or heating profiles. Such freezing profiles can be programmed to control nucleation, eg ice formation, eg to reduce intracellular ice formation.

[0693] In some embodiments, frozen cell features, including any cells and compositions as described, such as cell compositions with a specific cell concentration or density, are frozen in the presence of a cryoprotectant and/or filled into a container with a specific volume or surface to volume ratio , include improved, increased and/or accelerated reproduction; improved, increased and/or enhanced cell survival and reduced incidence of cell death, such as necrosis, programmed cell death and/or apoptosis; improved, enhanced and/or increased activity, eg cytolytic activity; and/or a reduction in the incidence of aging or latency after thawing compared to cells frozen by alternative methods.

[0694] In specific embodiments, cells are frozen at the cell density and/or surface area to volume ratio presented herein and have reduced cell death, e.g., necrosis and/or apoptosis, during and/or as a result of freezing , cryofreezing and/or cryopreservation, as compared to cells frozen at a different cell density and/or surface area to volume ratio, under the same or similar conditions. In specific embodiments, cells are frozen at the cell density and/or surface area to volume ratio presented herein and have reduced delayed cell death, e.g., fewer cells that die, e.g., due to necrosis, programmed death cells or apoptosis, within 48 hours after freezing, cryopreservation and/or cryopreservation, for example, after thawing frozen cells. In specific embodiments, at least or about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% fewer cells die during and/or as a result of freezing and/or cryopreservation compared to cells frozen at a different cell density and/or surface area to volume ratio, under the same or similar conditions. In specific embodiments, less than 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.1%, or 0.01% of cells frozen at a given cell density and/or the ratio of surface area to volume, die during or as a result of freezing, cryopreservation and/or cryopreservation.

[0695] In some embodiments, cells are frozen at the cell density and/or surface area to volume ratio described herein and have a reduced incidence of aging or latency during and/or as a result of freezing, cryopreservation, and/or cryopreservation compared to cells frozen at a different cell density and/or a different surface area to volume ratio under the same or similar conditions. In specific embodiments, at least or about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% fewer cells represent are senescent and/or latent cells compared to cells frozen at a different cell density and/or a different surface area to volume ratio under the same or similar conditions. In specific embodiments, cells are frozen at a given cell density and/or surface area to volume ratio, and less than 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.1 % or 0.01% of cells become senescent and/or latent as a result of freezing, cryopreservation and/or cryopreservation.

[0696] In specific embodiments, the cells are frozen, e.g., cryopreserved, at the cell density and/or surface area to volume ratio described herein, and have improved, accelerated, and/or faster proliferation, e.g., in stimulating conditions, such as incubation with the stimulating reagent described herein after cells are thawed, as compared to cells frozen at a different cell density and/or surface area to volume ratio, under the same or similar conditions. In specific embodiments, cells proliferate at a rate that is accelerated and/or faster by, about, or at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60% , 70%, 80%, 90%, 100%, 150%, 200%, 1x, 1.5x, 2x, 3x, 4x, 5x, or 10x compared to cells frozen at a different cell density and/or a different surface area to volume ratio, under the same or similar conditions. For example, in some embodiments, the thawed cells reach a multiplication threshold, such as a predetermined number, cell density, or a factor such as 2x multiplication, within 10%, within about 10%. , 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% less time than thawed cells that were frozen at a different cell density and/or different surface area to volume ratio, under the same or similar conditions.

[0697] In some embodiments, cells are frozen, e.g., cryo-freezed, at a certain cell density, and have improved, increased, and/or greater cytolytic activity, e.g., as measured by any assay for measuring cytolytic activity described herein, after cells are thawed, compared to cells frozen at a different cell density, such as a higher or lower density, under the same or similar conditions. In specific embodiments, the cytolytic activity is increased by, about, or at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 1x, 1.5x, 2x, 3x, 4x, 5x or 10x compared to cells frozen at different densities under the same or similar conditions.

VI. COMPOSITIONS AND COMPOSITIONS

[0698] The present invention also relates to compositions comprising cells, including pharmaceutical compositions and compositions, for example, a unit dosage form of the compositions, including the number of cells to be administered in a given dose or fraction thereof. Pharmaceutical compositions and formulations typically include one or more optional pharmaceutically acceptable carriers or excipients. In some embodiments, the composition includes at least one additional drug.

[0699] The term "pharmaceutical formulation" refers to a preparation that is in such a form as to ensure the effectiveness of the biological activity of the active ingredient contained therein, and which does not contain additional components that are unacceptably toxic to the individual to whom the formulation will be administered.

[0700] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than the active ingredient, that is non-toxic to an individual. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0701] In some aspects, the choice of carrier is determined in part by the particular cell and/or route of administration. Accordingly, there are a variety of 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 preservatives, or mixtures thereof, are typically present in an amount of from about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally 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; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (less than about 10 residues); 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 (for example, complexes Zn-protein); and/or non-ionic surfactants such as polyethylene glycol (PEG).

[0702] Buffering agents, 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 buffering agents is used. Buffering agents, or mixtures thereof, are typically present in an amount of from about 0.001% to about 4% by weight of the total composition. Methods for preparing pharmaceutical compositions suitable for administration are known. Illustrative methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins; 21st ed. (May 1, 2005).

[0703] The compositions may include aqueous solutions. The composition or composition may also contain more than one active ingredient that can be used for a particular sign, disease or condition being treated using cells, preferably ingredients with activity that complements the activity of cells, where the respective activities do not adversely affect each other. Such active ingredients are suitably 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 and/or vincristine.

[0704] In some embodiments, the composition includes cells in an amount effective to reduce the burden of the disease or condition, and/or in an amount that does not lead to CRS or severe CRS in the individual, and/or does not cause any of the other outcomes for methods as described in the present description.

[0705] The pharmaceutical composition, in some embodiments, contains 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, in some embodiments, is monitored by periodically evaluating treated individuals. The desired dose can be delivered by a single bolus of cells, by multiple boluses of cells, or by administering cells by continuous infusion.

[0706] Cells and compositions can be administered using standard methods, formulations and/or administration devices. Cell administration may be autologous or heterologous. For example, immunoreactive cells or progenitors can be obtained from one individual and administered to the same individual or another compatible individual. Peripheral blood-derived immunoreactive cells or their progeny (eg, derived in vivo , ex vivo , or in vitro ) can be administered by localized injection, including catheter, systemic injection, local injection, intravenous injection, or parenteral administration. When a therapeutic composition (eg, a pharmaceutical composition containing genetically modified immunoreactive cells) is administered, it is typically presented in a single dose injectable form (solution, suspension, emulsion).

[0707] Formulations include formulations for oral, intravenous, intraperitoneal, subcutaneous, intrapulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term "parenteral", within the scope of the invention, includes intravenous, intramuscular, subcutaneous, rectal, vaginal and intraperitoneal administration. In some embodiments, the cells are administered to an individual using peripheral systemic delivery via intravenous, intraperitoneal, or subcutaneous injection.

[0708] Compositions, in some embodiments, are in the form of sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which can, in some aspects, 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 within an appropriate viscosity range to provide longer periods of contact with specific tissues. Liquid or viscous compositions may contain carriers, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyol (eg glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

[0709] Sterile injectable solutions can be obtained by incorporating the cells into a solvent, for example, in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions may contain adjuvants such as wetting, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing agents, preservatives, flavoring agents and/or coloring agents, depending on the desired route of administration and preparation. In some aspects, standard guidelines may be consulted to obtain suitable preparations.

[0710] Various additives that enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol and sorbic acid. Sustained absorption of an injectable pharmaceutical form can be obtained using absorption delaying agents such as aluminum monostearate and gelatin.

[0711] Formulations to be used for in vivo administration are generally sterile. Sterility can easily be achieved, for example, by filtration through membranes for sterilization by filtration.

[0712] In some embodiments, the T cell therapeutic composition contains between about 10 million cells per ml and about 70 million cells per ml, or between about 10 million viable cells per ml and about 70 million viable cells per ml, including each. In some embodiments, the T cell therapeutic composition contains between about 15 million cells or viable cells per ml and about 60 million cells or viable cells per ml, including each. In some embodiments, the T cell composition contains more than 10 million cells or viable cells per ml. In some embodiments, the T cell therapeutic composition contains more than 15 million cells, or more than 15 million cells per ml.

[0713] In some embodiments, the product further comprises information indicating that the container, such as a vial or a pouch such as an IV pouch, or a syringe, contains the target number of units.

[0714] In some embodiments of the product, the container is the first container and the product further comprises additional containers, where each of the additional containers contains a unit dose containing the target number of T cell composition units. In some embodiments, the additional containers contain between about 10 million cells or viable cells per ml and about 70 million cells or viable cells per ml, between about 15 million cells or viable cells and about 60 million cells or viable cells per ml, more than 10 million cells or viable cells per ml, more than 15 million cells or viable cells per ml, including each, or a combination thereof. In some embodiments, the composition further comprises a cryoprotectant and/or the article of manufacture further comprises instructions for thawing the composition prior to administration to an individual.

[0715] In some embodiments, cells are suspended in a freezing solution, for example, after a washing step to remove plasma and platelets. In some aspects, any of a variety of known freezing solutions and parameters may be used. One example includes the use of PBS containing 20% DMSO and 8% human serum albumin (HSA) or other suitable cell freezing media. Then they are diluted with medium 1:1, so that the final concentration of DMSO and HSA were 10% and 4%, respectively.

[0716] In some aspects, any of a variety of known freezing solutions and parameters may be used. In some embodiments, the cell sample may contain a cryopreservation or glazing medium or solution containing a cryoprotectant. Suitable cryoprotectants include, but are not limited to, dimethyl sulfoxide (DMSO), glycerol, glycol, propylene glycol, ethylene glycol, propanediol, polyethylene glycol (PEG), 1,2-propanediol (PROH), or a mixture thereof. In some examples, the cryopreservation solution may contain one or more non-cell-penetrating cryopreservatives, including, but not limited to, polyvinylpyrrolidone, hydroxyethyl starch, polysaccharide, monosaccharide, alginate, trehalose, raffinose, dextran, human serum albumin, ficoll, lipoproteins, polyvinylpyrrolidone, hydroxyethyl starch, autologous plasma, or a mixture thereof. In some embodiments, the cells are suspended in a freezing solution with a final concentration of cryoprotectant between about 1% and about 20%, between about 3% and about 9%, or between about 6% and about 9% by volume, each included. In specific embodiments, the final concentration of cryoprotectant in the freezing solution is about 3%, about 4%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5% , about 8%, about 8.5%, about 9%, about 9.5% or about 10% by volume.

[0717] In some embodiments, the cryoprotectant is DMSO. In specific embodiments, cells are suspended in freezing solution with a final concentration of DMSO between about 1% and about 20%, between about 3% and about 9%, or between about 6% and about 9% by volume, each included. In specific embodiments, the final concentration of DMSO in the freezing solution is about 3%, about 4%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5% , about 8%, about 8.5%, about 9%, about 9.5% or about 10% by volume.

[0718] In specific embodiments, the cells are suspended in a freezing solution at a density between about 1×ten⁶ cells/ml and approximately 1×ten^eightcells/ml, between approximately 1×ten⁶ cells/ml and approximately 2×ten⁷ cells/ml, between approximately 1×ten⁷ cells/ml and approximately 5×ten⁷ cells/ml, or between approximately 1×ten⁷ cells/ml up to 5×10⁷ cells/ml, including each. In specific embodiments, the cells are suspended in a freezing solution at a density of approximately 1×ten⁶cells/ml, approximately 2×ten⁶cells/ml, approximately 5×ten⁶cells/ml, approximately 1×ten⁷cells/ml, approximately 1.5×ten⁷cells/ml, approximately 2×ten⁷cells/ml, approximately 2.5×ten⁷cells/ml, approximately 2.5×ten⁷cells/ml, approximately 2.5×ten⁷cells/ml, approximately 3×ten⁷cells/ml, approximately 3.5×ten⁷cells/ml, approximately 4×ten⁷cells/ml, approximately 4.5×ten⁷cells/ml, or approximately 5×ten⁷cells/ml. In specific embodiments, the cells are suspended in a freezing solution at a density between about 1.5×ten⁷cells/ml and approximately 6×ten⁷cells/ml, inclusive. In specific embodiments, the cells are suspended in a freezing solution at a density of at least about 1 x 10⁷cells/ml. In specific embodiments, the cells are suspended in a freezing solution at a density of at least about 1.5 x 107 cells/ml. In some embodiments, the cells are viable cells.

[0719] In some embodiments, transfer to the cryopreservation medium is associated with one or more processing steps, which may include washing the sample, e.g., the modified cell composition, e.g., to remove the medium and/or replace the cell medium with an appropriate cryopreservation buffer, or medium for subsequent freezing. In specific embodiments, the transfer to the cryopreservation medium is fully automated at the clinical scale level in a closed and sterile system. In specific embodiments, transfer to cryopreservation medium is carried out using the CliniMACS system (Miltenyi Biotec).

[0720] In some embodiments, cells are frozen, eg, cryopreserved, before, during, or after said methods for cell processing and/or modification. In some embodiments, the freezing and subsequent thawing step removes granulocytes and, to some extent, monocytes in the cell population. Cells are then typically frozen to −80° C. at a rate of 1° per minute and stored in the vapor phase in a liquid nitrogen storage vessel.

[0721] In some embodiments, the composition is enclosed in a bag suitable for cryopreservation (eg, CryoMacs® freeze bags, Miltenyi Biotec). In some embodiments, the composition is enclosed in a vial suitable for cryopreservation (eg CellSeal® vials, Cook Regentec).

VII. PRODUCTS

[0722] The present invention also relates to articles, such as kits and devices, for administering cells to individuals in accordance with the presented methods of adoptive cell therapy, and for storing and administering cells and compositions.

[0723] The articles include one or more containers, typically a plurality of containers, a packaging material, and a label or package insert on or associated with the container or containers and/or packaging, typically including instructions for administering the cells to an individual.

[0724] The containers typically contain the cells to be administered, for example, one or more of their unit doses. The product typically includes a plurality of containers, each containing a single unit dose of cells. A single dose may be the number or number of cells to be administered to the individual in the first dose, or double the number (or more) of cells to be administered in the first or any one or more consecutive dose(s). It may be the lowest dose or the lowest possible dose of cells that is administered to an individual in conjunction with the route of administration. In some embodiments, a unit dose is the minimum number of cells, or the number of cells, or the minimum number of reference units, or the target number of reference units, or reference units within the target range, that can be administered in a single dose to any individual having a particular disease or condition. , or any individual, in accordance with the methods in the present description. In some embodiments, the number of cells per unit dose is the number of cells, or the number of recombinant receptor-expressing or CAR-expressing cells, or the number of such cells of a particular phenotype, e.g., CD8+, apoptosis marker negative (e.g., annexin V- or caspase 3-), and CD8+, which is desirable to enter a specific individual at a dose, for example, the individual from which the cells originate. In some embodiments, the target number of reference units or reference units in the target range per unit dose is the number of reference units from the composition as a function of the number of cells, or the number of recombinant receptor-expressing or CAR-expressing cells, or the number of such cells of a particular phenotype, e.g., CD8+ , an apoptosis marker is negative (e.g., annexin V- or caspase 3-) and CD8+, and a recombinant receptor-dependent activity (e.g., antigen-specific activity) of the composition, and is desirably administered to a particular individual at a dose, e.g., the individual from which the cells are derived. In some embodiments, the cells are obtained from or in need of an individual to be treated using the methods described herein.

[0725] In some embodiments, the product contains a unit dose of cells containing a target number of reference units (RU) within a given range, for example, in accordance with the formula, as described elsewhere in the present description. Exemplary unit doses of target reference units (RU) or target total cells include any as described throughout this specification.

[0726] In some embodiments, each of the containers individually contains a single dose of cells, for example, including the same or substantially the same number of cells, or the number of recombinant receptor-expressing or CAR-expressing cells, or the number of such cells of a particular phenotype, for example, CD8+, a marker apoptosis negative (eg, annexin V– or caspase 3–). In some embodiments, each of the containers individually contains a unit dose of cells, for example, including the same or substantially the same number of target reference units or the number of reference units within the target range.

[0727] Suitable containers include, for example, bottles, vials, syringes, and flexible bags such as infusion bags. In specific embodiments, the containers are pouches, eg, flexible pouches, such as pouches suitable for infusing cells into individuals, eg, flexible plastic or PVC pouches, and/or IV solution pouches. The bags, in some embodiments, can be sealed and/or can be sterilized so as to ensure the sterility of the solution and delivery of cells and compositions. In some embodiments, containers, such as bags, have a capacity of or about, or at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1000 ml, such as between 10 or about 10 and 100 or about 100 or between 10 or about 10 and 500 or about 500 ml, each included. In some embodiments, the containers, e.g., pouches, are composed of and/or made from a material that is stable and/or allows stable storage and/or maintenance of cells at one or more of various temperatures, such as low temperatures, e.g. or approximately, or equal to or approximately equal to -20°C, -80°C, -120°C, 135°C and/or temperatures suitable for cryopreservation and/or other temperatures such as temperatures suitable for thawing cells, and body temperature, such as 37° C. or about 37° C., for example, to allow defrosting, for example, at the location of the individual or treatment, for example, at the site immediately prior to treatment.

[0728] Containers can be made from a variety of materials such as glass or plastic. In some embodiments, the container has one or more openings, for example, openings for sterile access, for example, for connecting tubing or cannulation with one or more tubing, for example, for intravenous or other infusion, and/or for connection for transfer to other containers to and from other containers such as cell culture and/or storage bags or other containers. Exemplary containers include infusion bags, intravenous solution bags, vials, including vials with stoppers that can be pierced with an injection needle.

[0729] The product may further include a package insert or label with one or more sections of identification information and/or instructions for use. In some embodiments, the information or instructions indicate that the content can or should be used to treat a particular condition or disease and/or provide instructions for doing so. The label or package insert may indicate that the contents of the product are for the treatment of a disease or condition. In some embodiments, the label or package insert provides instructions for treating an individual, e.g., an individual from which the cells are derived, by a method that includes administering a first and one or more subsequent doses of cells, for example, in accordance with any of the embodiments of the presented ways. In some embodiments, the instructions indicate the administration, in the first dose, of one single dose, e.g., the contents of one individual container from the article, followed by one or more consecutive doses at a specified time point or within a specified time window, and/or after detecting the presence or absence, or amount, or level of one or more factors or outcomes in an individual.

[0730] In some embodiments, the instructions indicate the administration of one or more unit doses to an individual.

[0731] In some embodiments, the label or package insert or package contains an identifier to indicate the specific identity of the individual from which the cells originate and/or to which the cells are intended to be administered. In the case of autologous transfer, the identity of the individual from whom the cells originate is the same as the identity of the individual to whom the cells are intended to be administered. Thus, the identification information may indicate that the cells are intended for administration to a particular patient, eg the one from whom the cells were originally obtained. Such information may be present on the packaging material and/or label in the form of a barcode or other coded identifier, or may indicate the name and/or other identifying characteristics of the individual.

[0732] The product, in some embodiments, includes one or more, usually a plurality, containers containing compositions containing cells, for example, in the form of their individual unit doses, and further includes one or more additional containers with the composition contained therein. , comprising an additional agent, such as a cytotoxic or other drug, for example, intended for administration in combination, for example, simultaneously or sequentially, in any order, with cells. Alternatively or additionally, the product may further comprise a different or the same container containing a pharmaceutically acceptable buffer. It may further include other materials such as other buffers, diluents, filters, tubing, needles and/or syringes.

[0733] The term "package insert" is used to refer to instructions commonly included in commercial packages of therapeutic products that contain information regarding the indications, use, dosage, administration, combination therapy, contraindications and / or warnings regarding the use of such therapeutic products.

VIII. DEFINITIONS

[0734] Unless otherwise specified, all technical terms, nomenclature and other technical and scientific terms or terminology, within the scope of the invention, are intended to have the same meaning as is generally accepted by a person skilled in the art to which the claimed subject matter belongs. In some instances, terms with well-known meanings are defined herein for clarity and/or reference, and the inclusion of such definitions herein should not necessarily be construed as representing significant differences from what is commonly known in the art.

[0735] Within the scope of the invention, singular forms include plural reference objects unless the context clearly requires otherwise. For example, the singular form means "at least one", or "one or more". It is understood that aspects and variants described herein include "consisting of" and/or "primarily consisting of" aspects and variants.

[0736] Throughout this description, various aspects of the claimed subject matter 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 subject matter. Accordingly, a description of a range should be considered as specifically describing all possible subranges, as well as individual numerical values within that range. For example, when a range of values is presented, it is understood that every intermediate value, between the upper and lower limits of that range, and any other specified or intermediate value within that specified range, is included in the claimed subject matter. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and also included in the claimed subject matter, limited to any specifically excluded limit in the specified range. When the specified range includes one or both of the limits, ranges excluding either or both of these included limits are also included in the described or claimed subject matter. This applies regardless of the range width.

[0737] The term "approximately", within the scope of the invention, refers to the usual range of error for the corresponding value, already known to the person skilled in the art. Reference to an "approximate" value or parameter in this specification includes (and describes) embodiments that pertain to that value or parameter per se. For example, a description referring to "about X" includes a description of "X".

[0738] 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.

[0739] 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 applied to a surface marker, the term refers to the presence of surface expression, as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker, and detecting said antibody, where the staining is detectable by flow cytometry at a level well above staining, detected by the same method for an isotype-matched control, under otherwise identical conditions, and/or at a level substantially similar to that of a cell known to be positive for that marker, and/or at a level well above that of cells are known to be negative for this marker.

[0740] 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 significant detectable presence on or in the cell of a particular marker, typically a surface marker. When applied to a surface marker, the term refers to the absence of surface expression, as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker, and detection of said antibody, where the staining is not detected by flow cytometry at a level well above staining, detected by the same method for an isotype-matched control, under otherwise identical conditions, and/or at a level well below that of a cell known to be positive for that marker, and/or at a level substantially similar to that of cells are known to be negative for this marker.

[0741] 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 inserted into the genome of the host cell into which it is 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".

IX. ILLUSTRATIVE EMBODIMENTS

1. An article containing:

(a) a container containing a unit dose of a T cell therapeutic composition comprising T cells containing a recombinant receptor, optionally a chimeric antigen receptor (CAR) that specifically binds to an antigen, wherein the unit dose contains the target number of reference units (RU ) within a given range, where RU in a given composition is determined by the formula

RU=A x B, where:

A is the number of cells, or a multiple, proportion or transformation, of some phenotype present in a given composition, or is the average or weighted average of the number of cells, or a multiple, proportion or transformation, of two or more phenotypes in a given composition; and

B is a parameter value, or a fraction thereof, or a multiple, or a transformation that indicates the degree of, or correlates with, the degree of recombinant receptor dependent, optionally CAR dependent, activity in the composition; and

(b) instructions for administering the composition, optionally one or more unit doses thereof, to an individual, optionally an individual having or suspected of having a disease or condition.

2. The product of embodiment 1, where A is the number of cells, or a multiple thereof, or proportion, or transformation, of a given phenotype present in a given composition.

3. The product of embodiment 1, where A is the average or weighted average of the number of cells, or its multiple, proportion or conversion, of two or more phenotypes in a given composition.

4. An article of any one of embodiments 1-3, wherein the target number of units is less than a threshold number of units, optionally a safe number of reference units, where the safe number of reference units is, in relation to a group of individuals analyzed after treatment with using a T cell therapeutic composition comprising T cells expressing a recombinant receptor, optionally CAR, the lowest number of drug reference units administered to an individual among those individuals in the group who continued to develop an adverse event.

5. The article of embodiment 4, wherein the adverse event is a serious adverse event, optionally severe neurotoxicity, optionally a grade equal to or greater than grade 4 or grade 5, or at least long-term grade 3 neurotoxicity.

6. The product of Embodiment 4 or Embodiment 5 where the target number of reference units is less than the safe number of reference units by an amount corresponding to a safety factor and/or by an amount in the range of 1.5 to 3 times , optionally approximately 2-fold, or a multiple of the standard deviation in a group of individuals who did not experience an adverse event, optionally, neurotoxicity grade 0-2, optionally, where the multiple lies in the range from 1.5- to 3-fold .

7. The product of any of embodiments 1-6, wherein the target number of reference units is equal to or greater than the reference effective amount of reference units, where the reference effective amount is, in relation to a group of individuals analyzed after treatment with a therapeutic T cell composition containing recombinant receptor, optionally CAR, number of drug units administered to one or more individuals within a group for which the desired therapeutic outcome is indicated, optionally a partial response or complete response (CR).

8. An article containing:

(a) a container containing a unit dose of a T cell therapeutic composition comprising T cells containing a recombinant receptor, optionally a chimeric antigen receptor (CAR), that specifically binds to an antigen, wherein:

the unit dose contains or contains approximately (i) the target number of total recombinant receptor-expressing cells, or the target number of total CD3+ recombinant receptor-expressing cells, or the target number of total CD8+ recombinant receptor-expressing cells, or (ii) the target number of reference units (RU) within a given the range where the target number of reference RUs is equal to or less than the threshold number of RUs, where the unit dose does not contain more than the threshold RU value,

where the amount of RU in a given composition is determined by the formula:

RU=A x B, where

A is the number of cells, or a multiple, proportion or transformation, of some phenotype present in a given composition, or is the average or weighted average of the number of cells, or a multiple, proportion or transformation, of two or more phenotypes in a given composition; and

B is a parameter value, or a multiple thereof, or a transformation that indicates the degree of or correlates with the degree of recombinant receptor dependent, optionally CAR dependent, activity in a given T cell composition; and

(b) instructions for administering the composition, optionally one or more unit doses thereof, to an individual, optionally an individual having or suspected of having a disease or condition.

9. The product of embodiment 8, where A is the number of cells, or a multiple thereof, or proportion, or transformation, of a given phenotype present in a given composition.

10. The article of embodiment 8, wherein A is the average or weighted average of the number of cells, or a multiple thereof, proportion or conversion, of two or more phenotypes in a given composition.

11. The product of any of embodiments 8-10, where:

the target amount is the target amount of recombinant receptor-expressing CD3+ cells that are negative (-) for an apoptosis marker and CD3+, optionally, where the apoptosis marker is annexin V or active caspase 3; or

the target amount is the target amount of recombinant receptor-expressing CD8+ cells that are negative (-) for the apoptosis marker and CD8+, optionally, where the apoptosis marker is annexin V or active caspase 3.

12. An article of any one of embodiments 1-11, wherein the target number of cells in (i) is: between and between about 5.0×10 ⁶ and 2.25×10 ⁷ , 5.0×10 ⁶ and 2.0 ×10 ⁷ , 5.0×10 ⁶ and 1.5×10 ⁷ , 5.0×10 ⁶ and 1.0×10 ⁷ , 5.0×10 ⁶ and 7.5×10 ⁶ , 7.5× 10 ⁶ and 2.25×10 ⁷ , 7.5×10 ⁶ and 2.0×10 ⁷ , 7.5×10 ⁶ and 1.5×10 ⁷ , 7.5×10 ⁶ and 1.0×10 ⁷ , 1.0×10 ⁷ and 2.25×10 ⁷ , 1.0×10 ⁷ and 2.0×10 ⁷ , 1.0×10 ⁷ and 1.5×10 ⁷ , 1.5×10 ⁷ and 2.25×10 ⁷ , 1.5×10 ⁷ and 2.0×10 ⁷ , 2.0×10 ⁷ and 2.25×10 ⁷ recombinant receptor expressing cells, including each optionally recombinant receptor expressing cells, which are CD3+ or CD8+ or which are negative (-) for the apoptosis marker and CD8+, optionally, where the apoptosis marker is annexin V or active caspase 3.

13. An article of any one of embodiments 1-12, wherein the target number of cells in (i) is:

between at least or at least about 5x10 ⁶ , 6x10 ⁶ , 7x10 ⁶ , 8x10 ⁶ , 9x10 ⁶ , 10x10 ⁶ , and about 15x10 ⁶ expressing the recombinant receptor, including each optionally expressing the recombinant receptor cells that are CD8+, or that are negative (-) for an apoptosis marker and CD8+, optionally, where the apoptosis marker is annexin V or active caspase 3;

between at least or at least about 5.55×10 ⁶ , 6.66×10 ⁶ , 7.77×10 ⁶ , 8.99×10 ⁶ , 1.0×10 ⁷ , 1.1×10 ⁷ and approximately 1.67×10 ⁷ expressing the recombinant receptor, including each optionally expressing the recombinant receptor cells that are CD8+, or which are negative (-) for the apoptosis marker and CD8+, optionally, where the apoptosis marker is annexin V or active caspase 3;

between at least or at least about 6.25×10 ⁶ , 7.5×10 ⁶ , 8.75×10 ⁶ , 1.0×10 ⁷ , 1.13×10 ⁷ , 1.25×10 ⁷ and approximately 1.9 x 10 ⁷ expressing the recombinant receptor, including each optionally expressing the recombinant receptor cells that are CD8+ or that are negative (-) for the apoptosis marker and CD8+, optionally, where the apoptosis marker is annexin V or active caspase 3;

between at least or at least about 7.14×10 ⁶ , 8.5×10 ⁶ , 1.0×10 ⁷ , 1.14×10 ⁷ , 1.29×10 ⁷ , 1.42×10 ⁷ and about 2.14 x 10 ⁷ expressing the recombinant receptor, including each optionally expressing the recombinant receptor cells that are CD8+ or that are negative (-) for the apoptosis marker and CD8+, optionally, where the apoptosis marker is annexin V or active caspase 3.

14. An article of any one of embodiments 1-13, wherein the target number of cells in (i) is between at least or at least about 5×10 ⁶ , 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9 x 10 ⁶ , 10 x 10 ⁶ and approximately 15 x 10 ⁶ recombinant receptor expressing cells that are negative (-) for an apoptosis marker and CD8+, including each optionally where the apoptosis marker is annexin V or active caspase 3.

15. An article of any one of embodiments 1-13, wherein the target number of cells in (i) is between at least or at least about 6.25×10 ⁶ , 7.5×10 ⁶ , 8.75×10 ⁶ , 1.0×10 ⁷ , 1.13×10 ⁷ , 1.25×10 ⁷ , and approximately 1.9×10 ⁷ recombinant receptor-expressing CD8+ cells, including each.

16. The product of any of embodiments 8-15, wherein the target reference amount of RU is less than a threshold number of units or is less than a reference safe amount of RU, where the reference safe amount of RU is, in relation to a group of individuals analyzed after treatment with using a T cell therapeutic composition comprising T cells expressing a recombinant receptor, optionally CAR, the lowest number of drug reference units administered to an individual among those individuals in the group who continued to develop an adverse event.

17. The product of Embodiment 16, wherein the adverse event is a serious adverse event, optionally a severe grade 4 or grade 5 neurotoxicity equal to or greater than grade 5 neurotoxicity, or at least grade 3 long-term neurotoxicity.

18. The product of Embodiment 16 or Embodiment 17, wherein the target reference amount of RUs is less than the safety number of reference units by an amount corresponding to a safety factor or at least 2 times.

19. The article of any of embodiments 8-18, where the target number of reference units is or exceeds the reference effective amount of reference units, where the reference effective amount is, in relation to a group of individuals analyzed after treatment using a composition of T-cells containing a recombinant receptor , optionally CAR, number of drug reference units administered to one or more individuals within a group for which the desired therapeutic outcome is shown, optionally, complete response (CR).

20. An article of any one of embodiments 1-19, wherein A is the number of cells of some phenotype present in the composition and B is a parameter value that indicates the degree of or correlates with the degree of recombinant receptor dependent activity in the composition.

21. The product of any one of Embodiments 1-20, where A and/or B is a quantity or value transformation, respectively, where the transformation includes a logarithmic transformation, a power transformation, or a logit transformation.

22. An article of any one of embodiments 1-21, wherein A is the number of cells of some phenotype present in the composition and B is a multiple or transformation of a parameter value indicative of the degree of or correlated with the degree of CAR-dependent activity in the composition T-cells, optionally, where B is the logarithmic transformation of the value.

23. The product of Embodiment 21 or Embodiment 22, where the logarithmic transformation is decimal log (log ₁₀ (x)), natural log (ln(x)) or binary log (log ₂ (x)).

24. An article of any one of embodiments 1-23, wherein A is the number of viable cells in the composition and/or is the number of cells that are not apoptotic, do not have a factor indicative of early apoptosis or apoptosis, are not in the early stages apoptosis, or are not in the late stages of apoptosis, and/or is the number of cells in a certain state of differentiation, and/or is the number of cells having a memory cell/stem cell trait, or represents its multiple or transformation.

25. The article of manufacture of any of embodiments 1-24, wherein the phenotype includes positive expression of a surface marker that is one or more of CD3, CD4, or CD8, and/or includes positive expression of a recombinant receptor, optionally CAR, or a surrogate marker for the expression of a recombinant receptor.

26. The product of embodiment 25, wherein the phenotype is CD3+ CAR, CD4+/CAR+, CD8+/CAR+.

27. The article of manufacture of any one of embodiments 1-26, wherein the phenotype includes the absence of a factor indicative of apoptosis or one or more steps in the apoptotic cascade or pathway, optionally expression of an apoptosis marker.

28. The article of manufacture of any one of embodiments 1-27, wherein the phenotype comprises negative expression of an apoptosis marker, optionally an early apoptosis or late apoptosis marker.

29. The product of embodiment 28, wherein the apoptosis marker is surface phosphatidylserine and/or detected using annexin V, or is an active caspase or a caspase proform, optionally an active caspase 3 or a caspase 3 proform.

30. The article of any of embodiments 1-29, wherein the phenotype includes annexin-.

31. The product of any of embodiments 1-30, where the phenotype includes an indicator of the production of one or a combination of cytokines, optionally not specific for the antigen or recombinant receptor and / or polyclonal production, where one or more cytokines is IL-2, IL- 13, IL-17, IFN-gamma or TNF-alpha.

32. The article of embodiment 31, wherein the production index is measured in an assay, optionally an intracellular cytokine staining assay, comprising incubating a sample from a T cell composition with a polyclonal agent, an antigen specific agent, or a recombinant receptor binding agent, optionally CAR.

33. The article of Embodiment 31 or Embodiment 32, wherein the agent is or contains PMA and ionomycin, or is or contains a T-cell receptor or a T-cell receptor complex agonist.

34. An article of manufacture of any of embodiments 1-33, wherein the phenotype includes negative expression of an activation marker, wherein the activation marker is selected from CD25, CD127, LAG3, Ki67, and combinations thereof.

35. The article of manufacture of any of embodiments 1-34, wherein the phenotype includes negative expression of a depletion marker, wherein the depletion marker is a PD1 or FOXP3 gene product, or a combination thereof.

36. The article of any one of embodiments 1-35 wherein the phenotype comprises a naïve or memory cell phenotype, optionally wherein the memory cell phenotype comprises an effector memory T cell phenotype, a central memory T cell phenotype, or an effector memory T cell phenotype expressing CD45RA (Temra).

37. An article of any one of embodiments 1-36, where A is total T cells, total CD3+ cells, total CD4+ or CD8+ cells, total CD3+CAR+ cells, total CD8+CAR+ cells, total CD4+ CAR+, or total number of living or viable cells from any of the above, or its multiple or converted value.

38. The article of any one of embodiments 1-37, where A is total CD3+ cells, total CD8+, total CD3+CAR+ cells, total CD8+CAR+ cells, or total live or viable cells from any of the above , or its multiple or converted value.

39. The article of any one of embodiments 1-38, wherein A is the total number of apoptosis marker negative (-) cells representing CD3+ CAR+ cells, the total number of apoptosis marker negative (-) cells representing CD4+ CAR+, total the number of cells negative (-) for the apoptosis marker, which are CD8+ CAR+ cells, or its multiple or converted value, where the apoptosis marker is annexin V or active caspase 3.

40. An article of any one of embodiments 1-39, where A is the total number of apoptosis marker-CD3+ CAR+ cells or the total number of apoptosis marker-CD8+ CAR+ cells, optionally, where the apoptosis marker is annexin V or active caspase 3.

41. A product containing:

(a) a container containing a unit dose of a T cell therapeutic composition comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds to an antigen, wherein the unit dose contains the target dose of the T cell therapeutic composition, where:

(i) if the value of a parameter that indicates the degree of or correlates with the degree of recombinant receptor-dependent activity of the composition is equal to or greater than a threshold value, the target dose is the first number (or lies in the first range of numbers) of cells of a given phenotype from the composition;

(ii) if the parameter value is less than the threshold value, the target dose is the second number (or lies in the second range of numbers) of cells of a given phenotype from the composition

where the first number (or first range) is lower than the second number (or second range); and

(b) instructions for administering the composition, optionally one or more unit doses thereof, to an individual, optionally an individual having or suspected to have a disease or condition.

42. The product of Embodiment 41, wherein the recombinant receptor dependent activity threshold is less than the reference safety level, where the reference safety level is, as applied to a group of individuals analyzed after treatment with a T cell therapeutic composition comprising T cells, expressing a recombinant receptor, optionally a CAR, the lowest level of CAR-dependent activity of the therapeutic composition administered to an individual among those individuals in the group who continued to develop an adverse event.

43. The article of embodiment 42, wherein the adverse event is a serious adverse event, optionally a severe grade 4 or grade 5 neurotoxicity equal to or greater than grade 5 neurotoxicity, or at least grade 3 long-term neurotoxicity.

44. The product of Embodiment 42 or Embodiment 43, where the threshold value is less than the safety reference level by an amount corresponding to a safety factor or at least 2 times.

45. The article of any one of embodiments 41-44, wherein the first amount is lower than the second amount by more than or more than about 1.5 times, 2.0 times, 3.0 times, 4.0 times, 5 .0 times, 6.0 times, 7.0 times, 8.0 times, 9.0 times, 10.0 times or more.

46. A product containing:

(a) a container containing a unit dose of a T cell therapeutic composition comprising T cells containing a recombinant receptor, optionally a chimeric antigen receptor (CAR), that specifically binds to an antigen, wherein:

the unit dose contains the target dose of the T cell therapeutic composition; and

the T cell therapeutic composition is above the lower limit (LSL) and below the upper limit (USL) for B, where B is a parameter value that indicates the degree of, or correlates with, the degree of recombinant receptor-dependent activity of the composition; and

(b) instructions for administering the composition, optionally one or more unit doses thereof, to an individual, optionally an individual having or suspected to have a disease or condition.

47. The product of embodiment 46, where the product is released to treat an individual only if the composition is below the USL for B.

48. The article of manufacture of any of embodiments 41-47, wherein the recombinant receptor dependent activity is indicative of the production or accumulation of a pro-inflammatory cytokine, optionally one or a combination of TNF-alpha, IFN-gamma, IL-2, and IL-10.

49. The product of any of embodiments 1-48, where the parameter is a measure of one or more factors, or a normalized value thereof.

50. The article of embodiment 49, wherein the indicator is determined in an assay comprising culturing or incubating for a fixed time, optionally 24 hours, a given composition or a sample thereof in the presence of an antigen, antigen-expressing cells, and/or a specific binding agent with a recombinant receptor, optionally CAR.

51. The product of embodiment 50, where the assay is an ELISA.

52. The product of any of embodiments 49-51, where the factor score is:

(i) the concentration, relative concentration, amount or relative amount of the factor; or

(ii) the amount or relative amount of the factor per unit of cells administered from the composition, or

(iii) the amount or relative amount of the factor per unit of cells administered from the composition per unit of time, optionally one hour; or

(iv) a level indicative of any of (i)-(iii).

53. The article of any of embodiments 49-52, wherein one or more of the factors is one or a combination of soluble factors, optionally one or a combination of cytokines, chemokines, or soluble receptors, optionally soluble costimulatory receptors.

54. The article of any of embodiments 49-53, wherein one or more of the factors is one or a combination of pro-inflammatory cytokines, Th2 cytokines, and Th17 cytokines.

55. The product of any of embodiments 49-54, wherein one or more of the factors is one or a combination of IL-2, IFN-gamma, TNF-alpha, IL4, IL-5, IL-10, IL-13, GM –CSF, sCD137, MIP1a and M1Pb.

56. The product of any of embodiments 49-55, wherein one or more of the factors is one or a combination of IL-2, IFN-gamma, TNF-alpha, and IL-10.

57. An article of any of embodiments 53-56 wherein one or more factors is a combination of any two or more of the above soluble factors and the parameter is the arithmetic or geometric mean of the two or more factors.

58. The article of any one of embodiments 49-57, wherein the parameter is the arithmetic or geometric mean, optionally of amount or concentration, of at least two of TNF-alpha, IFN-gamma, and IL-2, or of TNF- alpha, IFN-gamma and IL-2.

59. The product of any one of embodiments 49-58, where the parameter is a normalized value of the index, where the normalization is compared to a reference factor index.

60. The product of Embodiment 59, wherein the reference score is the average of scores among a plurality, optionally at least 10, at least 15, at least 20, of reference chimeric antigen receptor (CAR) T cell therapeutic compositions , in which:

(i) for each of the reference T cell therapeutic compositions, an acceptable safety profile was observed or determined as a result after administration to an individual, optionally where the individual has a disease or condition with antigen expression or association with an antigen; and/or

(ii) for each of the reference T cell therapeutic compositions, the desired efficacy was observed or determined as a result after administration to an individual, optionally where the individual has a disease or condition with antigen expression or association with an antigen.

61. The article of embodiment 60, wherein the acceptable safety profile is no observable grade 2 or higher neurotoxicity, or no grade 3 or higher neurotoxicity.

62. The product of Embodiment 60 or Embodiment 61, where the acceptable safety profile is no observable Grade 3 or higher neurotoxicity.

63. The product of any of embodiments 60-62, where efficacy is a partial response or is a complete response (CR).

64. The product of Embodiment 59, where the benchmark is the score, in the same assay, of a factor in a T cell reference composition that is prepared in the same manner as the T cell therapeutic composition but does not express a recombinant receptor, optionally a CAR , does not recognize a specific antigen and/or does not express any recombinant receptor, optionally any CAR.

65. The product of embodiment 64, where this parameter is normalized to control for patient-specific variability in a score of one or more factors.

66. The product of Embodiment 64 or Embodiment 65, where the parameter is the normalized value of a factor score, compared to the same score in the same assay, for a control factor, where the control factor level in the T cell therapeutic composition is known to be absent or observed no sign or correlation, or significant correlation with the outcome of an adverse event or toxicity, or their likelihood or risk, where the outcome of an adverse event or toxicity is not necessarily severe neurotoxicity.

67. The product of Embodiment 66, wherein the control factor is a factor that is not statistically correlated and/or correlated with the occurrence of an adverse event among a plurality of individuals who continued to develop an adverse event after administration of the T cell composition, optionally the control factor is is or contains one or a combination of IL-5, IL-13, GM-CSF and IL-6, optionally wherein the control factor score is the arithmetic or geometric mean of two or more of the above.

68. The product of any of embodiments 1-67, where the parameter does not include cytolytic activity or its index.

69. The article of any of embodiments 1-68, wherein the parameter does not include recombinant receptor dependent or antigen specific cytolytic activity or an index thereof.

70. The product of any of embodiments 1-40 and 49-69, where:

the phenotype is CD8+ CAR+ cells or an apoptosis marker - CD8+ CAR+ cells, optionally, where the apoptosis marker is annexin V or active caspase 3; and

parameter is a measure for a pro-inflammatory cytokine, which is optionally one or a combination of TNF-alpha, IL-2 and IFN-gamma, or is its normalized value.

71. An article of any one of embodiments 1-70, where the adverse event is a grade 4 or 5 neurotoxicity and the threshold number of units is:

is or is approximately 1.75×10 ⁷ if A is negative (-) for apoptosis marker and CD8+CAR+ and B is TNF-alpha or a normalized value thereof;

is or is approximately 2.19×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha or a normalized value thereof;

is or is approximately 1.25×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma or its normalized value;

is or is approximately 1.56×10 ⁷ if A is CD8+CAR+ and B is IFN-gamma or its normalized value;

is or is approximately 1.5×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IL-2 or a normalized value thereof;

is or is approximately 1.88×10 ⁷ if A is CD8+CAR+ and B is IL-2 or its normalized value;

is or is approximately 1.5×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IFN-gamma or a normalized value thereof;

is or is approximately 1.88×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof;

is or is approximately 2.0×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IL-2 or a normalized value thereof;

is or is about 2.5×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha and IL-2 or its normalized value;

is or is approximately 1.25×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma and IL-2 or a normalized value thereof;

is or is about 1.56×10 ⁷ if A is CD8+CAR+ and B is IFN-gamma and IL-2 or its normalized value;

is or is approximately 1.75×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha, IFN-gamma and IL-2 or a normalized value thereof;

is or is approximately 2.19×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma and IL-2 or its normalized value,

optionally, where the apoptosis marker is annexin V or active caspase 3.

72. An article of any one of embodiments 1-71, where the adverse event is a Grade 4 or 5 neurotoxicity and the target reference unit range is:

is between or approximately between 2.0×10 ⁵ and 1.75×10 ⁷ inclusive if A is negative (-) for apoptosis marker and CD8+CAR+ and B is TNF-alpha or a normalized value thereof;

is between or approximately between 2.5×10 ⁵ and 2.19×10 ⁷ inclusive if A is CD8+CAR+ and B is TNF-alpha or a normalized value thereof;

is between or approximately between 4×10 ⁵ and 1.25×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma or a normalized value thereof;

is between or approximately between 5x10 ⁶ and 1.56x10 ⁷ inclusive if A is CD8+CAR+ and B is IFN-gamma or a normalized value thereof;

is between or approximately between 2.0×10 ⁵ and 1.5×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IL-2 or a normalized value thereof;

is between or approximately between 2.5x10 ⁵ and 1.88x10 ⁷ inclusive if A is CD8+CAR+ and B is IL-2 or a normalized value thereof;

is between or approximately between 3.0×10 ⁵ and 1.5×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IFN-gamma or its normalized value;

is between or approximately between 3.75×10 ⁵ and 1.88×10 ⁷ inclusive if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof;

is between or approximately between 3.0×10 ⁵ and 2.0×10 ⁷ inclusive if A is negative (–) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IL-2 or its normalized value;

is between or approximately between 3.75×10 ⁵ and 2.5×10 ⁷ inclusive if A is CD8+CAR+ and B is TNF-alpha and IL-2 or a normalized value thereof;

is between or approximately between 4.0×10 ⁵ and 1.25×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma and IL-2 or its normalized value;

is between or approximately between 5.0×10 ⁵ and 1.56×10 ⁷ inclusive if A is CD8+CAR+ and if B is IFN-gamma and IL-2 or its normalized value;

is between or approximately between 4.0×10 ⁵ and 1.75×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha, IFN-gamma and IL –2 or its normalized value;

is between or approximately between 5.0×10 ⁵ and 2.19×10 ⁷ inclusive if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma and IL-2 or a normalized value thereof,

optionally, where the apoptosis marker is annexin V or active caspase 3.

73. An article of any one of embodiments 1-70, where the adverse event is at least Grade 3 long-term neurotoxicity and the threshold number of units is:

is or is approximately 1.0×10 ⁶ if A is negative (-) for apoptosis marker and CD8+CAR+ and B is TNF-alpha or a normalized value thereof;

is or is approximately 1.25×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha or a normalized value thereof;

is or is approximately 2.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma or its normalized value;

is or is approximately 2.5×10 ⁶ if A is CD8+CAR+ and B is IFN-gamma or its normalized value;

is or is approximately 3.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IL-2 or a normalized value thereof;

is or is approximately 3.75×10 ⁶ if A is CD8+CAR+ and B is IL-2 or its normalized value;

is or is approximately 1.5×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IFN-gamma or a normalized value thereof;

is or is about 1.88×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof;

is or is approximately 2.5×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IL-2 or a normalized value thereof;

is or is approximately 3.12×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha and IL-2; or its normalized value

is or is approximately 3.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma and IL-2 or a normalized value thereof;

is or is approximately 3.75×10 ⁶ if A is CD8+CAR+ and B is IFN-gamma and IL-2 or its normalized value;

is or is approximately 2.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha, IFN-gamma and IL-2 or a normalized value thereof;

is or is approximately 2.5×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma and IL-2 or its normalized value, optionally, where the apoptosis marker is annexin V or active caspase 3.

74. An article of any of embodiments 1-70 and 73, where the adverse event is at least Grade 3 long-term neurotoxicity and the target reference unit range is:

is between or approximately between 3.0×10 ⁵ and 1.0×10 ⁶ inclusive, if A is negative (-) for an apoptosis marker and CD8+CAR+, and B is TNF-alpha or a normalized value thereof;

is between or approximately between 3.75×10 ⁵ and 1.25×10 ⁶ inclusive if A is CD8+CAR+ and B is TNF-alpha or a normalized value thereof;

is between or approximately between 4×10 ⁵ and 2.0×10 ⁶ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma or a normalized value thereof;

is between or approximately between 5×10 ⁶ and 2.5×10 ⁶ inclusive if A is CD8+CAR+ and B is IFN-gamma or a normalized value thereof;

is between or approximately between 2.0×10 ⁵ and 3.0×10 ⁶ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IL-2 or a normalized value thereof;

is between or approximately between 2.5×10 ⁵ and 3.75×10 ⁶ inclusive if A is CD8+CAR+ and B is IL-2 or a normalized value thereof;

is between or approximately between 3.0×10 ⁵ and 1.5×10 ⁶ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IFN-gamma or its normalized value;

is between or approximately between 3.75×10 ⁵ and 1.88×10 ⁶ inclusive if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof;

is between or approximately between 3.0×10 ⁵ and 2.5×10 ⁶ , inclusive, if A is negative (–) for the apoptosis marker CD8+CAR+, and if B is TNF-alpha and IL-2 or its normalized value;

is between or approximately between 3.75×10 ⁵ and 3.12×10 ⁶ inclusive if A is CD8+CAR+ and B is TNF-alpha and IL-2 or a normalized value thereof;

is between or approximately between 4.0×10 ⁵ and 3.0×10 ⁶ inclusive if A is negative (–) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma and IL-2 or its normalized value;

is between or approximately between 5.0×10 ⁵ and 3.75×10 ⁶ inclusive if A is CD8+CAR+ and B is IFN-gamma and IL-2 or its normalized value;

is between or approximately between 4.0×10 ⁵ and 2.0×10 ⁶ inclusive if A is negative (–) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha, IFN-gamma and IL –2 or its normalized value;

is between or approximately between 5.0×10⁵ and 2.5×10⁶, inclusive, if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma and IL-2 or its normalized value,

optionally, where the apoptosis marker is annexin V or active caspase 3.

75. The article of any one of embodiments 1-74, wherein the T cell therapeutic composition contains between about 10 million cells per ml and about 70 million cells per ml, or between about 10 million viable cells per ml and about 70 million viable cells per ml , including each.

76. An article of any one of embodiments 1-75, wherein the T cell therapeutic composition contains between about 15 million cells or viable cells per ml and about 60 million cells or viable cells per ml, inclusive.

77. An article of any one of embodiments 1-76, wherein the T cell composition contains more than 10 million cells or viable cells per ml.

78. The article of any one of embodiments 1-77, wherein the T cell therapeutic composition contains more than 15 million cells or more than 15 million cells per ml.

79. The article of manufacture of any of embodiments 1-78, wherein the composition further comprises a cryoprotectant and/or the article further comprises instructions for thawing the composition prior to administration to an individual.

80. The article of any one of embodiments 1-79, wherein the disease or condition is cancer, optionally myeloma, lymphoma, or leukemia.

81. The article of embodiment 80, wherein the disease or condition is a B-cell malignancy, optionally a B-cell malignancy, selected from the group consisting of acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia ( CLL), non-Hodgkin's lymphoma (NHL), and diffuse large B-cell lymphoma (DLBCL).

82. The article of any of embodiments 1-81, wherein the antigen is CD19, CD22, ROR1, Ig-kappa, Her2, L1-CAM, CD20, mesothelin, CEA, hepatitis B surface antigen, antifolate receptor, CD23, CD24 , CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, ErbB3, ErbB4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R –alpha2, kdr, kappa light chain, Lewis Y, cell adhesion molecules L1, MAGE–A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligands, NY–ESO–1, MART–1, gp100, oncofetal antigen, TAG72, VEGF –R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, estrogen receptor, progesterone receptor, ephrinB2, CD123, CS–1, c–Met, GD–2, MAGE A3, CE7, Wilms tumor antigen 1 (WT–1) and cyclin A1 (CCNA1).

83. The article of manufacture of any of embodiments 1-82, wherein the recombinant receptor is a CAR.

84. The article of embodiment 83, wherein the CAR contains an extracellular antigen recognition domain that specifically binds to an antigen and an intracellular signaling domain containing ITAM, where optionally the intracellular signaling domain contains an intracellular domain from the CD3-zeta (CD3ζ) chain; and/or wherein the CAR further comprises a costimulatory signaling region, which optionally comprises a signaling domain from CD28 or 4-1BB.

85. The article of any of embodiments 1-84, wherein the T cells are CD4+ or CD8+.

86. The article of any of embodiments 1-85, wherein the T cells are primary T cells obtained from an individual.

87. An article of any of embodiments 1-86, wherein the article further comprises information indicating that the container contains the target number of units.

88. The article of manufacture of any of embodiments 1-87, wherein the container is the first container, and the article further comprises additional containers, where each of the additional containers contains a unit dose containing the target number of units from the T cell composition.

89. The article of embodiment 88 wherein the additional containers contain between about 10 million cells or viable cells per ml and about 70 million cells or viable cells per ml, between about 15 million cells or viable cells and about 60 million cells or viable cells per ml. ml, including each, or more than 10 million cells or viable cells per ml, more than 15 million cells or viable cells per ml, or a combination thereof.

90. The article of manufacture of any of embodiments 1-89, wherein the unit dose contains no more than 15×10 ⁶ CD8+CAR+ cells that are negative for annexin V or active caspase 3 or caspase 3 proform.

91. The article of manufacture of any of embodiments 1-90, wherein the unit dose further comprises an amount of CAR positive CD4+ cells, where the amount is in or about a 1:1 ratio of CD8+CAR+ cells.

92. The article composition of any of embodiments 1-91, wherein the T cell composition is prepared by a method in which:

frequency, (1) among CAR+ cells in the composition, (2) among CAR+CD3+ cells in the composition, and/or (3) among CAR+CD8+ cells in the composition, of cells of a phenotype indicating signs of biologically active cells and/or lack of apoptosis , or early or late stages of apoptosis, differs by no more than 40%, or no more than 30%, or no more than 20%, or no more than 10%, or no more than 5% of the average specified frequency during set of compositions of T-cells obtained by this method, and/or differs from such an average by no more than one standard deviation; or

the frequency, (1) among CAR+ cells in the composition, (2) among CAR+CD3+ cells in the composition, and/or (3) among CAR+CD8+ cells in the composition, of cells with a phenotype indicating no apoptosis, or early or late stages of apoptosis , differs by no more than 40%, or no more than 20%, or no more than 10%, among the plurality of T cell compositions obtained by this method

93. The product of embodiment 92, where the method includes:

(a) incubating a population of cells containing T cells with an agent containing a nucleic acid molecule encoding a recombinant receptor under conditions to introduce a nucleic acid encoding a recombinant receptor into cells in the population; and

(b) stimulating the cells, before, during and/or after said incubation, wherein the stimulation comprises incubating the cells in the presence of a stimulating condition that induces a primary signal, signaling, stimulating, activating and/or proliferating the cells.

94. The article of embodiment 93, wherein the method further comprises, before (a), isolating a population of cells from a biological sample.

95. The article of manufacture of embodiment 94, wherein the isolation comprises selecting cells based on surface expression of CD3 or based on surface expression of one or both of CD4 and CD8, optionally by positive or negative selection.

96. The article of manufacture of Embodiment 94 or Embodiment 95, wherein isolation comprises immunoselection based on affinity.

97. The article of manufacture of Embodiment 94-96, wherein the biological sample is or contains a whole blood sample, buffy coat sample, peripheral blood mononuclear cell (PBMC) sample, unfractionated T-cell sample, lymphocyte sample, leukocyte sample, apheresis product, or leukapheresis.

98. The article of any one of embodiments 93-97, wherein the stimulatory condition comprises incubation with a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex, and/or one or more intracellular signaling domains of one or more costimulatory molecules.

99. The article of manufacture of Embodiment 98, wherein the stimulatory reagent contains a primary agent that specifically binds to a member of the TCR complex and a secondary agent that specifically binds to a costimulatory T cell molecule.

100. The product of Embodiment 98 or Embodiment 99, wherein the primary agent specifically binds to CD3 and/or a costimulatory molecule selected from the group consisting of CD28, CD137 (4-1-BB), OX40, or ICOS.

101. The article of embodiment 99 or embodiment 100, wherein the primary and secondary agents contain antibodies and/or are present on the surface of a solid support, optionally a bead.

102. The product of any of embodiments 98-101, where:

a stimulating reagent is a reagent for which it is determined that the recombinant receptor dependent activity or antigen specific activity, optionally recombinant receptor dependent or antigen specific dependent production or accumulation of a pro-inflammatory cytokine, differs by no more than 40% or no more than 30 %, or no more than 20%, or no more than 10%, or no more than 5%, of the average pro-inflammatory cytokine production or accumulation among the plurality of T cell compositions produced by this method using a stimulatory reagent, and /or differs from such mean by no more than one standard deviation; and/or

a stimulating reagent is a reagent for which it is determined that the recombinant receptor dependent activity or antigen specific activity, optionally recombinant receptor dependent or antigen specific dependent production or accumulation of a pro-inflammatory cytokine, differs by no more than 40% or no more than 30 %, or not more than 20%, or not more than 10%, or not more than 5%, among the many compositions of T cells obtained by this method; and/or

a stimulating reagent is a reagent for which it is determined that the recombinant receptor dependent activity or antigen specific activity, optionally recombinant receptor dependent or antigen specificity dependent production or accumulation of a pro-inflammatory cytokine, for a cell composition obtained using a stimulating reagent, differs by no more than by more than 40%, not more than 30%, not more than 20% or not more than 10% or not more than 5% of the control composition, where the control composition and cell composition are obtained using the same method, including obtaining from the same population of cells, except that the control composition is implemented in the presence of a control stimulatory reagent, or a standard number of units of recombinant receptor-dependent activity.

103. The product of Embodiment 102 wherein the control stimulatory reagent, when used in the method, results in a T cell composition in which the recombinant receptor dependent activity or antigen specific activity is within an acceptable range of variability.

104. An article of any one of embodiments 93-103 wherein cell stimulation is performed or initiated prior to incubation, optionally for 18-24 hours at or about 37 degrees.

105. The article of any one of embodiments 93-104, wherein the stimulatory condition comprises a cytokine selected from IL-2, IL-15, and IL-7.

106. The article of any of the embodiments 93-105, wherein stimulation of the cells is carried out after incubation, optionally, for a period of time to reach the threshold concentration.

107. The article of any one of embodiments 93-106, further comprising (c) filling the container with all or part of the T cell composition, and optionally another solution, to a concentration between about 10 million cells and about 70 million cells per ml, inclusive.

108. The article of embodiment 107, wherein the container is filled with another solution and the solution contains a cryoprotectant, optionally DMSO.

109. The article of embodiment 107 or embodiment 108, wherein the concentration is between about 15 and about 60 million cells per ml, inclusive.

110. The article of any of embodiments 107-109, wherein the concentration is greater than 10 million cells per ml.

111. An article of any one of embodiments 107-110, wherein the concentration is greater than 15 million cells per ml.

112. The article of any of embodiments 107-111, wherein the concentration of DMSO is or is about or does not exceed 7.5%.

113. The article of embodiment 107 or embodiment 108, wherein the concentration is greater than 60 million cells per ml.

114. The product of any of embodiments 107-108 and 113, wherein the DMSO concentration is greater than 7.5%, optionally between or about between 7.5% and 9.0%, inclusive.

115. The article of manufacture of any of embodiments 93-114, wherein the agent containing the nucleic acid molecule encoding the recombinant receptor is a viral vector, optionally a lentiviral vector or a gamma-retroviral vector.

116. The product of any of embodiments 107-115, where the filling is carried out automatically, optionally in a closed system.

117. The article of any one of embodiments 93-116, further comprising freezing the cells in the container or storing the container at less than or about less than 80ºC.

118. A method of treatment comprising administering to an individual suffering from a disease or condition, a single dose of a T cell composition containing cells containing a recombinant receptor, optionally a chimeric antigen receptor (CAR), that specifically binds to an antigen associated with the disease or condition, where unit dose contains a target number of reference units (RU) within a given range, where RU in a given composition is determined by the formula

RU=A x B, where:

A is the number of cells, or a multiple, proportion or transformation, of some phenotype present in a given composition, or is the average or weighted average of the number of cells, or a multiple, proportion or transformation, of two or more phenotypes in a given composition; and

B is a parameter value, or a multiple thereof, or a transformation that indicates the degree of or correlates with the degree of recombinant receptor dependent, optionally CAR dependent, activity in the composition.

119. The method of embodiment 118, where A is the number of cells, or a multiple thereof, or proportion, or transformation, of a given phenotype present in a given composition.

120. The method of embodiment 118, wherein A is the average or weighted average of the number of cells, or a multiple thereof, proportion or conversion, of two or more phenotypes in a given composition.

121. The method of any of embodiments 118-120, wherein the target number of units is less than a threshold number of RUs, optionally a safe number of reference units, where the safe number of reference units is, in relation to a group of individuals analyzed after treatment with using a T cell therapeutic composition comprising T cells expressing a recombinant receptor, optionally CAR, the lowest number of drug reference units administered to an individual among those individuals in the group who continued to develop an adverse event.

122. The method of Embodiment 121, wherein the adverse event is a serious adverse event, optionally severe neurotoxicity, optionally a grade equal to or greater than grade 4 or grade 5, or at least long-term grade 3 neurotoxicity.

123. The method of Embodiment 121 or Embodiment 122, wherein the target number of reference units is less than the safe number of reference units by an amount corresponding to a safety factor and/or by an amount in the range of 1.5- to 3-fold , optionally approximately 2-fold, or a multiple of the standard deviation in a group of individuals who did not experience an adverse event, optionally, neurotoxicity grade 0-2, optionally, where the multiple lies in the range from 1.5- to 3-fold .

124. The method of any of embodiments 118-123, wherein the target number of reference units is equal to or greater than the reference effective amount of reference units, where the reference effective amount is, in relation to a group of individuals analyzed after treatment using a therapeutic T cell composition containing recombinant receptor, optionally CAR, number of drug units administered to one or more individuals within a group for which the desired therapeutic outcome is indicated, optionally a partial response or complete response (CR).

125. A method of treatment comprising administering to an individual suffering from a disease or condition, a single dose of a T cell composition containing cells containing a recombinant receptor, optionally a chimeric antigen receptor (CAR), that specifically binds to an antigen associated with the disease or condition, where the unit dose contains or contains approximately (i) the target number of total recombinant receptor-expressing cells or the target number of total CD8+ recombinant receptor-expressing cells, or (ii) the target number of reference units (RU) within a given range, where the target number is equal to or less than the threshold number of RU , where the unit dose does not contain more than the threshold value RU,

where the amount of RU in a given composition is determined by the formula:

RU=A x B, where

A is the number of cells, or a multiple, proportion or transformation, of some phenotype present in a given composition, or is the average or weighted average of the number of cells, or a multiple, proportion or transformation, of two or more phenotypes in a given composition; and

B is a parameter value, or a multiple thereof, or a transformation that indicates the degree of or correlates with the degree of recombinant receptor dependent, optionally CAR dependent, activity in a given T cell composition.

126. The method of embodiment 125, wherein A is the number of cells, or a multiple thereof, or proportion, or transformation, of a given phenotype present in a given composition.

127. The method of embodiment 125, wherein A is the average or weighted average of the number of cells, or a multiple thereof, proportion or conversion, of two or more phenotypes in a given composition.

128. The method of any of embodiments 118-127, wherein the target amount is the target amount of recombinant receptor-expressing CD3+ or CD8+ cells that are negative (–) for an apoptosis marker and CD3+ or CD8+, optionally, where the apoptosis marker is is annexin V or active caspase 3.

129. The method of any of embodiments 125-128, wherein the target number of cells in (i) is: between and between about 5.0×10 ⁶ and 2.25×10 ⁷ , 5.0×10 ⁶ and 2.0 ×10 ⁷ , 5.0×10 ⁶ and 1.5×10 ⁷ , 5.0×10 ⁶ and 1.0×10 ⁷ , 5.0×10 ⁶ and 7.5×10 ⁶ , 7.5× 10 ⁶ and 2.25×10 ⁷ , 7.5×10 ⁶ and 2.0×10 ⁷ , 7.5×10 ⁶ and 1.5×10 ⁷ , 7.5×10 ⁶ and 1.0×10 ⁷ , 1.0×10 ⁷ and 2.25×10 ⁷ , 1.0×10 ⁷ and 2.0×10 ⁷ , 1.0×10 ⁷ and 1.5×10 ⁷ , 1.5×10 ⁷ and 2.25×10 ⁷ , 1.5×10 ⁷ and 2.0×10 ⁷ , 2.0×10 ⁷ and 2.25×10 ⁷ recombinant receptor expressing cells, including each optionally recombinant receptor expressing cells, which are CD8+, or which are negative (-) for the apoptosis marker and CD8+, optionally, where the apoptosis marker is annexin V or active caspase 3.

130. The method of any of embodiments 125-129, wherein the target number of cells in (i) is:

between at least or at least about 5x10 ⁶ , 6x10 ⁶ , 7x10 ⁶ , 8x10 ⁶ , 9x10 ⁶ , 10x10 ⁶ , and about 15x10 ⁶ expressing the recombinant receptor, including each optionally expressing the recombinant receptor cells that are CD8+, or that are negative (-) for an apoptosis marker and CD8+, optionally, where the apoptosis marker is annexin V or active caspase 3;

between at least or at least about 5.55×10 ⁶ , 6.66×10 ⁶ , 7.77×10 ⁶ , 8.99×10 ⁶ , 1.0×10 ⁷ , 1.1×10 ⁷ and approximately 1.67×10 ⁷ expressing the recombinant receptor, including each optionally expressing the recombinant receptor cells that are CD8+, or which are negative (-) for the apoptosis marker and CD8+, optionally, where the apoptosis marker is annexin V or active caspase 3;

between at least or at least about 6.25×10 ⁶ , 7.5×10 ⁶ , 8.75×10 ⁶ , 1.0×10 ⁷ , 1.13×10 ⁷ , 1.25×10 ⁷ and approximately 1.9 x 10 ⁷ expressing the recombinant receptor, including each optionally expressing the recombinant receptor cells that are CD8+ or that are negative (-) for the apoptosis marker and CD8+, optionally, where the apoptosis marker is annexin V or active caspase 3;

between at least or at least about 7.14×10 ⁶ , 8.5×10 ⁶ , 1.0×10 ⁷ , 1.14×10 ⁷ , 1.29×10 ⁷ , 1.42×10 ⁷ and about 2.14 x 10 ⁷ expressing the recombinant receptor, including each optionally expressing the recombinant receptor cells that are CD8+ or that are negative (-) for the apoptosis marker and CD8+, optionally, where the apoptosis marker is annexin V or active caspase 3;

131. The method of any of embodiments 125-130, wherein the target number of cells in (i) is between at least or at least about 5×10 ⁶ , 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9x10 ⁶ , 10x10 ⁶ , and approximately 15x10 ⁶ recombinant receptor-expressing cells that are negative (-) for an apoptosis marker and CD8+, including each, optionally, where the apoptosis marker is annexin V or active caspase 3.

132. The method of any of embodiments 125-130, wherein the target number of cells in (i) is between at least or at least about 6.25×10 ⁶ , 7.5×10 ⁶ , 8.75×10 ⁶ , 1.0×10 ⁷ , 1.13×10 ⁷ , 1.25×10 ⁷ , and approximately 1.9×10 ⁷ recombinant receptor-expressing CD8+ cells, including each.

133. The method of any of embodiments 118-132, wherein the target reference amount of RU is less than the reference safe amount of RU, where the reference safe amount of RU is, in relation to a group of individuals analyzed after treatment with a T cell therapeutic composition containing T-cells expressing the recombinant receptor, optionally CAR, the smallest number of drug reference units administered to an individual among those individuals in the group who continued to develop an adverse event.

134. The method of Embodiment 133, wherein the adverse event is a serious adverse event, optionally severe grade 4 or grade 5 neurotoxicity equal to or greater than grade 5, or at least long-term grade 3 neurotoxicity.

135. The method of Embodiment 133 or Embodiment 134, wherein the target reference amount of RUs is less than the safety number of reference units by an amount corresponding to a safety factor or at least 2 times.

136. The method of any of embodiments 118-135, wherein the target number of reference units is or exceeds the reference effective amount of reference units, where the reference effective amount is, in relation to a group of individuals analyzed after treatment with a T cell composition containing the recombinant receptor , optionally CAR, number of drug reference units administered to one or more individuals within a group for which the desired therapeutic outcome is shown, optionally, complete response (CR).

137. The method of any of embodiments 118-136, wherein A is the number of cells of some phenotype present in the composition and B is a parameter value that indicates the degree of or correlates with the degree of recombinant receptor dependent activity in the composition.

138. The method of any one of embodiments 118-137, wherein A and/or B is a quantity or value transformation, respectively, where the transformation includes a logarithmic transformation, a power transformation, or a logit transformation.

139. The method of any of embodiments 118-138, wherein A is the number of cells of some phenotype present in a given composition and B is a multiple or transformation of a parameter value indicative of the degree of or correlated with the degree of CAR-dependent activity in the composition T-cells, optionally, where B is the logarithmic transformation of the value.

140. The method of Embodiment 138 or Embodiment 139, wherein the logarithmic transformation is decimal log(log ₁₀ (x)), natural log(ln(x)) or binary log(log ₂ (x)).

141. The method of any of embodiments 118-140, wherein A is the number of viable cells in the composition and/or is the number of cells that are not apoptotic, do not have a factor indicative of early apoptosis or apoptosis, are not in the early stages apoptosis, or are not in the late stages of apoptosis, and/or is the number of cells in a certain state of differentiation, and/or is the number of cells with memory cells/stem cells, or is its multiple or transformation.

142. The method of any of embodiments 118-141, wherein the phenotype includes positive expression of a surface marker that is one or more of CD3, CD4, or CD8, and/or includes positive expression of a recombinant receptor, optionally CAR, or a surrogate marker for the expression of a recombinant receptor.

143. The method of embodiment 142, wherein the phenotype is CD3+ CAR, CD4+/CAR+, CD8+/CAR+.

144. The method of any one of embodiments 118-143, wherein the phenotype includes the absence of a factor indicative of apoptosis or one or more steps in the apoptotic cascade or pathway, optionally expression of an apoptosis marker.

145. The method of any of embodiments 118-144, wherein the phenotype comprises negative expression of an apoptosis marker, optionally an early apoptosis or late apoptosis marker.

146. The method of embodiment 145, wherein the apoptosis marker is surface phosphatidylserine and/or detected using annexin V, or is an active caspase or a caspase proform, optionally an active caspase 3 or a caspase 3 proform.

147. The method of any of embodiments 118-146 wherein the phenotype includes annexin-.

148. The method of any of embodiments 118-147, wherein the phenotype includes an indicator of the production of one or a combination of cytokines, optionally not specific for an antigen or recombinant receptor and/or polyclonal production, where one or more cytokines is IL-2, IL- 13, IL-17, IFN-gamma or TNF-alpha.

149. The method of Embodiment 148, wherein the production index is measured in an assay, optionally an intracellular cytokine staining assay, comprising incubating a sample from a T cell composition with a polyclonal agent, an antigen specific agent, or a recombinant receptor binding agent, optionally CAR.

150. The method of Embodiment 148 or Embodiment 149 wherein the agent is or contains PMA and ionomycin, or is or contains a T-cell receptor or a T-cell receptor complex agonist.

151. The method of any of embodiments 118-150 wherein the phenotype comprises negative expression of an activation marker, wherein the activation marker is selected from CD25, CD127, LAG3, Ki67 and combinations thereof.

152. The method of any of embodiments 118-151 wherein the phenotype comprises negative expression of a depletion marker, wherein the depletion marker is a PD1 or FOXP3 gene product, or a combination thereof.

153. The method of any one of embodiments 118-152, wherein the phenotype comprises a naïve or memory cell phenotype, optionally wherein the memory cell phenotype comprises an effector memory T cell phenotype, a central memory T cell phenotype, or an effector memory T cell phenotype expressing CD45RA (Temra).

154. The method of any of embodiments 118-153 wherein A is total T cells, total CD3+ cells, total CD4+ or CD8+ cells, total CD3+CAR+ cells, total CD8+CAR+ cells, total CD4+ CAR+, or total number of living or viable cells from any of the above, or its multiple or converted value.

155. The method of any of embodiments 118-154, wherein A is total CD3+ cells, total CD8+, total CD3+CAR+ cells, total CD8+CAR+ cells, or total live or viable cells from any of the above. , or its multiple or converted value.

156. The method of any of embodiments 118-155, wherein A is the total number of apoptosis marker negative (–) cells that are CD3+ CAR+ cells, the total number of apoptosis marker negative (–) cells that are CD4+ CAR+, total the number of cells negative (-) for the apoptosis marker, which are CD8+ CAR+ cells, or its multiple or converted value, where the apoptosis marker is annexin V or caspase.

157. The method of any of embodiments 118-156 wherein A is the total number of apoptosis marker-CD3+ CAR+ cells or the total number of apoptosis marker-CD8+ CAR+ cells, where the apoptosis marker is annexin V or caspase 3

158. A method of treatment comprising administering to an individual suffering from a disease or condition, a single dose of a T cell composition containing cells containing a recombinant receptor, optionally a chimeric antigen receptor (CAR), that specifically binds to an antigen associated with the disease or condition, where the unit dose contains the target dose of the T cell therapeutic composition:

(i) if a parameter value that indicates the degree of, or correlates with, the degree of recombinant receptor dependent, optionally CAR dependent, activity of the composition is equal to or greater than a threshold value, the target dose is the first number (or lies in the first range of numbers) of cells of the given phenotype from the composition;

(ii) if the parameter value is less than the threshold value, the target dose is the second number (or lies in the second range of numbers) of cells of a given phenotype from the composition

where the first count (or first range) is lower than the second count (or second range).

159. The method of Embodiment 158, wherein the recombinant receptor dependent activity threshold is less than the reference safety level, wherein the reference safety level is, as applied to a group of individuals analyzed after treatment with a T cell therapeutic composition comprising T cells, expressing a recombinant receptor, optionally a CAR, the lowest level of CAR-dependent activity of the therapeutic composition administered to the individual among those individuals in the group who continued to develop the adverse event.

160. The method of Embodiment 159, wherein the adverse event is a serious adverse event, optionally severe grade 4 or grade 5 neurotoxicity equal to or greater than grade 5 neurotoxicity, or at least grade 3 long-term neurotoxicity.

161. The method of Embodiment 159 or Embodiment 160, wherein the threshold value is less than the safety reference level by an amount corresponding to a safety factor or at least 2 times.

162. The method of any of embodiments 159-161 wherein the first amount is lower than the second amount by more than 1.5 times, 2.0 times, 3.0 times, 4.0 times, 5 .0 times, 6.0 times, 7.0 times, 8.0 times, 9.0 times, 10.0 times or more.

163. The method of any of embodiments 118-162 wherein the recombinant receptor dependent activity is an indicator of the production or accumulation of a pro-inflammatory cytokine, optionally one or a combination of TNF-alpha, IFN-gamma, IL-2 and IL-10.

164. The method of any of embodiments 118-163, wherein the parameter is a measure of one or more factors, or a normalized value thereof.

165. The method of embodiment 164, wherein the index is determined in an assay comprising culturing or incubating for a fixed time, optionally 24 hours, a given composition or a sample thereof in the presence of an antigen, antigen-expressing cells, and/or a specific binding agent. with a recombinant receptor, optionally CAR.

166. The method of embodiment 165 wherein the assay is an ELISA.

167. The method of any of embodiments 164-166, wherein the factor score is:

(i) the concentration, relative concentration, amount or relative amount of the factor; or

(ii) the amount or relative amount of the factor per unit of cells administered from the composition, or

(iii) the amount or relative amount of the factor per unit of cells administered from the composition per unit of time, optionally one hour; or

(iv) a level indicative of any of (i)-(iii).

168. The method of any of embodiments 164-167, wherein one or more of the factors is one or a combination of soluble factors, optionally one or a combination of cytokines, chemokines, or soluble receptors, optionally soluble costimulatory receptors.

169. The method of any of embodiments 164-168 wherein one or more of the factors is one or a combination of pro-inflammatory cytokines, Th2 cytokines, and Th17 cytokines.

170. The method of any of embodiments 164-169 wherein one or more of the factors is one or a combination of IL-2, IFN-gamma, TNF-alpha, IL4, IL-5, IL-10, IL-13, GM –CSF, sCD137, MIP1a and M1Pb.

171. The method of any of embodiments 164-170, wherein one or more of the factors is one or a combination of IL-2, IFN-gamma, TNF-alpha, and IL-10.

172. The method of any of embodiments 168-171, wherein one or more factors is a combination of any two or more of the above soluble factors, and the parameter is the arithmetic or geometric mean of the scores for the two or more factors.

173. The method of any of embodiments 168-172, wherein the parameter is the arithmetic or geometric mean, optionally, of an amount or concentration, of at least two of TNF-alpha, IFN-gamma, and IL-2, or of TNF- alpha, IFN-gamma and IL-2.

174. The method of any one of embodiments 164-173, wherein the parameter is a normalized score value, where the normalization is compared to a reference factor score.

175. The product of Embodiment 174, wherein the reference score is the average of scores among a plurality, optionally at least 10, at least 15, at least 20, of reference chimeric antigen receptor (CAR) T cell therapeutic compositions , in which:

(i) for each of the reference T cell therapeutic compositions, an acceptable safety profile was observed or determined as a result after administration to an individual, optionally where the individual has a disease or condition with antigen expression or association with an antigen; and/or

(ii) for each of the reference T cell therapeutic compositions, the desired efficacy is observed or determined as a result after administration to an individual, optionally where the individual has a disease or condition with antigen expression or association with an antigen.

176. The method of Embodiment 175, wherein the acceptable safety profile is no observed grade 2 or higher neurotoxicity, or no grade 3 or higher neurotoxicity.

177. The method of Embodiment 175 or Embodiment 176, wherein the acceptable safety profile is no observable Grade 3 or higher neurotoxicity.

178. The method of embodiment 177, where the efficacy is a partial response or is a complete response (CR).

179. The method of Embodiment 178, wherein the benchmark is the score, in the same assay, of a factor in a reference T cell composition that is prepared in the same manner as the T cell therapeutic composition but does not express a recombinant receptor, optionally CAR , does not recognize a specific antigen and/or does not express any recombinant receptor, optionally any CAR.

180. The method of embodiment 179 wherein this parameter is normalized to control for patient-specific variability in a score of one or more factors.

181. The method of Embodiment 179 or Embodiment 180, where the parameter is the normalized value of the factor score, compared to the same score in the same assay, for a control factor, where the control factor level in the T cell therapeutic composition is known to be absent or observed no sign or correlation, or significant correlation with the outcome of an adverse event or toxicity, or their likelihood or risk, where the outcome of an adverse event or toxicity is not necessarily severe neurotoxicity.

182. The method of Embodiment 181, wherein the control factor is or contains a factor that is not statistically correlated and/or correlated with the occurrence of an adverse event among a plurality of individuals who continued to develop an adverse event after administration of the T cell composition, optionally the control factor is one or a combination of IL-5, IL-13, GM-CSF, and IL-6, optionally wherein the control factor score is the arithmetic or geometric mean of two or more of the above.

183. The method of any of embodiments 118-182, wherein the parameter does not include cytolytic activity or an index thereof.

184. The method of any of embodiments 118-183, wherein the parameter does not include recombinant receptor dependent or antigen specific cytolytic activity or an index thereof.

185. The method of any of embodiments 118-184, where:

the phenotype is CD8+ CAR+ cells or an apoptosis marker - CD8+ CAR+ cells, optionally, where the apoptosis marker is annexin V or active caspase 3; and

parameter is a measure for a pro-inflammatory cytokine, which is optionally one or a combination of TNF-alpha, IL-2 and IFN-gamma, or is its normalized value.

186. The method of any of embodiments 118-185, wherein the adverse event is grade 4 or 5 neurotoxicity and the threshold is:

is or is approximately 1.75×10 ⁷ if A is negative (-) for apoptosis marker and CD8+CAR+ and B is TNF-alpha or a normalized value thereof;

is or is approximately 2.19×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha or a normalized value thereof;

is or is approximately 1.25×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma or its normalized value;

is or is approximately 1.56×10 ⁷ if A is CD8+CAR+ and B is IFN-gamma or its normalized value;

is or is approximately 1.5×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IL-2 or a normalized value thereof;

is or is approximately 1.88×10 ⁷ if A is CD8+CAR+ and B is IL-2 or its normalized value;

is or is approximately 1.5×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IFN-gamma or a normalized value thereof;

is or is about 1.88×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof;

is or is approximately 2.0×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IL-2 or a normalized value thereof;

is or is approximately 2.5×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha and IL-2 or its normalized value;

is or is approximately 1.25×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma and IL-2 or a normalized value thereof;

is or is about 1.56×10 ⁷ if A is CD8+CAR+ and B is IFN-gamma and IL-2 or its normalized value;

is or is approximately 1.75×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha, IFN-gamma and IL-2 or a normalized value thereof;

is or is approximately 2.19×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma and IL-2 or its normalized value,

optionally, where the apoptosis marker is annexin V or active caspase 3.

187. The method of any of embodiments 118-186, wherein the adverse event is grade 4 or 5 neurotoxicity and the target reference unit range is:

is between or approximately between 2.0×10 ⁵ and 1.75×10 ⁷ , inclusive, if A is negative (-) for an apoptosis marker and CD8+CAR+, and B is TNF-alpha or a normalized value thereof;

is between or approximately between 2.5×10 ⁵ and 2.19×10 ⁷ inclusive if A is CD8+CAR+ and B is TNF-alpha or a normalized value thereof;

is between or approximately between 4×10 ⁵ and 1.25×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma or a normalized value thereof;

is between or approximately between 5×10 ⁶ and 1.56×10 ⁷ inclusive if A is CD8+CAR+ and B is IFN-gamma or a normalized value thereof;

is between or approximately between 2.0×10 ⁵ and 1.5×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IL-2 or a normalized value thereof;

is between or approximately between 2.5x10 ⁵ and 1.88x10 ⁷ inclusive if A is CD8+CAR+ and B is IL-2 or a normalized value thereof;

is between or approximately between 3.0×10 ⁵ and 1.5×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IFN-gamma or its normalized value;

is between or approximately between 3.75×10 ⁵ and 1.88×10 ⁷ inclusive if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof;

is between or approximately between 3.0×10 ⁵ and 2.0×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IL-2 or its normalized value;

is between or approximately between 3.75×10 ⁵ and 2.5×10 ⁷ inclusive if A is CD8+CAR+ and B is TNF-alpha and IL-2 or a normalized value thereof;

is between or approximately between 4.0×10 ⁵ and 1.25×10 ⁷ inclusive if A is negative (–) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma and IL-2 or its normalized value;

is between or approximately between 5.0×10 ⁵ and 1.56×10 ⁷ inclusive if A is CD8+CAR+ and if B is IFN-gamma and IL-2 or its normalized value;

is between or approximately between 4.0×10 ⁵ and 1.75×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha, IFN-gamma and IL –2 or its normalized value;

is between or approximately between 5.0×10 ⁵ and 2.19×10 ⁷ inclusive if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma and IL-2 or a normalized value thereof,

optionally, where the apoptosis marker is annexin V or active caspase 3.

188. The method of any of embodiments 118-185, wherein the adverse event is at least Grade 3 long-term neurotoxicity and the threshold is:

is or is approximately 1.0×10 ⁶ if A is negative (-) for apoptosis marker and CD8+CAR+ and B is TNF-alpha or a normalized value thereof;

is or is about 1.25×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha or a normalized value thereof;

is or is approximately 2.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma or its normalized value;

is or is approximately 2.5×10 ⁶ if A is CD8+CAR+ and B is IFN-gamma or its normalized value;

is or is approximately 3.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IL-2 or a normalized value thereof;

is or is approximately 3.75×10 ⁶ if A is CD8+CAR+ and B is IL-2 or its normalized value;

is or is approximately 1.5×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IFN-gamma or a normalized value thereof;

is or is about 1.88×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof;

is or is approximately 2.5×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IL-2 or a normalized value thereof;

is or is approximately 3.12×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha and IL-2; or its normalized value

is or is approximately 3.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma and IL-2 or its normalized value;

is or is approximately 3.75×10 ⁶ if A is CD8+CAR+ and B is IFN-gamma and IL-2 or its normalized value;

is or is about 2.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha, IFN-gamma and IL-2 or a normalized value thereof;

is or is approximately 2.5×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma and IL-2 or its normalized value, optionally, where the apoptosis marker is annexin V or active caspase 3.

189. The method of any of embodiments 118-185 and 188, wherein the adverse event is at least Grade 3 long-term neurotoxicity and the target reference unit range is:

is between or approximately between 3.0×10 ⁵ and 1.0×10 ⁶ inclusive, if A is negative (-) for an apoptosis marker and CD8+CAR+, and B is TNF-alpha or a normalized value thereof;

is between or approximately between 3.75×10 ⁵ and 1.25×10 ⁶ inclusive if A is CD8+CAR+ and B is TNF-alpha or a normalized value thereof;

is between or approximately between 4×10 ⁵ and 2.0×10 ⁶ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma or a normalized value thereof;

is between or approximately between 5×10 ⁶ and 2.5×10 ⁶ inclusive if A is CD8+CAR+ and B is IFN-gamma or a normalized value thereof;

is between or approximately between 2.0×10 ⁵ and 3.0×10 ⁶ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IL-2 or a normalized value thereof;

is between or approximately between 2.5×10 ⁵ and 3.75×10 ⁶ inclusive if A is CD8+CAR+ and B is IL-2 or its normalized value;

is between or approximately between 3.0×10 ⁵ and 1.5×10 ⁶ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IFN-gamma or its normalized value;

is between or approximately between 3.75×10 ⁵ and 1.88×10 ⁶ inclusive if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof;

is between or approximately between 3.0×10 ⁵ and 2.5×10 ⁶ inclusive if A is negative (–) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IL-2 or its normalized value;

is between or approximately between 3.75×10 ⁵ and 3.12×10 ⁶ inclusive if A is CD8+CAR+ and B is TNF-alpha and IL-2 or a normalized value thereof;

is between or approximately between 4.0x10 ⁵ and 3.0x10 ⁶ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma and IL-2 or its normalized value;

is between or approximately between 5.0×10 ⁵ and 3.75×10 ⁶ inclusive if A is CD8+CAR+ and B is IFN-gamma and IL-2 or its normalized value;

is between or approximately between 4.0×10 ⁵ and 2.0×10 ⁶ inclusive if A is negative (–) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha, IFN-gamma and IL –2 or its normalized value;

is between or approximately between 5.0×10⁵ and 2.5×10⁶, inclusive, if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma and IL-2 or its normalized value,

optionally, where the apoptosis marker is annexin V or active caspase 3.

190. The method of any of embodiments 118-189, wherein the T cell therapeutic composition contains between about 10 million cells per ml and about 70 million cells per ml, or between about 10 million viable cells per ml and about 70 million viable cells per ml , including each.

191. The method of any one of embodiments 118-190, wherein the T cell therapeutic composition contains between about 15 million cells or viable cells per ml and about 60 million cells or viable cells per ml, each including.

192. The method of any of embodiments 118-191 wherein the T cell composition contains more than 10 million cells or viable cells per ml.

193. The method of any of embodiments 118-192, wherein the T cell therapeutic composition contains more than 15 million cells or more than 15 million cells per ml.

194. The method of any one of embodiments 118-193, wherein the disease or condition is cancer, optionally myeloma, lymphoma, or leukemia.

195. The method of embodiment 194 wherein the disease or condition is a B-cell malignancy, optionally a B-cell malignancy, selected from the group consisting of acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia ( CLL), non-Hodgkin's lymphoma (NHL), and diffuse large B-cell lymphoma (DLBCL).

196. The method of any of embodiments 118-195 wherein the antigen is CD19, CD22, ROR1, Ig-kappa, Her2, L1-CAM, CD20, mesothelin, CEA, hepatitis B surface antigen, antifolate receptor, CD23, CD24 , CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, ErbB3, ErbB4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R –alpha2, kdr, kappa light chain, Lewis Y, cell adhesion molecules L1, MAGE–A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligands, NY–ESO–1, MART–1, gp100, oncofetal antigen, TAG72, VEGF –R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, estrogen receptor, progesterone receptor, ephrin B2, CD123, CS–1, c–Met, GD–2, MAGE A3, CE7, Wilms tumor antigen 1 (WT–1 ) and cyclin A1 (CCNA1).

197. The method of any of embodiments 118-196 wherein the recombinant receptor is a CAR.

198. The method of embodiment 197, wherein the CAR comprises an extracellular antigen recognition domain that specifically binds to an antigen and an intracellular signaling domain comprising ITAM, where optionally the intracellular signaling domain comprises an intracellular domain from the CD3-zeta (CD3ζ) chain; and/or wherein the CAR further comprises a co-stimulatory signaling region, which optionally comprises a signaling domain from CD28 or 4-1BB.

199. The method of any of embodiments 118-198 wherein the T cells are CD4+ or CD8+.

200. The method of any of embodiments 118-199 wherein the T cells are primary T cells, optionally wherein the T cells are autologous to the individual or allogeneic to the individual.

201. The method of any of embodiments 118-200, wherein the unit dose contains no more than 15 x 10 ⁶ CD8+CAR+ cells that are negative for annexin V or active caspase 3 or caspase 3 proform.

202. The method of any of embodiments 118-201, wherein the unit dose further comprises an amount of CAR positive CD4+ cells, where the amount is in a ratio of at least, or is in a ratio of, CD8+CAR+ cells of 1:1 or about 1: one.

203. The method of any of embodiments 118-202, wherein the method further comprises administering antilymphocyte chemotherapy prior to administration of the T cell composition, and/or wherein the individual is subjected to antilymphocyte chemotherapy prior to administration of the T cell composition.

204. The method of embodiment 203 wherein the antilymphocyte chemotherapy comprises administering fludarabine and/or cyclophosphamide to the individual.

205. The method of embodiment 204 wherein cyclophosphamide is administered in an amount of from or about 30 mg/kg to 60 mg/kg and/or fludarabine is administered in an amount of from or about 25 mg/m2 to 30 mg/m2.

206. The method of embodiment 204, where:

cyclophosphamide is administered in an amount of from or about 900 to 1000 mg, and/or fludarabine is administered in an amount of from or about 25 mg/m2 to 30 mg/m2; or

cyclophosphamide is administered at or about 300 mg/m2 and/or fludarabine is administered at or from about 30 mg/m2.

207. The method of embodiment 204 wherein cyclophosphamide is administered at or about 500 mg/m2 and/or fludarabine is administered at or from about 30 mg/m2.

208. The method of embodiment 204 wherein cyclophosphamide is administered at or about 500 mg/m2 and/or fludarabine is administered at or from about 60 mg/m2.

209. The method of any of embodiments 203-208 wherein the antilymphocyte drug is administered once daily for three consecutive days.

210. The method of any of embodiments 203-209 wherein the antilymphocyte drug is administered 5 days prior, 4 days prior, and 3 days prior to administration of the T cell composition.

211. The method of any of embodiments 203-210 wherein the antilymphocyte drug is administered once daily for 3-5 days, optionally once daily for 3 days.

212. The method of any of embodiments 204-211 wherein fludarabine and cyclophosphamide are administered sequentially.

213. The method of any of embodiments 204-211 wherein fludarabine and cyclophosphamide are administered simultaneously.

214. A method for analyzing a therapeutic composition containing a single dose of a T-cell composition, including:

(a) analyzing a sample from a T cell composition comprising T cells derived from an individual having the disease or condition and transduced with a nucleic acid encoding a recombinant receptor, optionally a chimeric antigen receptor (CAR), that specifically binds to an antigen associated with a disease or condition, where the analysis determines B for a cell composition, where B is a parameter value, or a multiple thereof, or a transformation that indicates the degree of or correlates with the degree of recombinant receptor-dependent activity in a given composition; and

(b) evaluating the activity of the cell composition based on B and/or evaluating whether the composition is above the lower specified limit (LSL) for B or below the upper specified limit (USL) for B.

215. A method for evaluating a T cell therapeutic composition, comprising evaluating a sample from a T cell composition containing T cells containing a recombinant receptor that specifically binds to an antigen associated with a disease or condition, by the activity of the cell composition based on B, and/ or judging whether the composition is above the lower set limit (LSL) for B or below the upper set limit (USL) for B.

216. The method of Embodiment 214 or Embodiment 215, wherein the product is released to treat an individual only if the composition is below USL for B.

217. The method of Embodiment 214 or Embodiment 215, where if B is greater than USL, recommend not administering the composition to the individual, or changing the number of cells administered to the individual to an adjusted unit dose, where the adjusted unit dose contains the target number of cells or the target number of reference units (RU) from a composition of T-cells, where RU in this composition is determined by the formula

RU=A x B, where:

A is the number of cells, or a multiple, proportion or transformation, of some phenotype present in a given composition, or is the average or weighted average of the number of cells, or a multiple, proportion or transformation, of two or more phenotypes in a given composition; and

B is a parameter value, or a fraction thereof, or a multiple, or a transformation that indicates the degree of or correlates with the degree of recombinant receptor dependent, optionally CAR dependent, activity in the composition.

218. The method of any of embodiments 214-217, wherein A is the number of cells, or a multiple thereof, or proportion, or transformation, of a given phenotype present in a given composition.

219. The method of any of embodiments 214-217, wherein A is the average or weighted average of the number of cells, or a multiple thereof, proportion or conversion, of two or more phenotypes in a given composition.

220. The method of any of embodiments 214-219, wherein the target number of units is less than a threshold number of units, optionally a safe number of reference units, where the safe number of reference units is, in relation to a group of individuals analyzed after treatment with using a T cell therapeutic composition comprising T cells expressing a recombinant receptor, optionally CAR, the lowest number of drug reference units administered to an individual among those individuals in the group who continued to develop an adverse event.

221. The method of embodiment 220, wherein the adverse event is a serious adverse event, optionally severe neurotoxicity, optionally a grade equal to or greater than grade 4 or grade 5, or at least long-term grade 3 neurotoxicity.

222. The method of Embodiment 220 or Embodiment 221, where the target number of reference units is less than the safe number of reference units by an amount corresponding to a safety factor and/or by an amount in the range of 1.5- to 3-fold , optionally approximately 2-fold, or a multiple of the standard deviation in a group of individuals who did not experience an adverse event, optionally, neurotoxicity grade 0-2, optionally, where the multiple lies in the range from 1.5- to 3-fold .

223. The method of any of embodiments 217-222, wherein the target number of reference units is equal to or greater than the reference effective amount of reference units, where the reference effective amount is, in relation to a group of individuals analyzed after treatment with a therapeutic T cell composition containing recombinant receptor, optionally CAR, number of drug units administered to one or more individuals within a group for which the desired therapeutic outcome is indicated, optionally a partial response or complete response (CR).

224. The method of any of embodiments 217-223 wherein the adjusted unit dose is less than, optionally, less than 1.5 times, less than 2 times, less than 3 times, less than 4 times the average unit dose for a group of individuals treated with a T cell therapeutic composition containing T cells expressing a recombinant receptor, optionally a CAR.

225. The method of any one of embodiments 214-224, wherein a sample from a T cell composition, optionally a cryopreserved sample, is evaluated after administration of the T cell composition to an individual.

226. The method of any of embodiments 214-225, wherein if B is greater than USL, the individual is determined to be at risk of toxicity.

227. The method of any of embodiments 214-225, wherein if B is greater than USL, the subject administered the composition is monitored and/or treated with an agent to alleviate or reduce the likelihood of a cytokine release syndrome or toxicity outcome following administration of the cell composition and , optionally, before the onset of a sign or symptom of the outcome of toxicity.

228. A method for assessing the risk of toxicity of a therapeutic composition of T cells, including:

(a) evaluating a sample from a T cell composition administered to an individual for a number of reference units (RU) within a given range, where the T cell composition contains T cells containing a recombinant receptor that specifically binds to an antigen associated with the disease, or state, where RU in this composition is determined by the formula:

RU=A x B, where:

A is the number of cells, or a multiple, proportion or conversion, of cells of a certain phenotype present in a given composition, or is the average or weighted average of the number of cells, or a multiple, proportion, or conversion, of two or more phenotypes in a given composition; and

B is a parameter value, or a fraction thereof, or a multiple, or a transformation that indicates the degree of, or correlates with, the degree of recombinant receptor-dependent, optionally CAR-dependent, activity in the composition; and

(b) comparing the amounts of RU with reference safe amounts of RU, where the comparison indicates whether or not the individual is at risk of experiencing an adverse event, optionally a serious adverse event, optionally severe neurotoxicity equal to or greater than grade 4 or grade 5, or at least long-term neurotoxicity degree 3.

229. The method of embodiment 228, where A is the number of cells, or a multiple thereof, or proportion, or transformation, of a given phenotype present in a given composition.

230. The method of embodiment 229, wherein A is the average or weighted average of the number of cells, or a multiple thereof, proportion or conversion, of two or more phenotypes in a given composition.

231. The method of any of embodiments 228-230, wherein the reference safe amount of RU is, in relation to a group of individuals analyzed after treatment using a T cell therapeutic composition containing T cells expressing a recombinant receptor, optionally CAR, the least amount reference units of drug administered to an individual, among those individuals in the group who continued to develop an adverse event.

232. The method of any of embodiments 228-231, wherein if the comparison indicates that the RU is higher than the reference safe RU, the individual administered the composition is monitored and/or treated with an agent to alleviate or reduce the likelihood of an outcome of toxicity or cytokine release syndrome , after administration of the cell composition and, optionally, before the onset of a sign or symptom of the outcome of toxicity.

233. A method for producing a therapeutic composition containing a single dose of a T-cell composition, including:

(a) analysis of a T cell composition comprising T cells derived from an individual having the disease or condition and transduced with a nucleic acid encoding a recombinant receptor, optionally a chimeric antigen receptor (CAR), that specifically binds to an antigen associated with the disease or a state where the assay determines B for a cell composition, where B is a parameter value, or a multiple thereof, or a transformation that indicates the degree of or correlates with the degree of recombinant receptor-dependent, optionally CAR-dependent, activity in the composition; and

(b) filling the container with all or part of the composition and optionally another solution to obtain a unit dose of the T cell composition, where the unit dose contains the target number of reference units (RU) from the T cell composition, where the RU in this composition is determined by the formula:

RU=A x B, where A is the number of cells, or a multiple, proportion or transformation, of some phenotype present in a given composition, or is the average or weighted average of the number of cells, or a multiple thereof, proportion or transformation, of two or more phenotypes in this composition.

234. A method for preparing a therapeutic composition containing a single dose of a T cell composition, comprising filling a container with all or part of the T cell composition containing T cells containing a recombinant receptor that specifically binds to an antigen associated with a disease or condition, and optionally, another solution to produce a unit dose of a T cell composition, where the unit dose contains the target number of reference units (RU) from the T cell composition, where the RU in the composition is determined by the formula:

RU=A x B, where

A is the number of cells, or a multiple, proportion or transformation, of some phenotype present in a given composition, or is the average or weighted average of the number of cells, or a multiple, proportion or transformation, of two or more phenotypes in a given composition; and

B is a parameter value, or a multiple thereof, or a transformation that indicates the degree of or correlates with the degree of recombinant receptor dependent, optionally CAR dependent, activity in the composition.

235. The method of Embodiment 233 or Embodiment 234, where A is the number of cells, or a multiple thereof, or proportion, or transformation, of a given phenotype present in a given composition.

236. The method of Embodiment 233 or Embodiment 234, wherein A is the average or weighted average of the number of cells, or a multiple thereof, proportion or conversion, of two or more phenotypes in a given composition.

237. The method of any of embodiments 217-236, wherein A is the number of cells of some phenotype present in the composition and B is a parameter value that indicates the degree of or correlates with the degree of recombinant receptor dependent activity in the composition.

238. The method of any of embodiments 217-237, wherein A and/or B is a quantity or value transformation, respectively, where the transformation includes a log transformation, a power transformation, or a logit transformation.

239. The method of any of embodiments 217-238, wherein A is the number of cells of some phenotype present in a given composition and B is a multiple or transformation of a parameter value indicative of the degree of or correlated with the degree of CAR-dependent activity in the composition T-cells, optionally, where B is the logarithmic transformation of the value.

240. The method of Embodiment 238 or Embodiment 239, where the logarithmic transformation is decimal log (log ₁₀ (x)), natural log (ln(x)) or binary log (log ₂ (x)).

241. The method of any of embodiments 217-240, wherein A is the number of viable cells in the composition and/or is the number of cells that are not apoptotic, do not have a factor indicative of early apoptosis or apoptosis, are not in the early stages apoptosis, or are not in the late stages of apoptosis, and/or is the number of cells in a certain state of differentiation, and/or is the number of cells having a memory cell/stem cell trait, or represents its multiple or transformation.

242. The method of any of embodiments 217-241, wherein the phenotype includes positive expression of a surface marker that is one or more of CD3, CD4, or CD8, and/or includes positive expression of a recombinant receptor, optionally CAR, or a surrogate expression marker for recombinant receptor.

243. The method of embodiment 242, wherein the phenotype is CD3+ CAR, CD4+/CAR+, CD8+/CAR+.

244. The method of any of embodiments 217-243, wherein the phenotype includes the absence of a factor indicative of apoptosis or one or more steps in the apoptotic cascade or pathway, optionally, expression of an apoptosis marker.

245. The method of any of embodiments 217-244, wherein the phenotype comprises negative expression of an apoptosis marker, optionally an early apoptosis or late apoptosis marker.

246. The method of embodiment 245, wherein the apoptosis marker is surface phosphatidylserine and/or detected using annexin V, or is an active caspase or a caspase proform, optionally an active caspase 3 or a caspase 3 proform.

247. The method of any of embodiments 217-246 wherein the phenotype includes annexin-.

248. The method of any of embodiments 217-247, where the phenotype includes an indicator of the production of one or a combination of cytokines, optionally not specific for the antigen or recombinant receptor and / or polyclonal production, where one or more cytokines is IL-2, IL- 13, IL-17, IFN-gamma or TNF-alpha.

249. The method of Embodiment 248, wherein the production index is measured in an assay, optionally an intracellular cytokine staining assay, comprising incubating a sample from a T cell composition with a polyclonal agent, an antigen specific agent, or a recombinant receptor binding agent, optionally CAR.

250. The method of Embodiment 248 or Embodiment 249, wherein the agent is or contains PMA and ionomycin, or is or contains a T-cell receptor or a T-cell receptor complex agonist.

251. The method of any of embodiments 217-250 wherein the phenotype comprises negative expression of an activation marker, wherein the activation marker is selected from CD25, CD127, LAG3, Ki67, and combinations thereof.

252. The method of any of embodiments 217-251 wherein the phenotype comprises negative expression of a depletion marker, wherein the depletion marker is a PD1 or FOXP3 gene product, or a combination thereof.

253. The method of any of embodiments 217-252, wherein the phenotype comprises a naïve or memory cell phenotype, optionally wherein the memory cell phenotype comprises an effector memory T cell phenotype, a central memory T cell phenotype, or an effector memory T cell phenotype expressing CD45RA (Temra).

254. The method of any of embodiments 217-253 wherein A is total T cells, total CD3+ cells, total CD4+ or CD8+ cells, total CD3+CAR+ cells, total CD8+CAR+ cells, total CD4+ CAR+, or total number of living or viable cells from any of the above, or its multiple or converted value.

255. The method of any of embodiments 217-253, wherein A is total CD3+ cells, total CD8+, total CD3+CAR+ cells, total CD8+CAR+ cells, or total living or viable cells from any of the above. , or its multiple or converted value.

256. The method of any of embodiments 217-253, wherein A is the total number of apoptosis marker negative (-) cells representing CD3+ CAR+ cells, the total number of apoptosis marker negative (-) cells representing CD4+ CAR+, total the number of cells negative (-) for the apoptosis marker, which are CD8+ CAR+ cells, or its multiple or converted value, where the apoptosis marker is annexin V or caspase.

257. The method of any of embodiments 217-256, wherein A is the total number of annexin-CD3+ CAR+ cells or the total number of annexin-CD8+ CAR+ cells.

258. The method of any of embodiments 214-257, wherein the recombinant receptor dependent activity is indicative of the production or accumulation of a pro-inflammatory cytokine, optionally one or a combination of TNF-alpha, IFN-gamma, IL-2, and IL-10.

259. The method of any of embodiments 214-258, wherein the parameter is a measure of one or more factors, or a normalized value thereof.

260. The method of embodiment 259, wherein the index is determined in an assay comprising culturing or incubating for a fixed time, optionally 24 hours, a given composition or a sample thereof in the presence of an antigen, antigen-expressing cells, and/or a specific binding agent. with a recombinant receptor, optionally CAR.

261. The method of embodiment 260 wherein the assay is an ELISA.

262. The method of any of embodiments 259-261, wherein the factor score is:

(i) the concentration, relative concentration, amount or relative amount of the factor; or

(ii) the amount or relative amount of the factor per unit of cells administered from the composition, or

(iii) the amount or relative amount of the factor per unit of cells administered from the composition per unit of time, optionally one hour; or

(iv) a level indicative of any of (i)-(iii).

263. The method of any of embodiments 259-262, wherein the one or more factors is one or a combination of soluble factors, optionally one or a combination of cytokines, chemokines, or soluble receptors, optionally soluble costimulatory receptors.

264. The method of any of embodiments 259-263, wherein the one or more factors is one or a combination of pro-inflammatory cytokines, Th2 cytokines, and Th17 cytokines.

265. The method of any of embodiments 259-264 wherein one or more of the factors is one or a combination of IL-2, IFN-gamma, TNF-alpha, IL4, IL-5, IL-10, IL-13, GM –CSF, sCD137, MIP1a and M1Pb.

266. The method of any of embodiments 259-265, wherein one or more of the factors is one or a combination of IL-2, IFN-gamma, TNF-alpha, and IL-10.

267. The method of any of embodiments 259-266, wherein one or more factors is a combination of any two or more of the above soluble factors, and the parameter is the arithmetic or geometric mean of the scores for the two or more factors.

268. The method of any of embodiments 259-267, wherein the parameter is the arithmetic or geometric mean, optionally, of an amount or concentration, of at least two of TNF-alpha, IFN-gamma, and IL-2, or of TNF- alpha, IFN-gamma and IL-2.

269. The method of any one of embodiments 259-268, wherein the parameter is a normalized score value, where the normalization is compared to a reference factor score.

270. The method of Embodiment 269 wherein the benchmark is the average of scores among a plurality of, optionally at least 10, at least 15, at least 20, of reference chimeric antigen receptor (CAR) T cell therapeutic compositions , in which:

(i) for each of the reference T cell therapeutic compositions, an acceptable safety profile was observed or determined as a result after administration to an individual, optionally where the individual has a disease or condition with antigen expression or association with an antigen; and/or

(ii) for each of the reference T cell therapeutic compositions, the desired efficacy is observed or determined as a result after administration to an individual, optionally where the individual has a disease or condition with antigen expression or association with an antigen.

271. The method of embodiment 270, wherein the acceptable safety profile is no observed grade 2 or higher neurotoxicity, or no grade 3 or higher neurotoxicity.

272. The method of embodiment 270 or embodiment 271, wherein the acceptable safety profile is no observable grade 3 or higher neurotoxicity.

273. The method of any of embodiments 270-272, wherein the efficacy is a partial response or is a complete response (CR).

274. The method of Embodiment 269, wherein the benchmark is the score, in the same assay, of a factor in a T cell reference composition that is prepared in the same manner as the T cell therapeutic composition but does not express a recombinant receptor, optionally CAR , does not recognize a specific antigen and/or does not express any recombinant receptor, optionally any CAR.

275. The method of embodiment 274 wherein this parameter is normalized to control for patient-specific variability in a score of one or more factors.

276. The method of Embodiment 274 or Embodiment 275, wherein the parameter is the normalized value of the factor score, compared to the same score in the same assay, for a control factor, where the control factor level in the T cell therapeutic composition is known to be absent or observed no sign or correlation, or significant correlation with the outcome of an adverse event or toxicity, or their likelihood or risk, where the outcome of an adverse event or toxicity is not necessarily severe neurotoxicity.

277. The method of embodiment 276, wherein the control factor is or contains a factor that is not statistically correlated and/or correlated with the occurrence of an adverse event among a plurality of individuals who continued to develop an adverse event after administration of the T cell composition, optionally the control factor is one or a combination of IL-5, IL-13, GM-CSF and IL-6, optionally wherein the control factor score is the arithmetic or geometric mean of two or more of the above.

278. The method of any of embodiments 214-277, wherein the parameter does not include cytolytic activity or an index thereof.

279. The method of any of embodiments 214-278, wherein the parameter does not include recombinant receptor dependent or antigen specific cytolytic activity or an index thereof.

280. The method of any of embodiments 217-279, where:

the phenotype is CD8+ CAR+ cells or an apoptosis marker - CD8+ CAR+ cells, optionally, where the apoptosis marker is annexin V or active caspase 3; and

parameter is a measure for a pro-inflammatory cytokine, which is optionally one or a combination of TNF-alpha, IL-2 and IFN-gamma, or is its normalized value.

281. The method of any of embodiments 217-280, where:

an adverse event is a grade 4 or 5 neurotoxicity and a threshold value of:

is or is approximately 1.75×10 ⁷ if A is negative (-) for apoptosis marker and CD8+CAR+ and B is TNF-alpha or a normalized value thereof;

is or is approximately 2.19×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha or a normalized value thereof;

is or is approximately 1.25×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma or its normalized value;

is or is approximately 1.56×10 ⁷ if A is CD8+CAR+ and B is IFN-gamma or its normalized value;

is or is approximately 1.5×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IL-2 or a normalized value thereof;

is or is approximately 1.88×10 ⁷ if A is CD8+CAR+ and B is IL-2 or its normalized value;

is or is approximately 1.5×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IFN-gamma or a normalized value thereof;

is or is about 1.88×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof;

is or is approximately 2.0×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IL-2 or a normalized value thereof;

is or is approximately 2.5×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha and IL-2 or its normalized value;

is or is approximately 1.25×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma and IL-2 or a normalized value thereof;

is or is about 1.56×10 ⁷ if A is CD8+CAR+ and B is IFN-gamma and IL-2 or its normalized value;

is or is approximately 1.75×10 ⁷ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha, IFN-gamma and IL-2 or a normalized value thereof;

is or is approximately 2.19×10 ⁷ if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma and IL-2 or its normalized value,

optionally, where the apoptosis marker is annexin V or active caspase 3.

282. The method of any of embodiments 217-281, wherein the adverse event is grade 4 or 5 neurotoxicity and the target reference unit range is:

is between or approximately between 2.0×10 ⁵ and 1.75×10 ⁷ , inclusive, if A is negative (-) for an apoptosis marker and CD8+CAR+, and B is TNF-alpha or a normalized value thereof;

is between or approximately between 2.5×10 ⁵ and 2.19×10 ⁷ inclusive if A is CD8+CAR+ and B is TNF-alpha or a normalized value thereof;

is between or approximately between 4×10 ⁵ and 1.25×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma or a normalized value thereof;

is between or approximately between 5×10 ⁶ and 1.56×10 ⁷ inclusive if A is CD8+CAR+ and B is IFN-gamma or a normalized value thereof;

is between or approximately between 2.0×10 ⁵ and 1.5×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IL-2 or a normalized value thereof;

is between or approximately between 2.5x10 ⁵ and 1.88x10 ⁷ inclusive if A is CD8+CAR+ and B is IL-2 or a normalized value thereof;

is between or approximately between 3.0×10 ⁵ and 1.5×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IFN-gamma or its normalized value;

is between or approximately between 3.75×10 ⁵ and 1.88×10 ⁷ inclusive if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof;

is between or approximately between 3.0×10 ⁵ and 2.0×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IL-2 or its normalized value;

is between or approximately between 3.75×10 ⁵ and 2.5×10 ⁷ inclusive if A is CD8+CAR+ and B is TNF-alpha and IL-2 or a normalized value thereof;

is between or approximately between 4.0×10 ⁵ and 1.25×10 ⁷ inclusive if A is negative (–) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma and IL-2 or its normalized value;

is between or approximately between 5.0×10 ⁵ and 1.56×10 ⁷ inclusive if A is CD8+CAR+ and if B is IFN-gamma and IL-2 or its normalized value;

is between or approximately between 4.0×10 ⁵ and 1.75×10 ⁷ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha, IFN-gamma and IL –2 or its normalized value;

is between or approximately between 5.0×10 ⁵ and 2.19×10 ⁷ inclusive if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma and IL-2 or a normalized value thereof,

optionally, where the apoptosis marker is annexin V or active caspase 3.

283. The method of any of embodiments 217-280, wherein the adverse event is at least Grade 3 long-term neurotoxicity and the threshold is:

is or is approximately 1.0×10 ⁶ if A is negative (-) for apoptosis marker and CD8+CAR+ and B is TNF-alpha or a normalized value thereof;

is or is about 1.25×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha or a normalized value thereof;

is or is approximately 2.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma or its normalized value;

is or is approximately 2.5×10 ⁶ if A is CD8+CAR+ and B is IFN-gamma or its normalized value;

is or is approximately 3.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IL-2 or a normalized value thereof;

is or is approximately 3.75×10 ⁶ if A is CD8+CAR+ and B is IL-2 or its normalized value;

is or is approximately 1.5×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IFN-gamma or a normalized value thereof;

is or is about 1.88×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof;

is or is approximately 2.5×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IL-2 or a normalized value thereof;

is or is approximately 3.12×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha and IL-2; or its normalized value

is or is approximately 3.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma and IL-2 or its normalized value;

is or is approximately 3.75×10 ⁶ if A is CD8+CAR+ and B is IFN-gamma and IL-2 or its normalized value;

is or is about 2.0×10 ⁶ if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha, IFN-gamma and IL-2 or a normalized value thereof;

is or is approximately 2.5×10 ⁶ if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma and IL-2 or its normalized value, optionally, where the apoptosis marker is annexin V or active caspase 3.

284. The method of any of embodiments 217-280 and 283, wherein the adverse event is at least Grade 3 long-term neurotoxicity and the target reference unit range is:

is between or approximately between 3.0×10 ⁵ and 1.0×10 ⁶ , inclusive, if A is negative (–) for an apoptosis marker and CD8+CAR+, and B is TNF-alpha or a normalized value thereof;

is between or approximately between 3.75×10 ⁵ and 1.25×10 ⁶ inclusive if A is CD8+CAR+ and B is TNF-alpha or a normalized value thereof;

is between or approximately between 4×10 ⁵ and 2.0×10 ⁶ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma or a normalized value thereof;

is between or approximately between 5×10 ⁶ and 2.5×10 ⁶ inclusive if A is CD8+CAR+ and B is IFN-gamma or a normalized value thereof;

is between or approximately between 2.0×10 ⁵ and 3.0×10 ⁶ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IL-2 or a normalized value thereof;

is between or approximately between 2.5×10 ⁵ and 3.75×10 ⁶ inclusive if A is CD8+CAR+ and B is IL-2 or its normalized value;

is between or approximately between 3.0×10 ⁵ and 1.5×10 ⁶ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IFN-gamma or its normalized value;

is between or approximately between 3.75×10 ⁵ and 1.88×10 ⁶ inclusive if A is CD8+CAR+ and B is TNF-alpha and IFN-gamma or a normalized value thereof;

is between or approximately between 3.0×10 ⁵ and 2.5×10 ⁶ inclusive if A is negative (–) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha and IL-2 or its normalized value;

is between or approximately between 3.75×10 ⁵ and 3.12×10 ⁶ inclusive if A is CD8+CAR+ and B is TNF-alpha and IL-2 or a normalized value thereof;

is between or approximately between 4.0x10 ⁵ and 3.0x10 ⁶ inclusive if A is negative (-) for the apoptosis marker CD8+CAR+ and if B is IFN-gamma and IL-2 or its normalized value;

is between or approximately between 5.0×10 ⁵ and 3.75×10 ⁶ inclusive if A is CD8+CAR+ and B is IFN-gamma and IL-2 or its normalized value;

is between or approximately between 4.0×10 ⁵ and 2.0×10 ⁶ inclusive if A is negative (–) for the apoptosis marker CD8+CAR+ and if B is TNF-alpha, IFN-gamma and IL –2 or its normalized value;

is between or approximately between 5.0×10⁵ and 2.5×10⁶, inclusive, if A is CD8+CAR+ and B is TNF-alpha, IFN-gamma and IL-2 or its normalized value,

optionally, where the apoptosis marker is annexin V or active caspase 3.

285. The method of any of embodiments 214-284 wherein the recombinant receptor is a CAR.

286. The method of embodiment 285, wherein the CAR comprises an extracellular antigen recognition domain that specifically binds to an antigen and an intracellular signaling domain comprising ITAM, where optionally the intracellular signaling domain comprises an intracellular domain from the CD3-zeta (CD3ζ) chain; and/or wherein the CAR further comprises a costimulatory signaling region, which optionally comprises a signaling domain from CD28 or 4-1BB.

287. The method of any of embodiments 214-286 wherein the T cells are CD4+ or CD8+.

288. The method of any of embodiments 214-287, wherein the T cells are primary T cells, optionally autologous or allogeneic for the individual.

289. A method of preparing a T cell therapeutic composition for cell therapy, comprising filling a container with all or part of the composition containing T cells to a concentration of between about 10 million cells and about 70 million cells per ml, inclusive, and optionally, with another solution.

290. The method of embodiment 289 wherein the T cells are obtained from an individual having the disease or condition and transduced with a nucleic acid encoding a recombinant receptor, optionally a chimeric antigen receptor (CAR), specific for an antigen associated with the disease or condition, or expressed in the disease or condition and/or the T cells contain a recombinant receptor, optionally a CAR.

291. The method of any of embodiments 233-290 wherein the container is filled using between about 15 and about 60 million cells per ml, inclusive.

292. The method of any of embodiments 233-291 wherein the container is filled using more than 10 million cells per ml.

293. The method of any of embodiments 233-292, wherein the container is filled with more than 15 million cells per ml.

294. The method of any of embodiments 233-293, wherein the filling is performed in automatic mode.

295. The method of any of embodiments 233-295 wherein the filling is carried out in a closed system.

296. The method of any of embodiments 233-295 wherein the container is filled with another solution and the solution contains a cryoprotectant, optionally DMSO.

297. The method of any of embodiments 233-295, further comprising freezing the cells in the container, or storing the container at less than or about less than 80°C.

298. The method of any of embodiments 233-298, wherein the recombinant receptor is a CAR.

299. The method of embodiment 298, wherein the CAR comprises an extracellular antigen recognition domain that specifically binds to an antigen and an intracellular signaling domain comprising ITAM, where optionally the intracellular signaling domain comprises an intracellular domain from the CD3-zeta (CD3ζ) chain; and/or wherein the CAR further comprises a costimulatory signaling region, which optionally comprises a signaling domain from CD28 or 4-1BB.

300. The method of any of embodiments 233-299, wherein the T cells are CD4+ or CD8+.

301. The method of any of embodiments 233-301, wherein the T cells are primary T cells, optionally autologous or allogeneic for the individual.

302. The method of any of embodiments 118-301, wherein the T cell composition is prepared by a method wherein:

frequency, (1) among CAR+ cells in the composition, (2) among CAR+CD3+ cells in the composition, and/or (3) among CAR+CD8+ cells in the composition, of cells of a phenotype indicating signs of biologically active cells and/or lack of apoptosis , or early or late stages of apoptosis, differs by no more than 40%, or no more than 30%, or no more than 20%, or no more than 10%, or no more than 5%, from the average indicated frequency in the set of compositions of T-cells obtained by this method, and/or differs from such an average by no more than one standard deviation; or

the frequency, (1) among CAR+ cells in the composition, (2) among CAR+CD3+ cells in the composition, and/or (3) among CAR+CD8+ cells in the composition, of cells with a phenotype indicating the absence of apoptosis, or early or late stages of apoptosis, differ by no more than 40%, or no more than 20%, or no more than 10%, among the plurality of T cell compositions obtained by this method

303. The method of embodiment 302, comprising:

(a) incubating a population of cells containing T cells with an agent containing a nucleic acid molecule encoding a recombinant receptor under conditions to introduce a nucleic acid encoding a recombinant receptor into cells in the population; and

(b) stimulating the cells, before, during and/or after said incubation, wherein the stimulation comprises incubating the cells in the presence of a stimulating condition that induces a primary signal, signaling, stimulating, activating and/or proliferating the cells.

304. The method of embodiment 303, further comprising, before (a), isolating a population of cells from a biological sample.

305. The method of embodiment 304, wherein the isolation comprises selecting cells based on surface expression of CD3 or based on surface expression of one or both of CD4 and CD8, optionally by positive or negative selection.

306. The method of embodiment 304 or embodiment 305, wherein the isolation comprises immunoselection based on affinity.

307. The method of embodiment 304-306 wherein the biological sample is or contains a whole blood sample, buffy coat sample, peripheral blood mononuclear cell (PBMC) sample, unfractionated T-cell sample, lymphocyte sample, leukocyte sample, apheresis product, or leukapheresis.

308. The method of any of embodiments 303-307, wherein the stimulatory condition comprises incubation with a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and/or one or more intracellular signaling domains of one or more costimulatory molecules.

309. The method of embodiment 308, wherein the stimulatory reagent comprises a primary agent that specifically binds to a member of the TCR complex and a secondary agent that specifically binds to a costimulatory T cell molecule.

310. The method of embodiment 308 or embodiment 309, wherein the primary agent specifically binds to CD3 and/or a costimulatory molecule selected from the group consisting of CD28, CD137 (4-1-BB), OX40, or ICOS.

311. The method of embodiment 306 or embodiment 310, wherein the primary and secondary agents contain antibodies and/or are present on the surface of a solid support, optionally a bead.

312. The method of any of embodiments 308-311, where:

a stimulating reagent is a reagent for which it is determined that the recombinant receptor dependent activity or antigen specific activity, optionally recombinant receptor dependent or antigen specific dependent production or accumulation of a pro-inflammatory cytokine, differs by no more than 40% or no more than 30 %, or no more than 20%, or no more than 10%, or no more than 5%, of the average pro-inflammatory cytokine production or accumulation among the plurality of T cell compositions produced by this method using a stimulatory reagent, and /or differs from such mean by no more than one standard deviation; and/or

a stimulating reagent is a reagent for which it is determined that the recombinant receptor dependent activity or antigen specific activity, optionally recombinant receptor dependent or antigen specific dependent production or accumulation of a pro-inflammatory cytokine, differs by no more than 40% or no more than 30 %, or not more than 20% or not more than 10%, or not more than 5%, among the many compositions of T-cells obtained by this method; and/or

a stimulating reagent is a reagent for which it is determined that the recombinant receptor dependent activity or antigen specific activity, optionally recombinant receptor dependent or antigen specificity dependent production or accumulation of a pro-inflammatory cytokine, of for a cell composition obtained using a stimulating reagent, differs not more than 40% or not more than 30%, or not more than 20% or not more than 10%, or not more than 5% of the control composition, where the control composition and cell composition are obtained using the same method , including derived from the same population of cells, except that the control composition is implemented in the presence of a control stimulatory reagent, or a standard number of units dependent on the recombinant receptor activity.

313. The method of embodiment 312 wherein the control stimulus reagent, when used in the method, results in a T cell composition in which the recombinant receptor dependent activity or antigen specific activity is within an acceptable range of variability.

314. The method of any of embodiments 303-313 wherein cell stimulation is performed or initiated prior to incubation, optionally for 18-24 hours at or about 37 degrees

315. The method of any of embodiments 303-314, wherein the stimulatory condition includes a cytokine selected from IL-2, IL-15, and IL-7.

316. The method of any of embodiments 303-315, wherein cell stimulation is performed after incubation, optionally, for a period of time to reach a threshold concentration.

317. The method of any of embodiments 303-316, further comprising (c) filling the container with all or part of the T cell composition, and optionally another solution, to a concentration between about 10 million cells and about 70 million cells per ml, inclusive.

318. The method of embodiment 317 wherein the container is filled with another solution and the solution contains a cryoprotectant, optionally DMSO.

319. The method of embodiment 317 or embodiment 318, wherein the concentration is between about 15 and about 60 million cells per ml, inclusive.

320. The method of any of embodiments 317-319 wherein the concentration is greater than 10 million cells per ml.

321. The method of any of embodiments 317-320 wherein the concentration is greater than 15 million cells per ml.

322. The method of any of embodiments 317-321, wherein the concentration of DMSO is or is about or does not exceed 7.5%.

323. The method of embodiment 317 or embodiment 318 wherein the concentration is greater than 60 million cells per ml.

324. The method of any of embodiments 317-320 and 323, wherein the concentration of DMSO is greater than 7.5%, optionally between or about between 7.5% and 9.0%, inclusive.

325. The method of any of embodiments 303-324, wherein the agent comprising the nucleic acid molecule encoding the recombinant receptor is a viral vector, optionally a lentiviral vector or a gamma-retroviral vector.

326. The method of any of embodiments 317-325, wherein the filling is carried out automatically, optionally in a closed system.

327. The method of any of embodiments 303-326, further comprising freezing the cells in the container, or storing the container at less than or about less than 80°C.

328. A unit dose of a T cell therapeutic composition containing a number of cells containing a recombinant receptor, optionally a chimeric antigen receptor (CAR), specific for an antigen associated with a disease or condition, or expressed in a disease or condition, where the number of cells is between and between approximately 5.0×10 ⁶ and 2.25×10 ⁷ , 5.0×10 ⁶ and 2.0×10 ⁷ , 5.0×10 ⁶ and 1.5×10 ⁷ , 5.0×10 ⁶ and 1.0×10 ⁷ , 5.0×10 ⁶ and 7.5×10 ⁶ , 7.5×10 ⁶ and 2.25×10 ⁷ , 7.5×10 ⁶ and 2.0×10 ⁷ , 7.5×10 ⁶ and 1.5×10 ⁷ , 7.5×10 ⁶ and 1.0×10 ⁷ , 1.0×10 ⁷ and 2.25×10 ⁷ , 1.0×10 ⁷ and 2 .0×10 ⁷ , 1.0×10 ⁷ and 1.5×10 ⁷ , 1.5×10 ⁷ and 2.25×10 ⁷ , 1.5×10 ⁷ and 2.0×10 ⁷ , 2, 0 x 10 ⁷ and 2.25 x 10 ⁷ recombinant receptor-expressing cells, optionally, recombinant receptor-expressing cells that are CD8+, or are negative (–) for an apoptosis marker and CD8+, including each, optionally, where the apoptosis marker is annexin V or active casp azu 3.

329. A unit dose of embodiment 328, where the number of cells is:

between at least or at least about 5x10 ⁶ , 6x10 ⁶ , 7x10 ⁶ , 8x10 ⁶ , 9x10 ⁶ , 10x10 ⁶ , and about 15x10 ⁶ expressing the recombinant receptor, including each optionally expressing the recombinant receptor cells that are CD8+ or that are negative (-) for an apoptosis marker and CD8+, optionally, where the apoptosis marker is annexin V or active caspase 3;

between at least or at least about 5.55×10 ⁶ , 6.66×10 ⁶ , 7.77×10 ⁶ , 8.99×10 ⁶ , 1.0×10 ⁷ , 1.1×10 ⁷ and about 1.67×10 ⁷ recombinant receptor-expressing cells, optionally recombinant receptor-expressing cells that are CD8+ or that are negative (–) for an apoptosis marker and CD8+, including each optionally, where the apoptosis marker is annexin V or active caspase 3;

between at least or at least about 6.25×10 ⁶ , 7.5×10 ⁶ , 8.75×10 ⁶ , 1.0×10 ⁷ , 1.13×10 ⁷ , 1.25×10 ⁷ and about 1.9 x 10 ⁷ expressing the recombinant receptor, optionally expressing the recombinant receptor, cells that are CD8+ or that are negative (–) for an apoptosis marker and CD8+, including each optionally, where the apoptosis marker is annexin V or active caspase 3;

between at least or at least about 7.14×10 ⁶ , 8.5×10 ⁶ , 1.0×10 ⁷ , 1.14×10 ⁷ , 1.29×10 ⁷ , 1.42×10 ⁷ and approximately 2.14 x 10 ⁷ recombinant receptor-expressing cells, optionally recombinant receptor-expressing cells that are CD8+ or are negative (–) for an apoptosis marker and CD8+, including each optionally, where the apoptosis marker is annexin V or active caspase 3.

330. A unit dose of embodiment 328 or embodiment 329, wherein the number of cells is between at least or at least about 5×10 ⁶ , 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9×10 ⁶ , 10×10 ⁶ and approximately 15×10 ⁶ expressing the recombinant receptor of CD8+ cells, including each.

331. A unit dose of embodiment 328 or embodiment 329, wherein the number of cells is between at least or at least about 6.25×10 ⁶ , 7.5×10 ⁶ , 8.75×10 ⁶ , 1.0 x10 ⁷ , 1.13 x 10 ⁷ , 1.25 x 10 ⁷ , and approximately 1.9 x 10 ⁷ recombinant receptor-expressing cells that are negative (–) for the apoptosis marker and CD8+, optionally, where the apoptosis marker is annexin V or active caspase 3 each included.

332. A single dose from any of embodiments 328-332 that does not contain more than a threshold number of reference units (RU), where the amount of RU in a given composition is determined by the formula:

RU=A x B, where

A is the number of cells, or a multiple or transformation thereof, of cells of a certain phenotype present in a given composition, or is the average or weighted average of the number of cells, or a multiple, proportion or transformation, of two or more phenotypes in a given composition; and

B is a parameter value, or a multiple thereof, or a transformation that indicates the degree of or correlates with the degree of recombinant receptor dependent, optionally CAR dependent, activity in a given T cell composition.

333. The unit dose of embodiment 332, where the threshold number of reference units is less than the reference safe number of units, where the reference safe number of units is, in relation to a group of individuals analyzed after treatment using a T cell therapeutic composition containing T cells expressing the recombinant receptor, optionally CAR, the smallest number of units of drug administered to an individual among those individuals in the group who continued to develop an adverse event.

334. The unit dose of Embodiment 333, where the adverse event is a serious adverse event, optionally severe grade 4 or grade 5 neurotoxicity equal to or greater than or greater than grade 5 neurotoxicity, or at least grade 3 long-term neurotoxicity.

335. A single dose from option 333 or option 334, where the threshold number of units is less than the safe number of reference units by the amount corresponding to the safety factor, or at least 2 times.

336. A unit dose of any of embodiments 332-335, where the threshold number of units is or exceeds the reference effective number of units, where the reference effective amount is, in relation to a group of individuals analyzed after treatment using a composition of T cells containing a recombinant receptor, optionally, CAR, the number of units of drug administered to one or more individuals among the group for which the desired therapeutic outcome is shown, optionally, complete response (CR).

337. A unit dose of any of embodiments 121-127 wherein at least 50%, at least 60%, at least 75%, at least 80%, or at least 90% of CD8+ cells and/or CD8 The +CAR+ cells in the composition are not positive for an apoptosis marker, are not apoptotic, or are not in the early stages of apoptosis, or are not in the late stages of apoptosis.

338. A single dose of any of embodiments 328-337 containing not more than 1.88×10 ⁷ CD8+ CAR+ T cells, optionally CD8+ CAR+ T cells, that are not apoptotic or not in the early stages of apoptosis, or are not in the late stages of apoptosis.

339. A single dose composition of any of embodiments 328-338 containing at least 5×10 ⁶ annexin-CD8+ CAR+ cells, but not more than 1.5×10 ⁷ annexin-CD8+ CAR+ cells.

340. A single dose composition of any of embodiments 328-339, wherein the T cell composition is prepared by a method in which:

frequency, (1) among CAR+ cells in the composition, (2) among CAR+CD3+ cells in the composition, and/or (3) among CAR+CD8+ cells in the composition, of cells of a phenotype indicating signs of biologically active cells and/or lack of apoptosis , or early or late stages of apoptosis, differs by no more than 40%, or no more than 30%, or no more than 20%, or no more than 10%, or no more than 5%, from the average specified frequency in a variety of compositions of T-cells obtained by this method, and/or differs from such an average by no more than one standard deviation; or

the frequency, (1) among CAR+ cells in the composition, (2) among CAR+CD3+ cells in the composition, and/or (3) among CAR+CD8+ cells in the composition, of cells with a phenotype indicating no apoptosis, or early or late stages of apoptosis , differs by no more than 40%, or no more than 20%, or no more than 10%, among the plurality of T cell compositions obtained by this method.

341. A unit dose of embodiment 340, where the method includes:

(a) incubating a population of cells containing T cells with an agent containing a nucleic acid molecule encoding a recombinant receptor under conditions to introduce a nucleic acid encoding a recombinant receptor into cells in the population; and

(b) stimulating the cells, before, during and/or after said incubation, wherein the stimulation comprises incubating the cells in the presence of a stimulating condition that induces a primary signal, signaling, stimulating, activating and/or proliferating the cells.

342. The unit dose of embodiment 341, wherein the method further comprises, before (a), isolating a population of cells from a biological sample.

343. The unit dose of embodiment 342, wherein the isolation comprises selecting cells based on surface expression of CD3 or based on surface expression of one or both of CD4 and CD8, optionally by positive or negative selection.

344. The unit dose of embodiment 342 or embodiment 343, wherein isolation comprises immunoselection based on affinity.

345. Unit dose of embodiment 342-344 wherein the biological sample is or contains a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T-cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or a product of leukapheresis.

346. The unit dose of any of embodiments 341-345, wherein the stimulatory condition comprises incubation with a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and/or one or more intracellular signaling domains of one or more costimulatory molecules.

347. The unit dose of embodiment 346 wherein the stimulatory reagent contains a primary agent that specifically binds to a member of the TCR complex and a secondary agent that specifically binds to a costimulatory T cell molecule.

348. A unit dose from embodiment 346 or embodiment 347, wherein the primary agent specifically binds to CD3 and/or a costimulatory molecule selected from the group consisting of CD28, CD137 (4-1-BB), OX40, or ICOS.

349. The unit dose of embodiment 347 or embodiment 348, wherein the primary and secondary agents contain antibodies and/or are present on the surface of a solid support, optionally a bead.

350. A single dose of any of embodiments 346-349, where:

a stimulating reagent is a reagent for which it is determined that the recombinant receptor dependent activity or antigen specific activity, optionally recombinant receptor dependent or antigen specific dependent production or accumulation of a pro-inflammatory cytokine, differs by no more than 40% or no more than 30 %, or no more than 20%, or no more than 10%, or no more than 5%, of the average pro-inflammatory cytokine production or accumulation among the plurality of T cell compositions produced by this method using a stimulatory reagent, and /or differs from such mean by no more than one standard deviation; and/or

a stimulating reagent is a reagent for which it is determined that the recombinant receptor dependent activity or antigen specific activity, optionally recombinant receptor dependent or antigen specific dependent production or accumulation of a pro-inflammatory cytokine, differs by no more than 40% or no more than 30 %, or not more than 20%, or not more than 10%, or not more than 5%, among the many compositions of T-cells obtained by this method; and/or

a stimulating reagent is a reagent for which it is determined that the recombinant receptor dependent activity or antigen specific activity, optionally recombinant receptor dependent or antigen specificity dependent production or accumulation of a pro-inflammatory cytokine, for a cell composition obtained using a stimulating reagent, differs by no more than than 40% or not more than 30%, or not more than 20% or not more than 10%, or not more than 5% of the control composition, where the control composition and cell composition are obtained using the same method, including derived from the same population of cells, except that the control composition is implemented in the presence of a control stimulatory reagent, or a standard number of units of recombinant receptor-dependent activity.

351. A single dose of embodiment 350 wherein the control stimulatory reagent, when used in the method, results in a T cell composition in which the recombinant receptor dependent activity or antigen specific activity is within an acceptable range of variability.

352. A single dose of any of embodiments 341-351, where cell stimulation is performed or initiated prior to incubation, optionally within 18-24 hours at or about 37 degrees

353. The unit dose of any of embodiments 341-352, wherein the stimulatory condition includes a cytokine selected from IL-2, IL-15, and IL-7.

354. A single dose of any of embodiments 341-353, where stimulation of cells is carried out after incubation, optionally, for a period of time to achieve a threshold concentration.

355. A unit dose of any of embodiments 341-354, further comprising (c) filling the container with all or part of the T cell composition, and optionally another solution, to a concentration between about 10 million cells and about 70 million cells per ml, inclusive .

356. Unit dose from embodiment 355, where the container is filled with another solution and the solution contains a cryoprotectant, optionally DMSO.

357. A single dose of option 355 or option 356, where the concentration is between about 15 and about 60 million cells per ml, inclusive.

358. A single dose of any of embodiments 355-357 wherein the concentration is greater than 10 million cells per ml.

359. A single dose of any of embodiments 355-358 wherein the concentration is greater than 15 million cells per ml.

360. The dosage unit of any of embodiments 355-359, wherein the concentration of DMSO is or is about or does not exceed 7.5%.

361. A unit dose from embodiment 355 or embodiment 356 where the concentration is greater than 60 million cells per ml.

362. A single dose of any of embodiments 355-358 and 361, wherein the DMSO concentration is greater than 7.5%, optionally between or about between 7.5% and 9.0%, inclusive.

363. The unit dose of any of embodiments 341-362, wherein the agent containing the nucleic acid molecule encoding the recombinant receptor is a viral vector, optionally a lentiviral vector or a gamma-retroviral vector.

364. The unit dose of any of embodiments 355-363, where the filling is carried out in automatic mode, optionally in a closed system.

365. A unit dose of any of embodiments 341-364, further comprising freezing the cells in the container or storing the container at less than or about less than 80°C.

366. A method for determining whether an individual is at risk of toxicity, comprising analyzing the number of recombinant receptor-expressing cells in the blood of an individual, where said individual has previously received a dose of recombinant receptor-expressing cells, where the individual is at risk of developing toxicity if:

(i) no more than four days after the start of administration, the number of recombinant receptor-expressing cells in the individual's blood is at least or about 2 recombinant receptor-expressing cells per microliter;

(ii) no more than five or six days after the start of administration, the number of recombinant receptor-expressing cells in the individual's blood is at least or about 5 recombinant receptor-expressing cells per microliter, or at least or about 10 recombinant receptor-expressing cells per microliter ; or

(iii) no more than seven days after the start of administration, the number of cells expressing the recombinant receptor in the blood of the individual is at least or about 15 cells expressing the recombinant receptor per microliter.

367. A method for determining whether an individual is at risk of toxicity, including:

(a) administering to an individual suffering from the disease or condition a dose of cells expressing the recombinant receptor; and

(b) after administration of the cells, assaying the amount of cells expressing the recombinant receptor in the individual's blood, where the individual is at risk of developing toxicity if:

(i) no more than four days after the start of administration, the number of recombinant receptor-expressing cells in the individual's blood is at least or about 2 recombinant receptor-expressing cells per microliter;

(ii) no more than five or six days after the start of administration, the number of recombinant receptor-expressing cells in the individual's blood is at least or about 5 recombinant receptor-expressing cells per microliter, or at least or about 10 recombinant receptor-expressing cells per microliter. microliter; or

(iii) no more than seven days after the start of administration, the number of cells expressing the recombinant receptor in the blood of the individual is at least or about 15 cells expressing the recombinant receptor per microliter.

368. The method of embodiment 366 or embodiment 367, where if it is determined that the individual is at risk of toxicity; stopping the administration of cells expressing the recombinant receptor, administering to the individual a lower dose of cells expressing the recombinant receptor; administering to the individual cells expressing another recombinant receptor; and/or administering to the individual an agent capable of treating, preventing, slowing down, or attenuating the development of toxicity.

369. The method of any one of embodiments 366-368, wherein if the individual is determined to be at risk of toxicity, the individual is administered an agent capable of treating, preventing, slowing, or attenuating the development of toxicity.

370. A method for evaluating a stimulating reagent for use in obtaining a therapeutic composition of T cells, including:

(i) assessing the composition of T cells obtained using the stimulatory reagent, depending on the recombinant receptor activity or phenotype; and

(ii) comparing the recombinant receptor dependent activity or phenotype with the same activity or phenotype obtained for the control composition, or comparing with a standard unit for the recombinant receptor dependent activity or phenotype,

where the stimulant is determined to be releasable for use in a method for producing a therapeutic T cell composition if the recombinant receptor dependent activity or phenotype of the cell composition obtained using the stimulating reagent differs by no more than 40% or no more than 30%, or not more than 20%, or not more than 10%, or not more than 5% of the same activity obtained for the control composition, or from the standard unit.

371. The method of embodiment 370 wherein the recombinant receptor dependent activity is an antigen specific activity.

372. The method of Embodiment 370 or Embodiment 371, wherein the control composition and T cell composition are prepared using the same method, including preparation from the same cell population, except that the control composition is implemented in the presence of a control stimulatory reagent.

373. The method of any of embodiments 370-372 wherein the recombinant receptor dependent activity is assessed and compared to the same activity obtained for a control composition or compared to a standard number of units of recombinant receptor dependent activity.

374. The method of any of embodiments 370-373 wherein the recombinant receptor dependent activity is recombinant receptor dependent or antigen specificity dependent production or accumulation of a pro-inflammatory cytokine.

375. The method of embodiment 364 wherein the pro-inflammatory cytokine is IL-2, TNF-alpha, IFN-gamma, or IL-10.

376. The method of any of embodiments 370-372 wherein the phenotype is assessed and compared to the same phenotype obtained for a control composition or compared to a standard unit for the phenotype.

377. The method of any of embodiments 370-372 and 376, wherein the phenotype includes a particular differentiation state and/or a memory/stem cell-like phenotype.

378. The method of any of embodiments 370-372, 376, and 377, wherein the phenotype includes positive expression of a surface marker of CD3, CD8, CD27, CD28 and/or positive expression of a recombinant receptor, optionally CAR, or a surrogate marker expression of the recombinant receptor.

379. The method of any of embodiments 370-372 and 376-378, wherein the phenotype is CD27+/CD28+/CD8+.

380. The method of any of embodiments 370-379, wherein the stimulatory reagent is capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and/or one or more intracellular signaling domains of one or more costimulatory molecules.

381. The method of any of embodiments 370-380, wherein the stimulatory reagent comprises a primary agent that specifically binds to a member of the TCR complex and a secondary agent that specifically binds to a costimulatory T cell molecule.

382. The method of embodiment 380 or embodiment 381, wherein the primary agent specifically binds to CD3 and/or a costimulatory molecule selected from the group consisting of CD28, CD137 (4-1-BB), OX40, or ICOS.

383. The method of embodiment 381 or embodiment 382, wherein the primary and secondary agents contain antibodies and/or are present on the surface of a solid support, optionally a bead.

384. A container containing the T cell therapeutic composition at a concentration between about 10 million cells and about 70 million cells per ml, and optionally another solution, wherein the T cell therapeutic composition contains T cells containing the recombinant receptor.

385. The container of embodiment 384, where the concentration is between about 15 and about 60 million cells per ml.

386. The container of embodiment 384 or 385, where the concentration is or is about 15 million cells per ml.

387. The container of embodiment 384 or 385 wherein the container is filled with another solution and the solution contains a cryoprotectant, optionally DMSO.

388. The container of any of embodiments 384-386, where the container is an ampoule or a sachet.

389. Container from any of embodiments 384-387, where the container is an ampoule, optionally a cryogenic ampoule, and the volume of the composition is not more than 20 ml, optionally, 1 ml - 20 ml, 1 ml - 15 ml, 1 ml - 10 ml, 1 ml - 5 ml, 1 ml - 2.5 ml, 2.5 ml - 20 ml, 2.5 ml - 15 ml, 2.5 ml - 10 ml, 2.5 ml - 5 ml, 5 ml - 20 ml, 5 ml - 15 ml, 5 ml - 10 ml, 10 ml - 20 ml, 10 ml - 15 ml or 15 ml - 20 ml.

390. A container from any of embodiments 384-388, where the container is a bag, optionally a freezer bag, and the volume of the composition is:

is between or between approximately 15 ml and 150 ml, 20 ml and 100 ml, 20 ml and 80 ml, 20 ml and 60 ml, 20 ml and 40 ml, 40 ml and 100 ml, 40 ml and 80 ml, 40 ml and 60 ml, 60 ml and 100 ml, 60 ml and 80 ml or 80 ml and 100 ml, each included; or

is at least or at least about 15 ml, at least or at least about 20 ml, at least or at least about 30 ml, at least or at least about 40 ml, at least or at least about 50 ml, at least or at least about 60 ml, at least or at least about 70 ml, at least or at least about 80 ml, or at least or at least about 90 ml; and/or

is not more than 100 ml.

391. The container of any of embodiments 384-390, where the ratio of surface area to volume of the composition in the container is:

is between or between about 0.1 cm ^-1 and 100 cm ^-1 ; 1 cm– ¹ and 50 cm– ¹ , 1 cm– ¹ and 20 cm– ¹ , 1 cm– ¹ and 10 cm– ¹ , 1 cm– ¹ and 7 cm– ¹ , 1 cm– ¹ and 6 cm– ¹ , 1 cm– ¹ and 3 cm– ¹ , 1 cm– ¹ and 2 cm– ¹ , 2 cm– ¹ and 20 cm– ¹ , 2 cm– ¹ and 10 cm– ¹ , 2 cm– ¹ and 7 cm– ¹ , 2 cm– ¹ and 6 cm– ¹ , 2 cm– ¹ and 3 cm– ¹ , 3 cm– ¹ and 20 cm– ¹ , 3 cm– ¹ and 10 cm– ¹ , 3 cm– ¹ and 7 cm– ¹ , 3 cm– ¹ and 6 cm– ¹ , 6 cm– ¹ and 20 cm– ¹ , 6 cm– ¹ and 10 cm– ¹ , 6 cm– ¹ and 7 cm– ¹ , 7 cm– ¹ and 20 cm– ¹ , 7 cm– ¹ and 10 cm– ¹ or 7 cm– ¹ and 20 cm– ¹ , each included; or

is, is approximately, or is at least 3 cm ^-1 , 4 cm ^-1 , 5 cm ^-1 , 6 cm ^-1 , 7 cm ^-1 , 10 cm ^-1 , 15 cm ^-1 , or 20 cm ^-1 .

392. The container of any of embodiments 384-3391, wherein T cells are obtained from an individual having a disease or condition and transduced with a nucleic acid encoding a recombinant receptor, optionally a chimeric antigen receptor (CAR), specific for an antigen associated with the disease or a condition, or expressed in a disease or condition, and/or the T cells contain a recombinant receptor, optionally a CAR.

393. The container of any of embodiments 97-105, wherein the recombinant receptor is a CAR.

394. The container of embodiment 106, wherein the CAR contains an extracellular antigen recognition domain that specifically binds to an antigen and an intracellular signaling domain containing ITAM, where optionally the intracellular signaling domain contains an intracellular domain from the CD3-zeta (CD3ζ) chain; and/or wherein the CAR further comprises a costimulatory signaling region, which optionally comprises a signaling domain from 4-1BB.

395. The container of any of embodiments 97-107, wherein the T cells are CD4+ or CD8+.

396. The method of any of embodiments 97-108, wherein the T cells are primary T cells.

397. The container of any of embodiments 97-109, wherein the T cell composition is prepared by a method in which:

frequency, (1) among CAR+ cells in the composition, (2) among CAR+CD3+ cells in the composition, and/or (3) among CAR+CD8+ cells in the composition, of cells of a phenotype indicating signs of biologically active cells and/or lack of apoptosis, or early or late stages of apoptosis, differs by no more than 40%, or no more than 30%, or no more than 20%, or no more than 10%, or no more than 5%, from the average indicated frequency during set of compositions of T-cells obtained by this method, and/or differs from such an average by no more than one standard deviation; or

the frequency, (1) among CAR+ cells in the composition, (2) among CAR+CD3+ cells in the composition, and/or (3) among CAR+CD8+ cells in the composition, of cells with a phenotype indicating the absence of apoptosis, or early or late stages of apoptosis, differs by no more than 40%, or no more than 20%, or no more than 10%, among the plurality of T cell compositions obtained by this method.

X. EXAMPLES

[0742] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1 Determination of parameters of CAR+ T cell compositions that are predictive of severe neurotoxicity

[0743] Various parameters were evaluated following treatment of individuals with relapsed or refractory acute lymphoblastic leukemia (ALL) using therapeutic compositions containing autologous T cells modified to express a chimeric antigen receptor (CAR) against CD19. Parameters assessed included therapeutic cell composition and dose parameters (including functional traits and numbers and frequencies of cells of different phenotypes), patient characteristics, and clinical and treatment parameters, including parameters indicative of response and toxicity-related outcomes.

[0744] Parameters were analyzed for individuals, after administration, and compared among individuals using a range of statistical analyses, including descriptive, graphical, non-parametric, and model-based analyses. Relationships between different parameters were evaluated. In particular, this study assessed the extent to which various parameters of individual patients and cell compositions or doses correlated (directly or inversely) with the likelihood of a therapeutic response and/or with the risk of an adverse outcome such as neurotoxicity, for example, severe neurotoxicity or grade 5 neurotoxicity and cerebral edema.

A. CAR+ T-cell therapy

[0745] Evaluated individuals (N=38) were administered a T cell composition containing autologous T cells expressing the anti-CD19 chimeric antigen receptor (CAR) for the treatment of adult ALL. At the start of treatment, out of 38 individuals, thirty-two (32) showed morphological evidence of disease in the bone marrow (at least 5% blast cells), and six (6) showed molecularly detectable disease by PCR. Subjects were 18 years of age or older, with recurrent or refractory CD19 morphologically positive disease (as defined by more than 5% of blast cells in the bone marrow) including extramedullary and Philadelphia chromosome (Ph+) positive disease, with one or more previous cycles of therapy salvage (including individuals treated with allogeneic hematopoietic stem cells), and had scores of 0-2 according to the US Eastern United Cancer Group (ECOG). Individuals had adequate organ function as shown by serum creatinine levels < 1.5 x upper limit of normal [0ULN] or creatinine excretion > 30 mL/min/1.73 m2, alanine aminotransferase < 5 x ULN, bilirubin < 2.0 mg/ dl, dyspnea grade < 1 and oxygen saturation > 92% when breathing room air, and left ventricular ejection fraction of > 40%. Individuals included individuals with prior treatment with blinatumomab. Exclusion criteria included isolated extramedullary disease or Burkitt's disease, active CNS involvement in disease (CNS3) or CNS pathology, active acute (grades II–IV) or extensive chronic graft-versus-host disease (GvHD), active infection, and prior gene therapy. Subjects including individuals treated with alemtuzumab within the past 6 months, clofarabine or cladribine within the past 3 months, GvHD drugs within the past 4 weeks, or salvage chemotherapy and therapeutic corticosteroids within the past week were also excluded.

[0746] Demographic and baseline characteristics of individuals included in the study are shown in Table E1A.

Table E1–A: Demographic and Baseline Characteristics

All individuals (N=38) Morphologically positive individuals (n=32) MRD+ individuals (n=6) Men, n(%) 28 (74) 25 (78) 3 (50) Age, median (range), years 39 (19–69) 28 (74) 34 (24–47) Age >30 years, n(%) 22 (58) 19 (59) 3 (50) White, n (%) 34 (89) 28 (88) 6 (100) Time from initial diagnosis (range), years 1.8
(0.5–21.5) 1.7
(0.5–10.6) 2.2
(0.6–21.5) Number of previous modes, median (range) 2 (1–7) 2 (1–7) 2 (1–3) Ph+, n (%) 4 (11) 4 (13) 0 (0) Previous treatment with blinatumomab, n (%) 4 (11) 16 (50) 3 (50) Prior HSCT treatment, n (%) 14 (37) 11 (34) 3 (50) Prior CNS disease, n (%) 8 (21) 7 (22) 1 (17) Previous CNS radiation exposure, n (%) 12 (32) 11 (34) 1 (17) Prior IT chemotherapy, n (%) 30 (79) 26 (81) 4 (67) Previous LD BM of blast cells, median (range), % 48 (0–98) 66 (6–98) 2 (0–4) Aggressive transitional treatment, n (%) ^a 19 (50) 17 (53) 2 (33)

IT, intrathecal; LD, antilymphocyte therapy.

^a Hyper CVAD Parts A or B, regimens that include fludarabine and/or cytarabine.

[0747] The administered T cell therapeutic compositions are prepared by a method comprising immunoaffinity-based selection of T cells (including CD4+ and CD8+ cells) from leukapheresis samples from individuals, followed by activation and transduction with a viral vector encoding an anti-CD19 CAR, expansion and cryopreservation. The CAR contained an anti-CD19 scFv derived from a mouse antibody, a CD28 region including an extracellular region, a transmembrane domain and a costimulatory region, and an intracellular CD3-zeta signaling domain. Cryopreserved cell compositions were thawed in situ prior to intravenous administration. Full volumes of cell compositions were administered, which were filled and cryopreserved.

[0748] Cells were administered at a first target dose of approximately 1×10 ⁶ CD3+CAR+ cells/kg (individual body weight). The target dose and dose range for administration was evaluated in part based on a comparison of toxicity outcomes for a group of individuals with morphologically confirmed disease, who were administered similar compositions, in a range of different numbers of CD3+CAR+ cells per administered dose. In particular, the relationship of CD3+CAR+/kg dose and what was present or not was assessed, including whether or not the individual continued to develop grade 3 neurotoxicity, and whether or not the target dose and dose range were below the lowest number of CD3+CAR+ cells/ kg administered to any of those individuals showing grade 3 or higher neurotoxicity is less than the safety margin of at least 1.5 x 10 ⁶ CD3+CAR+ cells/kg. See Figure 22. The dose did not take into account the number or frequency of CD4+ cells compared to CD8+ cells, or the number or frequency of other T cell subgroups. Cytotoxic activity against CAR+ cell antigen was evaluated as an indicator of activity. Some individuals were given a second dose between about 14 and 28 days after the initial dose. Prior to administration of autologous CAR-expressing cells, individuals were subjected to preparatory antilymphocyte chemotherapy, including a single dose of either cyclophosphamide alone (approximately 1–3 g/m2) or cyclophosphamide (30–60 mg/kg) and fludarabine (25 mg/m 2–30 mg / m2, injected once a day for three days).

[0749] Individuals were monitored for response and other outcomes, including bone marrow, peripheral blood, and cerebrospinal fluid (CSF) testing. Individuals were also assessed and monitored for neurotoxicity (neurological complications including symptoms of confusion, aphasia, encephalopathy, myoclonic seizures, convulsions, lethargy, and/or changes in mental status), divided into grades on a scale of 1-5, in accordance with the US National Cancer Institute - Common Criteria Toxicity Scale (CTCAE), version 4.03 (NCI–CTCAE v4.03). Common Toxicity Criteria (CTCAE) scale, version 4.03 (NCI–CTCAE v4.03). See Common Terminology for Adverse Events (CTCAE) Version 4, US Department 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) was also defined and monitored, graded based on severity.

[0750] Response outcomes (percentage of individuals showing complete response (CR) or complete response with incomplete brain recovery (CRi) (CR/CRi ratio) (with a median follow-up of 4 months)), and toxicities are summarized in Table E1-B and E1–C for individuals showing pathologically confirmed disease at the start of treatment. (NE=not assessable). As shown, 56% of individuals developed grade 3-5 neurotoxicity. Five (5) individuals developed Grade 5 neurotoxicity and, in particular, Grade 5 cerebral edema.

Table E1–B: Summary of treated individuals

Morphologically confirmed Cy Flu/Cy Ratio CR/CRi fifty% 100% NE 8% NE: 37% Severe CRS (grade 3 or higher) 17% 38% Grade 3–5 neurotoxicity 46% 88% Degree 5: 8% Degree 5: 38%

Table E1–C: Outcomes for 32 treated individuals with pathologically confirmed disease

(total treated=38 individuals)

CY Flu/Cy CY Total morphologically confirmed Total MRD Period of time Time period 1 Time period 2 Time period 3 Individuals, N fourteen eight ten 32 6 Ratio CR/CRi
# not estimable 6/14 (43%) 5/5 (100%); 3NE 4/8 (50%) Severe grade 3/4 CRS 1/14 (7%) 3/8 (38%) 3/10 (30%) 7 (22%) 2 (33%) Severe NTX grades 3–5 3/14(21%) 7/8(88%) 8/10(80%) 18 (56%) 2 (33%) Treatment-related mortality Degree 5 events: 0 Degree 5 events: 3
(all NTX events) Degree 5 events: 2
(both NTX events) Degree 5 events: 5 (16%) Degree 5 events: 0

An additional 6 treated individuals were positive for minimal residual disease (MRD+) at the time of treatment.

[0751] At least 15 cases of grade 5 neurotoxicity and at least 13 cases of cerebral edema associated with CAR–T cell therapy have been published in several different clinical studies, as reported in the publicly available information, including studies involving CAR– T-cells expressing different CARs with different antigen-binding domains and different co-stimulatory domains, obtained using different manufacturing methods and for the treatment of different diseases or indications.

B. Pharmacokinetics (PK) of CAR+ T cells in the blood

[0752] The analysis was performed on samples obtained from the blood of individuals at various time points after administration of the first and, where applicable, the second dose of cell compositions. Samples were analyzed by flow cytometry for the presence and number of CAR-expressing cells in the blood.

[0753] As shown in FIG. 1 and 2, faster peaks of CAR-expressing T cells were observed in the blood of individuals who continued to develop more severe neurotoxicity, and in particular those who developed grade 5 neurotoxicity and cerebral edema. For example, the results showed that these individuals had a detectable level of CAR+ cells per µl of blood within 4–7 days after the first dose of cells, with more than 20 CAR+ cells per µl of blood observed in the blood of all such individuals within seven days after administration. cells . In individuals with grade 3 or 4 neurotoxicity, the peak of CAR+ T cell expansion was generally observed later and with a lower maximum number of CAR+ T cells per microliter (FIG. 2).

C. Evaluation and comparison of clinical parameters, patient parameters, translation and CMC in different individuals

[0754] Various parameters were evaluated for different groups of patients, in accordance with the severity of the disease at the beginning of treatment and the outcome of treatment or the occurrence of toxicity, including various degrees of neurotoxicity. The presence or absence of correlations and significance were assessed through descriptive, graphical, non-parametric and model-based analyzes as described in this example and in example 2. Approximately 500 clinical and translational parameters were analyzed using univariate and multivariate logistic regression. Approximately 140 parameters of chemical properties, manufacturing process and quality control (CMC) were investigated in one-way and multi-way analyzes using non-parametric tests and methods based on partitioning (decision tree).

[0755] Various features of CMC were evaluated in samples from T cell compositions administered to individuals (thawed from samples of cryopreserved therapeutic compositions administered to individuals (CDP)). Samples were analyzed for presence, absence, level, normalized level, amount, and/or normalized amount for various parameters. Among the illustrative clinical and patient signs assessed were the patient's age, history of prior treatment, and whether the patient was subjected to antilymphocyte and/or maintenance chemotherapy prior to treatment, the occurrence and extent of neurotoxicity, and other outcomes of toxicity. Cell composition-specific and/or dose-related parameters included amounts and frequencies (or normalized values thereof) for different cell populations (at dose administered and/or administered per individual weight), cytokine production, cytotoxic activity in response to stimulation with CAR antigen, and other parameters indicative of function, such as activation or stimulation, and/or effector function, in response to various conditions. The degree of correlation of various parameters (individually and, in some cases, their combinations) of therapeutic compositions with neurotoxicity and other outcomes observed in individuals after infusion of CAR+ T cells was assessed.

[0756] The results of illustrative statistical analyzes of specific parameters for a subset of individuals with pathologically confirmed disease at the start of treatment were consistent with the finding that patient age, prior treatment history, and antilymphocyte and/or maintenance chemotherapy correlated (directly or inversely) with the occurrence of grade neurotoxicity. 5. For example, in this group of individuals, younger patients less than 30 years of age, patients who have been subjected to a lower number (equal to two or less) cycles of prior therapy, and patients who have been administered fludarabine within 3 months or who have been subjected to aggressive maintenance chemotherapy were more likely to experience grade 5 neurotoxicity in this study. The calculated odds ratios estimated for those experiencing cerebral edema after cell therapy treatment for illustrative clinical factors are shown in Table E1-D. Odds ratios were calculated by dividing the odds of experiencing grade 5 neurotoxicity associated with demographic or treatment history by the odds associated with no demographic or treatment history. An odds ratio greater than 1 indicates an increased likelihood or likelihood of grade 5 neurotoxicity.

Table E1–D: Illustrative Clinical Factors Associated with Cerebral Edema

Demographics/Treatment History odds ratio 95%CI p-value Introduction Flu/CY LD 7.25 1.14, 53.96 0.045 Administering high-intensity transitional chemotherapy 4.68 0.63.32.64 0.133 Age < 30 years 5.16 0.83, 55.93 0.112 < 2 previous lines of therapy 7.24 0.72, 980.23 0.208

[0757] No association with higher risk was observed for prior CNS radiation exposure, prior treatment with intrathecal (IT) chemotherapy, prior CNS disease, prior allogeneic cell therapy, higher ECOG functional status, or prior treatment with blinatumomab.

[0758] This study also observed that certain such parameters (e.g., young ( < 30 years) patient age and fewer previous treatment cycles) correlate with certain therapeutic composition-specific or dose-related features (including total CD8+CAR +AnnV negative cells administered to an individual per cell dose, and the level of antigen-specific accumulation of the pro-inflammatory cytokine, IL-2, as measured in cultures of thawed therapeutic cell composition in the presence of cells expressing CD19, a CAR antigen indicative of CAR-induced cell activity). Also among the exemplary traits evaluated were numbers or frequencies (or number/kg) of viable cells, and/or cells of different phenotypes, at individual doses administered to individuals. Illustrative such phenotypes included the expression of one or more surface markers, as assessed by flow cytometry. For example, phenotypes included CD3+, CD8+, CD4+ and/or CAR+. Also present among the phenotypes were viability, and phenotypes associated with or indicative of, or thought to be indicative of, functional, healthy, or biologically active cells. In some aspects, such phenotypes included the absence or slight presence or expression of markers indicative of apoptosis, apoptotic cells, or various, e.g., early or late stages of one or more of death or entry into the apoptotic pathway (e.g., annexin V, caspase 3) . In some aspects, such markers include the ability of cells to produce cytokines or other factors in a manner that is not specific to the CAR antigen, for example, by staining intracellular cytokines (ICS) in response to PMA/ionomycin and/or FOXP3. In some aspects, markers and phenotypes included markers and phenotypes associated with T cell activation, depletion, stem cell-like properties, naive T cells, longevity, T cell subgroups, memory phenotype(s), and memory T cell subgroup phenotypes. , such as T _CM , T _EM , T _SCM .

[0759] For example, features related to the therapeutic composition of cells included the total number of CD8+CAR+ T cells per dose administered, the total number or frequency (eg, among CD8+CAR+ T cells) of CD8+CAR+ T cells per dose administered, as are observed negative for a factor indicative of apoptosis, such as surface staining of annexin V (annexin V–) or cleavage with caspase 3 (indicative of non-apoptotic cells). In some examples, features related to the therapeutic composition of cells included the total number of CD3+CAR+ T cells per dose administered, the total number or frequency (e.g., among CD3+CAR+ T cells) of CD3+CAR+ T cells per dose as observed negative for a factor indicative of apoptosis, such as annexin V surface staining (annexin V–) or caspase 3 cleavage (indicative of non-apoptotic cells).

[0760] Also among the exemplary parameters were properties specific to therapeutic compositions and/or their preparation, including viable cell concentration (VCC), cell multiplicity between inoculation and collection of drug product; and number of copies of the vector (VCN) in the administered dose.

[0761] Also among the exemplary parameters were parameters generally associated with or indicative of function or activity, or other traits or abilities of the cells in the compositions. Among these were various features associated with cell stimulation outcomes, including various measures of proliferation and activation or activity, including those induced in a TCR and/or CAR dependent manner or in a TCR and/or CAR induced manner. Illustrative of such parameters related to function or activity were the rates or levels (such as amount or concentration, or level per cell) of production of one or more factors (such as various cytokines) by cells in the composition, in response to an antigen-specific CAR or other stimulation.

[0762] In intracellular cytokine (ICS) staining assays, flow methods were used to evaluate the ability of cells in a composition to produce various cytokines in response to a non-antigen specific stimulus. Cells were stimulated with PMA/ionomycin in the presence of a Golgi apparatus inhibitor. FoxP3 expression was assessed by a QPCR assay targeting the Treg-specific demethylation region (TSDR) in the FOXP3 gene. FOXP3 levels indicative of the Treg phenotype were not observed.

[0763] Several parameters indicative of CAR-directed antigen-specific activities and functions (such as CAR antigen-dependent cytokine secretion and cytotoxicity) of the compositions were evaluated in co-culture assays. Cells of the therapeutic composition to be evaluated were incubated in the presence of CD19 expressing target cells. For cytokine assays, cumulative amounts (pg/ml) of cytokine(s) (e.g., IL-2, IFN-gamma, TNF-alpha, IL-6, sCD137, MIP1b, MIP1a, IL-10, IL-4, IL- 13, IL-5 or GM-CSF) was evaluated after incubation of target cells and the number of different compositions, including the same number of transduced or CAR+ cells, in the same volume, for a period of approximately 22-24 hours. Such assays provide an indication of antigen-specific cytokine secretion per CAR+ cell per dose. In other assays, cytolytic activity against CD19-expressing target cells was assessed after incubation with T cells.

[0764] In some instances, measures of activity or functional effect in response to a non-specific CAR antigen stimulus, such as the production of a cytokine or other factor following stimulation with an anti-CD3/anti-CD28 antibody, such as in the form of a magnetic bead coating, were assessed. In such assays, cultures containing fewer CAR+ cells among the T cells in culture may have a higher relative score.

[0765] Table E2 lists a plurality of features, selected in an unbiased manner among the features that were evaluated for therapeutic compositions of cells and/or their doses administered to individuals in studies. These features were assessed by univariate analysis for a subgroup (22) of individuals who showed morphologically confirmed disease at the start of treatment. Table E2 lists p values indicating the degree to which each trait was observed to correlate (either directly (+) or inversely (–)), based on univariate analysis, with grade neurotoxicity and/or cerebral edema (when comparing individuals with grade 5 neurotoxicity/cerebral edema with other individuals). Among 15 individuals, the degree to which each feature correlated or did not correlate with response outcome (as assessed by comparing individuals achieving a complete response (CR) or no response (NR)) was also assessed; Table E2 also lists the p values for this comparison.

[0766] The parameters listed in Table E2 are ranked from lowest p value to highest p value, in descending order, based on a one-way statistical comparison between a given trait and brain neurotoxicity/edema (grade 5/CE neurotoxicity, compared to neurotoxicity grade 4 or lower). For parameters for which the p-values in relation to the degree of neurotoxicity were less than or equal to 0.2, Table E2 additionally indicates whether the observed relationship with neurotoxicity was a direct ((+) indicating that it was observed that the average level or parameter score was greater in individuals with grade 5 neurotoxicity and cerebral edema compared to individuals with grade 4 neurotoxicity or lower), or was inverse ((–) indicating that it was observed that the mean level or parameter score was lower in individuals with grade 5).

[0767] FIG. 3A - FIG. 3U shows graphs for a subgroup of parameters for which relationships (direct or inverse) between parameter and grade 5 neurotoxicity were observed, showing results for individual individuals in different neurotoxicity or response groups. The observed p-values for these plots, in relation to neurotoxicity, were 0.05 or lower.

[0768] FIG. 3V is a graph comparing the number of CD3+CAR+ cells/kg (dose) administered to individuals and whether the individual developed grade 5 neurotoxicity (right side) and/or achieved a complete response (left side). While an association between the two variables was observed, CD3+CAR+ cells/kg was not a strong predictor of grade 5 neurotoxicity, and no clear margin of safety was observed. These results are consistent with the interpretation that dose determination based solely on the amounts or frequencies of CD3+CAR+ per dose, without taking into account other parameters such as the abundance of biologically active (eg, non-apoptotic) cells and/or CD8 versus CD4 cells, may not be adequate to prevent the risk of severe neurotoxicity, and that it is advantageous to develop dosing methods that take into account one or more prognostic parameters, as observed in the present description.

[0769] FIG. 4, 5A and 5B show plots and p-values for measures of antigen-specific secretion of individual cytokines (TNF-alpha, IFN-gamma, IL-2), for individuals in different neurotoxicity or response groups.

[0770] As shown, it was observed that a number of parameters evaluated for therapeutic compositions of cells and doses significantly correlated (directly or inversely) with the development of grade 5 neurotoxicity after administration of the composition to an individual.

Table E2: Evaluation of Cell Therapeutic Composition Parameters and/or Doses and Correlation with Neurotoxicity and Response Outcomes

Grade of neurotoxicity (grade 5 versus grade 4 or lower) Response (CR or CRi) Number of injected annexin-CD8+ CAR-expressing cells p-value 0 (+) p-value 0.164 ICS: IL13 from CD8+CAR+ p-value 0.001(-) p-value 0.912 Number of annexin-CD8+ CAR-expressing cells/kg p-value 0.001 (+) p-value 0.13 Frequency of CD127+ cells among CD4+CAR+ p-value 0.004(-) p-value 0.426 Total infused cells per kg p-value 0.007 (+) p-value 0.824 Number of injected active caspase-negative CD8+ CAR-expressing cells p-value 0.007 (+) p-value 0.498 ICS: IL13 from CD4+CAR+ p-value 0.011(-) p-value 0.654 Frequency of annexin V+ cells among CD8+CAR+ cells p-value 0.012(-) p-value 0.056 FOXP3 by qPCR p-value 0.012 (+) p-value Number of injected annexin V-CD3+ CAR-expressing cells p-value 0.012 (+) p-value 0.056 Number of administered annexin V– CD3+CAR cells/kg p-value 0.015 (+) p-value 0.1 Viable Cell Concentration (VCC) p-value 0.017 (+) p-value 0.859 Total number of infused cells p-value 0.017 (+) p-value 0.859 ICS: IL2 from CD4+CAR+ p-value 0.019(-) p-value 0.738 Frequency of annexin V+ cells among CD3+CAR+ p-value 0.024(-) p-value 0.164 Frequency of FOXP3+ among CD4+CAR+ p-value 0.03 (+) p-value 0.912 CD3+ frequency p-value 0.033 (+) p-value 0.806 Total number of injected CD8+ CAR-expressing cells/kg p-value 0.037 (+) p-value 0.738 Non-antigen/CAR specific IL-10 production per total cell count (CD3/28 stim.) p-value 0.04 (+) p-value one ICS: TNFA from CD4+CAR+ p-value 0.044(-) p-value 0.953 ICS: TNFA from CD8+CAR+ p-value 0.044(-) p-value 0.498 Non-antigen/CAR specific IL-6 production per total cell count (CD3/28 stim.) p-value 0.045 (+) p-value NaN Non-antigen/CAR specific IFN-gamma production per total cell count (CD3/28 stim.) p-value 0.055 (+) p-value 0.25 Frequency of PD1 Expressing Cells Among CD4+CAR+ Cells p-value 0.055 (+) p-value 0.076 Index of IL10 production after CAR antigen-specific culture p-value 0.057 (+) p-value 0.722 Frequency of annexin V+ among CD4+CAR+ p-value 0.067(-) p-value 0.164 Frequency of PD1+ cells among CD8+CAR+ p-value 0.067 (+) p-value 0.36 Frequency of CD3+CAR+ among cells per dose p-value 0.08(-) p-value 0.738 Injected CD3+CAR+ cells/kg p-value 0.08 (+) p-value 0.574 Total number of CD8CAR cells per dose p-value 0.08 (+) p-value 0.654 Non-antigen/CAR specific production of sCD137 per total cell count (CD3/28 stim.) p-value 0.085 (+) p-value 0.592 Frequency of effector memory T cells among CD8+CAR+ p-value 0.094 (+) p-value 0.912 Number of injected CD3+CAR+ cells negative for active caspase cleavage 3 p-value 0.094 (+) p-value 0.738 Frequency of naïve phenotype cells among CD8+CAR+ cells p-value 0.094 (+) p-value 0.654 Frequency of CD27+ CD28+ cells among CD8+CAR+ cells p-value 0.111(+) p-value 0.203 CD4+CAR+ frequency per cell per dose p-value 0.126(-) p-value 0.906 Frequency of effector memory cell phenotype cells among CD3+CAR+ p-value 0.13(+) p-value one Number of administered annexin V– CD4+CAR+ cells/kg p-value 0.13 (+) p-value 0.13 TNF-alpha production index after CAR antigen-specific culture p-value 0.15 (+) p-value 0.654 Number of injected annexin V– CD4+CAR+ cells p-value 0.15 (+) p-value 0.1 sCD137 production rate after CAR antigen-specific culture p-value 0.168 (+) p-value 0.635 Index of IFNG production after CAR antigen-specific culture p-value 0.174 (+) p-value one Non-antigen/CAR specific IL-4 production per total cell count (CD3/CD28 stim.) p-value 0.174 (+) p-value 0.912 Total number of injected CD3+CAR+ cells p-value 0.174 (+) p-value 0.824 Number of vector copies (per genome) p-value 0.177(-) p-value 0.498 MIP1B production rate after CAR antigen-specific culture p-value 0.199 (+) p-value 0.824 Non-antigen/CAR specific MIP1b production per total cell count (CD3/CD28 stim.) p-value 0.199 (+) p-value 0.824 ICS: IL2 from CD8+CAR+ p-value 0.199(-) p-value 0.426 Index of IL-2 production after CAR antigen-specific culture p-value 0.199 (+) p-value 0.824 Frequency of CD27+ CD28+ among CD3+CAR+ cells p-value 0.199 (+) p-value 0.25 CD4/CD8 ratio p-value 0.199(-) p-value 0.824 Multiplication frequency: from inoculation 0 to pre-harvest p-value 0.199(-) p-value 0.498 Endotoxin p-value 0.217 p-value 0.192 Non-antigen/CAR specific TNFa production per total cell count (CD3/CD28 stim.) p-value 0.227 p-value 0.738 ICS: IFNG from CD8+CAR+ p-value 0.227 p-value 0.301 CD8+ Frequency per dose p-value 0.227 p-value 0.824 Non-antigen/CAR specific IL-2 production per total cell count (CD3/CD28 stim.) p-value 0.257 p-value 0.738 Frequency of CD127+ among CD8+CAR+ cells p-value 0.257 p-value 0.1 Frequency of naïve phenotype cells among CD3+CAR+ p-value 0.257 p-value 0.574 Frequency of LAG3+ among CD4+CAR+ p-value 0.263 p-value 0.301 CD4+ frequency per dose p-value 0.263 p-value 0.824 Non-antigen/CAR specific MIP1a production per total cell count (CD3/CD28 stim.) p-value 0.29 p-value 0.738 Frequency of CD27–CD28+ cells among CD8+CAR+ p-value 0.29 p-value 0.426 Index of MIP1a production in CAR antigen-specific culture p-value 0.325 p-value one ICS: IFNG from CD4+CAR+ p-value 0.325 p-value 0.426 Temra frequency from CD4+CAR+ p-value 0.325 p-value 0.824 Frequency of CD27– CD28– from CD4+CAR+ p-value 0.325 p-value 0.301 Index of cytolytic activity p-value 0.351 p-value 0.906 Index of IL-4 production in CAR antigen-specific culture p-value 0.363 p-value 0.912 Index of IL-6 production in CAR antigen-specific culture p-value 0.391 p-value 0.514 Frequency of CD27–CD28– among CD8+CAR+ p-value 0.403 p-value 0.426 Frequency of CD27+ CD28– among CD3+CAR+ p-value 0.403 p-value one ICS: IL17 from CD4+CAR+ p-value 0.446 p-value 0.498 CD27–CD28+ among CD3+CAR+ p-value 0.446 p-value 0.912 Frequency of CD25+ among CD4+CAR+ p-value 0.456 p-value 0.514 Index of GMCSF production in CAR antigen-specific culture p-value 0.491 p-value one Frequency of naive cells among CD4+CAR+ p-value 0.491 p-value 0.738 CD25+ frequency among CD8+CAR+ p-value 0.502 p-value 0.738 Number of vector copies (per CAR+ genome) p-value 0.531 p-value 0.941 CD27–CD28– among CD3+CAR+ p-value 0.538 p-value 0.574 Number of injected CD4CAR cells/kg p-value 0.538 p-value 0.498 Number of injected CD4CAR cells p-value 0.538 p-value 0.25 Tem phenotype frequency among CD8+CAR+ cells p-value 0.587 p-value 0.426 Phenotype frequency CD27+ CD28– among CD8+CAR+ p-value 0.602 p-value 0.824 Cell viability in a therapeutic composition p-value 0.628 p-value 0.813 Tcm phenotype frequency among CD8+CAR+ cells p-value 0.638 p-value 0.426 Frequency of CD8+CAR+ per dose p-value 0.682 p-value 0.953 Ki67+ phenotype among CD8+CAR+ cells p-value 0.709 p-value one Tem phenotype among CD4+CAR+ p-value 0.794 p-value 0.906 Non-antigen/CAR specific GMCSF production per total cell count (CD3/CD28 stim.) p-value 0.801 p-value 0.654 ICS: IL17 from CD8+CAR+ p-value 0.801 p-value 0.912 Ki67+ frequency among CD4+CAR+ cells p-value 0.881 p-value 0.407 Frequency of LAG3+ among CD8+CAR+ cells p-value 0.911 p-value 0.813 Index of IL-13 production in CAR antigen-specific culture p-value 0.914 p-value one Frequency of Tcm phenotype among CD4+CAR+ p-value 0.914 p-value 0.574 Phenotype CD27+ CD28– among CD4+CAR+ p-value 0.914 p-value 0.301 Phenotype CD27–CD28+ among CD4+CAR+ p-value 0.914 p-value 0.912 FOXP3+ phenotype among CD8+CAR+ p-value 0.97 p-value 0.594 Phenotype CD27+ CD28+ among CD4+CAR+ p-value 0.97 p-value 0.953 Non-antigen/CAR specific IL-13 production per total cell count (CD3/CD28 stim.) p-value 0.971 p-value 0.574 Frequency of Tcm phenotype among CD3+CAR+ p-value 0.971 p-value 0.25 Tem frequency among CD3+CAR+ p-value 0.971 p-value 0.426 Index of IL-5 production in CAR antigen-specific culture p-value one p-value 0.906 Non-antigen/CAR specific IL-5 production per total cell count (CD3/CD28 stimul.) p-value one p-value 0.553

[0771] It was observed that the relationships observed in this study in relation to the number of copies of the vector (per cell or per genome) were considered as surrogate indicators of cell endurance and / or a set of variables associated with the relative representation of modified or unmodified cells in the composition and / or the total cell density in the composition. For example, in some embodiments, cells from blood samples that were generally less healthy at the start of the process had a lower likelihood of surviving during the transduction phase, and/or cell death during transduction could increase transduction efficiency. Thus, in some aspects, in some cases, higher viral vector copy numbers per composition, such as those introduced via transduction with a lentiviral or gamma-retroviral vector, may indicate compositions in which a greater percentage of cells were unhealthy at the start of the process and, in certain aspects may have properties that correlate, for example, inversely with neurotoxicity, for example, inversely with neurotoxicity. In some embodiments, a higher transduction frequency may result in a composition having a higher frequency of modified (eg, CAR+) cells during cryopreservation, storage, and thawing. In some aspects, when the dose is based on numbers of modified cells (or subtypes thereof, e.g., CAR+CD3+), such compositions with a higher frequency of modified cells may have a lower overall cell density, which, in specific embodiments, correlates with a decrease in the level or the frequency of biologically active or non-apoptotic cells.

[0772] The results of this one-way analysis identified several dose and/or therapeutic cell composition parameters that were observed to be significantly correlated (directly or inversely) with the occurrence of grade 5 neurotoxicity/cerebral edema. Among these parameters were parameters indicative of the number, number per patient weight, and/or frequency of cells having a phenotype typically indicative of biologically active cells, such as non-apoptotic CAR+ T cells or non-apoptotic CAR+CD8+ T cells introduced into dose. A number of parameters correlated with grade 5 neurotoxicity were observed not to be significantly correlated with treatment response outcome (herein, whether or not an individual achieves a complete response or no response). In various cases, it has been observed that a number of individuals achieving CR have levels of these parameters that are lower (in the case of a direct correlation) than the threshold level corresponding to the lowest amount (in the case of a direct correlation) observed among individuals who developed an undesirable effect. , such as grade 5 neurotoxicity. For example, the results indicated that numbers of biologically active or non-apoptotic CD3+ and/or CD8+ cells expressing CAR can be used to determine a dose within the safety and efficacy margins to reduce the risk of neurotoxicity and be within the range, in which responses are observed. No association has been identified between lethal neurotoxicity and T cell differentiation status or other phenotypes.

[0773] It has been observed that various measures of the number(s) or normalized numbers of cells that are negative for surface staining for apoptosis markers (including annexin V) at doses administered to an individual (or the frequency of such apoptosis marker negative cells among product cells, e.g. , among CAR+CD3+ or CAR+CD8+), correlate with neurotoxicity. As a rule, it was observed that the results for annexin V and caspase-3 are similar. While such parameters can also be considered to be related to the function or activity of the CAR-T cell composition, and thus would be expected to be related to efficacy, these variables have not generally been observed to be significantly correlated with treatment response outcome. , in this case, with whether or not the individual achieves a complete response or no response (see, for example, FIG. 2-5).

[0774] Results were consistent when using the frequency or number of non-apoptotic (and/or biologically active) T-cells or CAR+ cells, or subgroup(s) thereof (as shown by various measures) as a parameter to predict Grade 5 neurotoxicity or cerebral edema brain, and/or the severity of neurotoxicity, and using such parameter(s) to determine the appropriate dose and/or appropriate safety margins for CAR-T therapy.

Example 2 Multivariate Analysis of CAR+ T Cell Composition Parameters that are Predictive for Severe Neurotoxicity and/or Cerebral Edema

[0775] Univariate analyzes typically account for one covariance at a time. Multivariate analyzes take into account multiple covariances at once and/or in combination, and can generally take into account covariance, i.e. interaction between covariances.

[0776] Multivariate analyzes were performed to identify combinations of parameters (among parameters individually assessed in univariate analysis) that were highly correlated with and/or had high predictive value for clinical and/or toxicity outcomes.

[0777] A recursive tree analysis, splitting the factors into two parts, was performed to analyze different combinations of features. Specifically, the pooled dataset from the univariate analysis, including approximately 250 covariances (parameters), was examined using a split-based method (algorithmically, using a decision tree) for binary classification. Usually, using classical regression methods, functional (linear and non-linear) relationships are obtained, as a rule, more suitable for physicochemical data and/or testing hypotheses. Using partitioning techniques, algorithmic models ("trees") are typically obtained, which may, in some embodiments, be more suitable for biological data and exploratory analysis. Compared to data collection methods such as support vector machine (SVM) and artificial neural networks (ANN), partition models can provide advantages in ease of interpretation.

[0778] Partition models typically include nested containers/bins of if-then-else conditional statements, such as predictive mean responses. In some embodiments, because partitioning methods do not accept a prior model, they are more useful in evaluating noisy measurements or individual measurements, non-linear data, and datasets with mixed data types (i.e., predictor and response variables that are categorical and/or continuous).

[0779] In general, tree-based methods are non-parametric and, compared to other multivariate methods, are better equipped to handle noisy measurements and individual measurements, a combination of continuous and categorical variables, and non-linear relationships.

[0780] In the method in this study, data were taken into account for a total group of 38 individuals and, separately, for a group of 24 individuals with morphologically confirmed disease. Correlations with neurotoxicity were assessed by grouping patients in three different ways: according to whether or not individuals developed grade 5 neurotoxicity, whether or not they developed grade 4 or 5 neurotoxicity, and whether or not they developed grade 4, 5 or long-term neurotoxicity ( 10 days or more) grade 3 neurotoxicity (3p). Model complexity was minimized by using combinations of at most two covariances (one primary predictor and one secondary predictor) at a time and maintaining a split group size of at least five observations.

[0781] Conducted an iterative search, in which several alternative models were obtained using the (same) best primary predictor and multiple secondary predictors, provided that the number of false positives and false negatives was satisfactorily small. The next best primary predictor was then searched for and the steps were repeated. Several alternative models were evaluated and found that the best models (the models with the fewest false positives and false negatives) consistently used similar covariances.

[0782] Using this method, it was observed that specific parameter pairs have greater predictive power for the severity of neurotoxicity (eg, grade 5 neurotoxicity). Among these pairings were combinations of amounts, or normalized amounts, of specific cell populations (such as CAR+CD3+ non-apoptotic or CAR+CD8+ non-apoptotic) at the administered dose and an indicator of CAR antigen-specific activity in the therapeutic composition, such as pro-inflammatory cytokines. Among these pairs, there was a combination of the number of apoptosis marker-negative CD8+ CAR-expressing cells administered to an individual, in combination with a parameter indicative of TNF-alpha production, as measured by accumulation in the supernatant after 18-hour co-culture of a sample of a therapeutic composition of cells containing 0, 25x106 CD3+CAR+ cells co-cultured with CD19 expressing cells in a 1:1 ratio (see Figures 6A and 6B). Similar findings were obtained for other pro-inflammatory cytokines such as IFN-gamma and IL-2 instead of TNF-alpha.

[0783] In this study, for these combinations of variables, no strong predictive response (as measured by CR versus no CR), inverse or forward, was observed (see, for example, Figures 9, 11, 13). In addition, several individuals who achieved CR or CRi in the study described in Example 1 were dosed with a cell product containing amounts (or normalized amounts) of the apoptosis marker-CD8+CAR+ cells that were below the smallest number (or normalized amount) in any dose administered to individuals who continued to develop grade 5 neurotoxicity/cerebral edema. Similarly, several individuals who continued to achieve CR were administered cell compositions, as determined by the assay, having levels of production of a CAR antigen-specific cytokine (eg, TNF-a, IFN-g, IL-2) that were lower than the lowest observed among compositions administered to any individuals who experienced grade 5 neurotoxicity/cerebral edema (see, for example, Figures 4 and 5).

[0784] The results of the analysis after another study were consistent with the usefulness of the identified pairs of covariances for determining dose and predicting neurotoxicity. Tracking results of similar compositions of anti-CD19 CAR-expressing cells administered in another clinical study in a subgroup (11 of 31) of individuals (selected by the presentation of a number of different responses), with relapsed or non-responsive adult ALL, were analyzed for certain parameters that appeared to be correlated or inversely correlated with neurotoxicity outcomes in the above study. Of 31 individuals, 77% experienced CR or CRi, with rates of 61% for relapse and 27% for stem cell transplant = (SCT) among individuals with CR/CRi. 42% experienced grade 3 or higher CRS and 35% showed grade 3 or 4 neurotoxicity; none of the 31 individuals experienced grade 5 neurotoxicity or cerebral edema.

[0785] Certain parameters were evaluated and compared for individuals and cell compositions in this other study, generally as described above, including the number of injected apoptosis marker-negative CD8+ CAR-expressing cells and the same antigen-specific activity parameter (same TNF-alpha accumulation score). in an assay involving co-culture with CD19 expressing cells as used above). The results are shown in FIG. 7 and FIG. 6b. For the dose administered to each of 11 individuals evaluated in another similar clinical study - of which none showed grade 5 neurotoxicity or cerebral edema -: (i) the number of apoptosis marker-negative, CAR CD8+ expressing cells was below the least such cells at a dose administered to any individual with grade 5 neurotoxicity in the above study and/or (ii) the level of a parameter indicative of CAR-induced activity (e.g. TNF-alpha production) (B) was below the lowest level observed at doses for individuals with grade 5 in the above study. As shown in FIG. 6B, the overall range of the product profile for this assay among the majority of individuals in the other study evaluated was below that level indicative of CAR-induced activity, as indicated by the shaded oval.

[0786] Thus, among individuals in another study using a similar composition of CAR-expressing cells, none of whom developed grade 5 neurotoxicity, one or both of the covariances in the pair predictive of neurotoxicity were below the level observed for any patient experiencing toxicity. Similar results were observed when IFNg or IL-2 was evaluated instead of TNF-alpha. The results were consistent with the interpretation of using a combination of functional trait (degree of CAR antigen-specific activity, as assessed by secretion of pro-inflammatory cytokines after co-culture with antigen-expressing cells) and number (or number per weight) of non-apoptotic CAR+CD8+ cells to determine or evaluate an appropriate dose for minimizing the risk or severity of neurotoxicity.

[0787] The results of these studies were consistent with the benefits of using a composite method to determine an appropriate unit dose for administering CAR–T compositions. For example, the results are consistent with the usefulness of a method in which the unit dose is based on two or more parameters, and/or in which the release of the unit dose to be administered occurs only after accounting for any variability that could potentially contribute to each of those two or more. parameters. For example, the results are consistent with the benefits of administering a single dose and/or identifying an appropriate dose, wherein (A) refers to the number or number of cells of a particular phenotype (such as CD3+CAR+, CD8+CAR+, or CD8+CAR+ negative for an apoptosis marker) and others, or a composite number of cells of multiple phenotypes, in which (B) shows CAR-induced/CAR-dependent and/or antigen-specific activity, such as CAR-induced production of a pro-inflammatory cytokine. The results were consistent with the usefulness of a composite method in which (1) a target number of cells (A) is administered, provided that the activity score (B) is below a certain threshold number (based on correlations with toxicity), (2) a different target dose (A) administered, depending on whether B is above or below a given one or more threshold values (and optionally, where cells are not injected if B exceeds the upper limit of the safety threshold) and/or (3) the dose is determined as the target number of reference units, where the number of reference units is determined based on a function of A and B (eg, A x B, or a function of the converted value(s) or multiple(s) of A and B).

[0788] In addition, it has been observed that, in some cases, changing the storage conditions or handling, or batch of the type(s) of raw material or reagent(s) used in the method for preparing a composition of modified T cells - in an otherwise similar method of modification cells - appears to correlate, in the final therapeutic composition, with certain parameters that have been observed herein to be related to the severity of neurotoxicity and/or cerebral edema, e.g. in the study described above, e.g. in combination with another covariance. For example, changes in such storage or handling conditions, or batches of such materials or reagents, have been observed to correlate with changes in specific measures of antigen-specific activity in the therapeutic composition prepared by these methods, such as, for example, the production of a pro-inflammatory cytokine (e.g., TNF-alpha) in response for co-cultivation with cells expressing the CAR antigen.

[0789] The levels observed for exemplary parameter scores (a score of CAR antigen-specific TNF-alpha production and frequency of CD27+/CD28+ phenotype among CAR+ cells) were compared for two groups of therapeutic cell compositions produced by a method using two different batches of raw materials.

[0790] The results are shown in Figures 23A and 23B. As shown, the levels of these parameters were variable among the samples. A higher average level of each parameter was observed in cell compositions obtained using the method using one batch (batch 2) of raw materials, compared with cell compositions obtained using a comparable method using a different batch (batch 1) of the same reagent (TNFa: p=0.066); (CD27+/CD28+ frequency: p=0.018). Figure 23B further shows the degrees of neurotoxicity/cerebral edema developed in individuals treated with each individual cell composition. Among 15 individuals, higher degrees of neurotoxicity/cerebral edema were observed among individuals who were administered cell compositions prepared using Reagent Lot 2.

[0791] In another example, it was also observed that storage of reagents and/or manipulation of reagents affect the parameters among two groups of therapeutic compositions of cells obtained by a method using the same culture medium, using two different storage conditions and/or manipulations, in an otherwise similar way of modifying cells. Two groups of therapeutic compositions of cells obtained from the same medium, maintained under each condition, were evaluated for the production of TNF-alpha in response to co-cultivation with cells expressing the CAR antigen. As shown in Figure 23C, TNF-alpha production levels were variable among samples. A trend towards lower TNF-alpha production was observed in cell compositions prepared using media maintained under the first storage conditions compared to compositions prepared using the same media maintained under the second storage conditions.

[0792] The observations were consistent with the interpretation of what may provide benefits - for example, in identifying a safe and effective dose of a cell composition - accounting (for example, analysis before the release of the product and / or for a factor in the dosing method) the degree of variability of factors that may contribute into the types of cell/antigen-specific activity in the composition, in terms of compositions derived from cells originating from different individuals and/or in the presence of one or more different storage conditions, types of raw materials or reagents, or manipulations with them, for example, when using different batches of reagents or other types of raw materials. The results highlight certain advantages of the embodiments presented herein. In some aspects, it is advantageous to reduce the variability of such parameters and/or to confirm an acceptable range of variability among such different conditions/lots, for example, when introducing a new batch or a reagent subjected to storage or manipulation under different conditions, for example, using a release assay testing one or several parameters of the compositions of cells obtained using the method, including such changes, such as parameters, as observed in the present description, associated with the risk of neurotoxicity, so as to exclude variability of such parameters, for example, in different doses or products of cells.

[0793] In some embodiments, the methods, articles, compositions, doses, and dosing methods provided provide advantages in that they take into account, and, where appropriate, adjust or correct for, potential sources of variability, including variability derived from for reagent changes, and/or patient-to-patient variability. For example, in some embodiments, it may be advantageous to generate modified T cells by a method involving the use of a T cell stimulation/expansion reagent (or a batch thereof) that is confirmed in a release assay to be below or within an acceptable range of variability, compared to a threshold level of a parameter, e.g., specific activity, in relation to that parameter of a therapeutic composition (such as an indication of the amount or relative amount of a reagent required to induce a certain degree of antigen-specific activity in the final T-cell composition obtained by a T-cell modification method, for example, compared to a control reagent or standard unit, as applicable to that assay). In some embodiments, the provided embodiments include doses of cells in which the cells are obtained by a method that includes a release assay to confirm that any variability in such specific activity parameters (e.g., antigen-specific pro-inflammatory cytokine level) is within an acceptable range and/or below the upper the set limit. In some embodiments, the present methods include such a release assay. In some embodiments, the preparation of cell compositions is carried out using reagents and/or methods for which the variability of such parameters is within an acceptable range. In some embodiments, the present methods, compositions, and articles of manufacture provide advantages in that they use dosing and/or cell preparation methods that reduce the risk associated with the potential variability of such specific activity parameters, for example, by minimizing the variability of the second parameter, which, together with a specific activity parameter correlates with the risk of toxicity. For example, by minimizing the variability in the frequency of biologically active or healthy cells (eg, non-apoptotic cells) obtained by the method, the impact of changes in specific activity-related parameters on safety can be minimized. In some embodiments, a dose or dosing method comprising the feature(s) associated with the number or frequency of biologically active or non-apoptotic cells, such as biologically active or non-apoptotic modified cells, or modified CD8+ cells, reduces the risk associated with variability in antigen-specific activity parameters. . In specific aspects, this is achieved by dosing, including an upper limit for such cells and/or determining the dose based on reference units, for example, based on a formula that takes into account the number of biologically active cells.

[0794] The compositions administered to the individuals in the study in Example 1 were compared with compositions prepared by an alternative method of obtaining modified T cells from blood samples derived from individuals with recurrent or non-responsive NHL. The method involves separately obtaining and then mixing, approximately in the target ratio, CD4+ and CD8+ populations of modified T cells originating from the same individual. Each population was generated in a similar manner involving the use of various factors and reagents, including cytokines, for example during activation, transduction (including the use of a lentiviral vector) and/or cell expansion. The method further included filling containers with product, cryopreserving and thawing the cells at cell densities that were comparatively higher than those used in the method in the above study and were uniform across samples (see Example 4). In general, the method provided an increased degree of control over the phenotype, function and metabolic profile of the modified cells in the final composition. CAR included a 41BB-derived co-stimulatory domain, in contrast to the CD28 domain.

[0795] It has been observed that among samples obtained using this method (for example, a method using controlled ratios of T cell subgroups, cytokines and reagents to provide control of phenotype, function and metabolic status) and controlled and higher cell density during cryopreservation ), there was less individual-to-individual variability, compared to variability among samples analyzed in Example 1, for features of therapeutic cell compositions, as observed herein, that are highly predictive (alone and/or in combination) for the risk of severe neurotoxicity.

[0796] For example, alternative method (B) resulted in higher frequencies of biologically active or healthy cells among CD8+CAR+ cells, as measured by negativity for various markers of apoptosis among samples obtained using this method, compared with samples after administration, as described in example 1 (see, for example, FIG. 21A). In addition, there was a lower degree of variability in this parameter among samples obtained by this method from different individuals. As observed herein, the number of such cells was observed to correlate with grade 5 neurotoxicity and cerebral edema, while the number of CD3+CAR+ viable cells (ignoring the apoptotic state, compared to biologically active cells) was a poorer prognostic factor. factor and did not clearly define the margin of safety. In some embodiments, using a method with reduced variability in the frequency of such biologically active cells among modified cells reduces the risk that particular patients (e.g., patients having cells that are less prone to apoptosis or that are healthier) may inadvertently administer more a higher than intended dose of biologically active cells, dosing based on the number of modified T cells in general. In addition, methods with greater control of phenotype and function have been observed to reduce the degree of variability in the ability of cells obtained by this method to produce pro-inflammatory cytokines in an antigen-specific manner (see, eg, FIG. 21B). The results in the above study are consistent with the interpretation that, in particular, in combination with the amounts of biologically active modified cells, the dosing method taking into account such cell-specific parameters of activity (for example, such that a specific target range of such activity - or not more threshold - presented at a given dose) can be used to provide a dose capable of providing a desired clinical or therapeutic outcome while still remaining within the safety margin or reducing the risk of unwanted toxicity, such as neurotoxicity.

[0797] In some embodiments, the use of a method that consistently produces higher frequencies of biologically active modified cells allows the use of cell doses that are much lower (in terms of numbers of modified, e.g., CAR+ cells) than other dosing methods. in which the modified cells are more frequently positive for apoptotic markers or are otherwise less healthy. For example, based on the observations herein, and given the observation that available dosing methods generally do not take into account the frequency of apoptotic cells, in some embodiments, the numbers of modified (e.g., CAR+) T cells (e.g., modified CD8+ and/or CD4+ cells) are as low as 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 million cells.

Example 3: Definition and formula for target unit dose

[0798] Exemplary doses, dose ranges, and methods and formulas for dosing and dose determination have been identified and developed based on combinations of parameters identified in multivariate analyzes that appear to be predictive of the degree of neurotoxicity.

[0799] Evaluated data for all thirty-eight individuals or only for individuals with morphologically confirmed disease. Dosing data were available for 37/38 individuals; correlation with response (CR versus no CR) was considered only for patients with morphological burden of disease, and was not considered for individuals who developed grade 5 neurotoxicity and were not amenable to CR. For individuals, the number of reference units (RU) per administered dose was determined, where:

[0800] RU=A x B,

[0801] A was equal to the number of apoptosis marker-negative, CD8+, CAR-expressing cells, and

[0802] B was a parameter indicative of CAR antigen-specific activity among the cells in the composition. In some assays (see FIGS. 8-13), B was a measure indicative of the level of antigen-specific production of an individual cytokine (e.g., concentration per hour per number of cells injected), or the geometric or arithmetic mean of such a measure, for multiple cytokines.

[0803] In other assays (see FIGS. 14-20), B was an adjusted value indicative of levels of one or more cytokines based on that score, or a composite score normalized to the central trend of that score among individuals with grade 0 neurotoxicity -2.

[0804] Results are plotted on a logarithmic scale for subgroups of individuals grouped according to degree of neurotoxicity (where individuals are grouped by whether or not they experienced grade 4 or 5 neurotoxicity, or whether or not they experienced long-term grade 3 neurotoxicity (3p ), 4 or 5 neurotoxicity) and/or response rate (CR/no CR).

[0805] The results are shown in FIG. 8–20. Specifically, figures 8-13 show the results among 38 individuals.

[0806] Figures 14, 15 and 16 show the results of RU among 32 individuals with morphologically confirmed disease, where A was equal to the number of apoptosis marker negative, CD8+, CAR-expressing cells, B was an adjusted measure of antigen-specific production of individual cytokine (TNF-alpha , IFN-gamma, and IL-2, respectively) normalized by the central trend of the individual cytokine score among individuals with grade 0–2 neurotoxicity (where Figures 14A, 15A, and 16A show results for individuals grouped according to grade of neurotoxicity, and Figures 14B, 15B and 16B show results for individuals grouped according to response (CR versus no response CR)).

[0807] Separation according to different amounts of RU was observed between groups of individuals having different degrees of types of toxicity. The results were used to identify exemplary upper or cutoff reference units (based on the formula RU=A x B), and exemplary target RU amounts and target doses. In some examples, the threshold or target amount of RU is identified to be lower than the amount of RU determined among individuals who developed a particular degree of neurotoxicity, in some aspects, lower than the amount of RU corresponding to the safety window, for example, resulting in 1.5 -fold or 2-fold decrease in the number of RU. Plots grouping patients according to response were consistent with the finding that target RU values and target doses should be within acceptable response windows.

Example 4: Correlation of Total Cell Density in a Cryopreserved Composition and Composition Parameters Indicative of Cell Function and Other Traits

[0808] The contribution of cell density, eg, viable cell concentration (VCC), during cryopreservation and storage of T cell therapeutic formulations was investigated. It is known that an increase in cell density in specific compositions reduces cell survival. It has been observed in the present specification, however, that for certain cryopreserved cell compositions for therapeutic use, in particular those that are cryopreserved at relatively low cell densities (e.g., below 10% cell volume of total frozen volume), the increase in cell density during cryopreservation , storage and/or thawing of the composition, for example, a therapeutic product, may provide benefits, for example, in relation to health and/or biological activity and/or function of cells.

[0809] Cryopreserved cell therapeutic compositions administered to individuals in the clinical study described in Example 1 were cryogenically frozen at various densities below approximately 1% (cell volume as a percentage of total frozen volume) using DMSO. In the studies evaluating the various parameters above, samples from cell compositions were subjected to cryopreservation, storage, and thawing under conditions that mimic the conditions under which administered cell compositions were stored, transported, and thawed, including similar time periods.

[0810] To assess the contribution of cell density during cryopreservation, storage and thawing, compositions of cells from different individuals subjected to cryogenic freezing at different cell densities (1×10 ⁶ cells per ml, 8×10 ⁶ cells per ml and 15×10 ⁶ cells per ml) and using various concentrations of DMSO (between approximately 3% and 9% DMSO by volume). The cryopreserved cell compositions were then thawed and cell activity (as measured by antigen-specific cytotoxicity) in the compositions was assessed.

[0811] The results showed the observation that an increase in cellular activity was generally associated with higher cell concentrations and/or DMSO concentrations during cryopreservation. The results were consistent with the interpretation that cryopreservation conditions may affect the biological activity of T cell therapeutic compositions.

[0812] In an additional study, cell compositions containing cells expressing anti-CD19 CAR were cryogenically frozen at various concentrations (e.g., 1 x 10 ⁶ cells/ml, 2 x 10 ⁶ cells/ml, 5 x 10 ⁶ cells/ml 10 x 10 ⁶ cells/ml, 20 x 10 ⁶ cells/ml, 50 x 10 ⁶ cells/ml and 100 x 10 ⁶ cells/ml) and 7.5% DMSO and stored at 80 degrees for about 15 hours, thawed , and then analyzed for viability (viable cell concentration and diameter (VCC), and apoptosis markers (eg, annexin V and activated caspase-3) by flow cytometry.

[0813] The observed loss in viable cell concentration (% VCC difference), in this study, during the cryogenic freezing and thawing period, was in the range of 10-45%. No general trend was observed among concentrations for these traits.

[0814] Apoptosis marker results showed a general trend for both CD4+ CAR-expressing cells and CD8+ CAR-expressing cells toward higher levels of cell apoptosis marker+ cells observed after thawing for compositions in which cells were frozen and stored at more low concentrations of cells. In particular, the results showed that over the tested DMSO concentration range, e.g., at 7.5% DMSO, in some embodiments, total cell densities between 10 or about 10 and 60 or about 60 million, or at or about 15 and at or about 60 or 70 million cells per ml during cryopreservation, storage and thawing were particularly preferred to improve cell health and/or increase the frequency of biologically active cells in the composition after cryopreservation and thawing.

[0815] The results are consistent with the utility of higher cell densities or cell concentrations for CD8+ cells and/or CD4+ cells in the cryopreservation of T cell therapeutic products.

Example 5: Evaluation and comparison of neuropathology in different individuals

[0816] Neuropathology assessments at autopsy were performed for four individuals with acute lymphoblastic leukemia (ALL) who developed severe neurotoxicity, including grade 4 or 5 neurotoxicity, and/or cerebral edema after treatment with therapeutic compositions containing T cells modified to express a chimeric antigen receptor (CAR). Specifically, autopsy studies were performed on 2 individuals who experienced grade 5 cerebral edema (n=2) and 2 individuals who experienced severe neurotoxicity in the absence of cerebral edema (n=2). The results generally support the conclusion that B-ALL brain involvement was not observed to be a factor in cerebral edema. In two individuals with cerebral edema, neither congenital immunocytes, nor B-ALL malignant tumor cells, nor CD19+ malignant tumor cells were observed in the CNS. In addition, it was observed that in patients with cerebral edema, the edema tended to be vasogenic, not cytotoxic. In patients with cerebral edema, leakage of perivascular fibrin and red blood cells suggested complete damage to the blood-brain barrier (BBB). BBB was examined in these individuals. Endothelial damage was also observed. Apparently, however, there was no marked infiltration of T-cells, which is consistent with the conclusion that cerebral edema did not develop as a result of infiltration and/or activation of CAR-T cells in the brain or CNS.

[0817] In addition, in the brains of individuals who developed cerebral edema, perivascular and more diffuse patterns of damage/activation of astrocytes and microglia, indicative of BBB, were observed. The observation was consistent with the conclusion that microglial activation contributed to the development of cerebral edema in individuals subjected to CAR-T cell therapy. In addition, irreversible damage to astrocytes (clasmatodendrosis) was observed in individuals who developed cerebral edema, in contrast to the proliferation of astrocytes observed in individuals who developed grade 4 neurotoxicity. No significant edema was observed in individuals for whom cerebral edema was indicated. in peripheral tissues such as the lung, liver or kidney. Complete damage to the BBB and resulting vasogenic edema was not observed in individuals who developed grade 4 neurotoxicity.

[0818] At autopsy, for individuals who did not develop cerebral edema (n=2), a diffuse infiltration of CD8+ T cells was observed, inconsistent with a simple response to focal injury. T cells were present in the CNS in these individuals. No evidence of blood-brain barrier pathology was observed in these individuals in the absence of cerebral edema.

Example 6 Administration of Anti-CD19 CAR Expressing Cells to Individuals with recurrent and refractory non-Hodgkin's lymphoma (NHL)

[0819] Therapeutic compositions of CAR+ T cells containing autologous T cells expressing a chimeric antigen receptor (CAR) specific for CD19 were administered to individuals with B cell malignancies. The administered T cell therapeutic compositions were prepared by a method involving immunoaffinity-based enrichment of CD4+ and CD8+ cells from leukapheresis samples from individuals to be treated. Isolated CD4+ and CD8+ T cells were individually activated and independently transduced with a lentiviral vector encoding anti-CD19 CAR. Modified CD4+ and CD8+ T cells were independently expanded in the presence of cytokines to a threshold cell count. After expansion, the cells were formulated and cryopreserved in low volume. The CAR contained an anti-CD19 scFv, a mouse antibody-derived, an immunoglobulin-derived spacer, a transmembrane domain derived from CD28, a costimulatory region derived from 4-1BB, and an intracellular signaling domain from CD3-zeta. Cryopreserved cell compositions were thawed prior to intravenous administration. A therapeutic dose of T cells was administered in the form of a specific cell composition by introducing a CD4+ CAR+ cell population and a CD8+ CAR+ cell population, administered at a target ratio of approximately 1:1.

A. Individuals and treatment

Example 6.A.1

[0820] The studies described in this example 6.A.1 describe the treatment and evaluation of individuals at a specific time point (6.A.1) in an ongoing clinical study of such therapy in patients with B-cell malignancies. Specifically, a cohort (full cohort) (at this time point, fifty-five (55)) of human adults with relapsed or refractory (R/R) aggressive non-Hodgkin's lymphoma (NHL), including diffuse large B-cell lymphoma (DLBCL), new-onset or transformed from indolent lymphoma (NOS), primary mediastinal large B-cell lymphoma (PMBCL), and grade 3b follicular lymphoma (FLG3B) after failure of 2 lines of therapy. Among the treated individuals, there were those with an Eastern United Cancer Group (ECOG) score between 0 and 2 (median follow-up 3.2 months). The complete cohort did not include individuals with mantle cell lymphoma (MCL). No individual was excluded based on previous allogeneic stem cell transplantation (SCT), secondary central nervous system (CNS) involvement, or ECOG score of 2, and there was no minimum absolute lymphocyte count (ALC) requirement for apheresis.

[0821] Outcomes were separately assessed for a core subgroup of individuals within the full cohort (individuals within the full cohort except for individuals with poor functional status (ECOG 2), DLBCL transformed from marginal zone cell lymphoma (MZL) and/or chronic lymphocytic leukemia (CLL, Richter's transformation) (core cohort)). At this time point, in Example 1.A.1, outcomes were assessed for 44 individuals within this core cohort.

[0822] The demographic and background characteristics of individuals from the complete and core cohorts at this time point in Example 6.A.1 are shown in Table E3.

Table E3. Demographic and background characteristics Characteristic FULL, N=55 COW, N=44 Median 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 after 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 Grade 3B 12) 12) Molecular subtype, n (%) Double/triple rearrangement of genes 15 (27) 12 (27) double expression 6 (11) 4 (9) Patient characteristics, n (%) Chemotherapy resistance ^† 42 (76) 34 (77) ECOG 0–1 48 (87) 44 (100) ECOG2 7(13) 0 Prior 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 to last chemotherapy-containing regimen or relapse <12 months after autologous SCT

[0823] As shown in Table E4, individuals 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 with level dose 1 (DL-1) containing 5×10 ⁷ total CAR-expressing T cells (n=30 for individuals assessed in Example 6.A.1), a double dose of DL1, in which each dose was administered at an interval of approximately fourteen ( 14) days (n=6 for the individuals assessed in Example 6.A.1, including one individual who was inadvertently given two doses of DL2 on a two-dose schedule due to a dosing error), or a single dose with a dose level of 2 ( DL-2) containing 1×10 ⁸ (DL-2) total CAR expressing T cells (n=18 for individuals evaluated in Example 6.A.1). Beginning three (3) days prior to CAR+ T cell infusion, individuals were subjected to anti-lymphocyte chemotherapy using fludarabine (flu, 30 mg/m2) and cyclophosphamide (Cy, 300 mg/m2).

Table E4: Dosing levels and number of T cell subsets for a cell composition containing anti-CD19 CAR T cells

Dose level (DL) Dose of helper T cells (T _H ) (CD4+CAR+) The dose of cytotoxic T cells (T _C ) (CD8+CAR+) Dose of total T-cells (CD3+CAR+) DL1 25×10 ⁶ 25×10 ⁶ 50×10 ⁶ DL2 50×10 ⁶ 50×10 ⁶ 100×10 ⁶

Example 6.A.2

[0824] For example 6.A.2, at a subsequent time point in the clinical study described in this example above, the results were analyzed at a second time point. At this time point in the analysis in example 6.A.2, 74 patients were treated (51 men, 23 women). Individuals included sixty-nine (69) individuals in the complete DLBCL cohort (including 67 DLBCL NOS (45 new-onset, 14 FL-transformed, 8 CLL-or-MZL-transformed), 1 Grade 3B FL, and 1 PMBCL), and 5 individuals in the cohort MCL. Among individuals in the complete (DLBCL) cohort, median age was 61 years (range 26, 82), median previous treatment cycles was 3 (range 1, 12), 46 (67%) were refractory to chemotherapy, 32 (46%) had any prior transplantation, and at least 16 (23%) patients had double/triple gene rearrangement lymphoma. Forty-nine (49) individuals in the core cohort were evaluated at this time point in 6.A.2.

B. Security

[0825] Evaluated the presence or absence of therapy-induced adverse events (TEAE) after CAR-T-cell therapy. Individuals were also assessed and monitored for neurotoxicity (neurological complications, including symptoms of confusion, aphasia, encephalopathy, myoclonic seizures, convulsions, lethargy, and/or altered mental status) divided into grades on a scale of 1-5, according to the US National Cancer Institute – The Common Criteria Toxicity Scale (CTCAE), version 4.03 (NCI–CTCAE v4.03). Common Toxicity Criteria (CTCAE) scale, version 4.03 (NCI–CTCAE v4.03). See Common Terminology for Adverse Events (CTCAE) Version 4, US Department 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) was also defined and monitored, graded based on severity. See Lee et al, Blood. 2014;124(2):188–95.

Example 6.B.1

[0826] Example 6.B.1 describes the results based on the time point analysis in Example 6.B.1.

[0827] In 84% of individuals from the full cohort in the analysis in example 6.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 individuals from the complete cohort did not experience any degree of CRS or neurotoxicity. No differences were observed in the incidence of CRS, neurotoxicity (NT), sCRS, or severe neurotoxicity (sNT) between dose levels. Table E5 summarizes the incidence of adverse events of cytokine release syndrome (CRS) and neurotoxicity in patients 28 days after administration of at least one dose of CAR-T cells. As shown in Table E5 , sCRS (grades 3-4) was not observed in any of the individuals who received a single dose of DL2 or a double dose of DL1. Severe neurotoxicity or severe CRS (grades 3-4) was observed in 16% (9/55) of individuals in the full cohort and in 18% (8/44) of individuals in the core subgroup. 11% (n=6) of individuals were administered tocilizumab, 24% (n=13) of individuals were administered dexamethasone. Among individuals with ECOG2 in the full cohort, the observed rates of CRS and neurotoxicity were 71% and 29%, respectively.

Table E5. Evaluation of the presence or absence of CRS adverse events and neurotoxicity for Example 6.B.1 COMPLETE COW 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 Degrees 1–2, n (%) 18 (33) 10 (33) 5 (26) 3 (50) 12 (27) Degrees 3–4, n (%) 12) 13) 0 0 12) Neurotoxicity Degrees 1–2, n (%) 3 (6) 13) 2 (11) 0 2(5) Degrees 3–4, n (%) 9 (16) 6 (20) 2 (11) 1 (17) 8 (18) ^† Inclu ^† Including one patient treated with DL2 dosing schedule 2–due to dosing error

Example 6.B.2

[0828] Example 6.B.2 describes the evaluation at a time point in Example 6.B.2. Up to this time point, adverse event (AE) data were collected for antilymphocyte therapy (LD) up to 90 days after administration of CAR-expressing T cells. At the second time point, 69 individuals in the DLBCL cohort (complete cohort) were evaluated for safety, where 38 had a single dose schedule of DL1, 25 had a single dose schedule of DL2, and 6 had a dual dose schedule 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 TEAE grade 5 diffuse alveolar lesion was observed.

[0829] No acute infusion toxicity was observed, and it was observed that the majority of individuals in the complete cohort, 64% (44/69), did not have CRS or NT, indicating that outpatient delivery of CAR-expressing T cells may be possible. The frequencies of CAR-T cell-associated toxicities, including CRS and NT, did not differ between dosing levels. The safety profile was observed to be similar across cohorts and dosage levels. Among 25 individuals in the full cohort (36%) who experienced CRS or NT of any degree, 21 (30%) had CRS and 14 (20%) had NT. None of the individuals had CRS grade 3 and only one (1%, 1/69) had CRS grade 4 and required treatment at the ICU; the other 29% (20/69) had CRS grades 1–2. Of the 20% of individuals with NT, 6% (4/69) had NT grades 1–2 and 14% (10/69) had NT grades 3–4; 2 (3%) had seizures. Grade 5 CRS or Grade 5 NT was not observed. Cerebral edema was not observed. All CRS and NT events resolved except for one case of grade 1 tremor, which continued for the duration of the analysis. 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 individuals were observed to have NT, and only 10% (7/69) were observed to have CRS (all grades 1); NT preceded CRS in >70% of individuals. In total, thirteen (13) individuals (19%) required intervention for CRS or NT using anti-cytokine therapy (tocilizumab alone for 1 (1%), dexamethasone alone for 6 (9%), or both for 6 (9%) ), and only one required the support of any vasoconstrictor. The median doses of tocilizumab and dexamethasone were 1 and 6, respectively. The median duration of CRS and NT presence was 5 days and 11 days, respectively. Analysis of the core cohort (n=49) also showed similar rates of CRS and NT.

[0830] In this assessment, low rates of onset and late onset of CRS and/or NT at dosing were observed, which supports the feasibility of outpatient infusion, for example, with hospitalization at the first sign of fever, or with fever lasting longer than a certain period of time, due to it. At the time of evaluation in 6.B.2, four individuals were treated on an outpatient basis.

C. Response outcomes after treatment

[0831] Individuals were monitored for response, including assessment of tumor burden at 1, 3, 6, 7, 12, 18, and 24 months after CAR+ T cell injection.

Example 6.C.1

[0832] Example 6.C.1 describes the results based on the time point analysis in Example 6.A.1 and 6.B.1.

[0833] Response rates are listed in Table E6. High duration response rates were observed in a cohort of individuals that included individuals with severe prior treatment or poor prognosis and/or recurrent or refractory disease. For individuals across all doses 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 bovine cohort, the overall response rate (ORR) was 66%; the CR rate at three months was 50% among the bovine cohort. In the bovine cohort, ORR at 3 months was 58% (11/19) at dose level 1 and 78% at dose level 2; the CR rate at 3 months was 42% (8/19) for dose level 1 and 56% (5/9) for dose level 2, consistent with the estimated effect of dose-dependent response on treatment outcome. In addition, the results were consistent with the relationship between dose and duration of response.

Table E6. Answer COMPLETE COW All dose levels DL1S DL2S DL1D ^c All dose levels DL1S DL2S DL1D ^a Best overall answer, N ^a 54 thirty eighteen 6 44 25 fifteen four 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 months f/u, ^nb 41 24 eleven 6 32 19 9 four 3-month 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) 3-month 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 DL1 1 dose; DL2S: Schedule DL2 1 dose; DL1D schedule: DL1 2 doses;
^a Includes patients with PD events, death, or re-staging within 28 days. Patients treated <28 days prior to data point acquisition were not included.
^b The denominator is the number of patients who received CAR-T cell therapy ≥ 3 months ago, before the data point, with efficacy estimated at month 3, or until PD or death was assessed.
^c Including one patient treated with DL2 dosing schedule 2 due to dosing error

[0834] Among individuals treated six months or more prior to a specific assessment time point, of ten (10) patients who had a response at three months, 9 (90%) maintained a response at six months. At this assessment time point, 97% of the responders in the core subgroup were alive and on track, with a median track time of 3.2 months. Long-term survival was observed in responders, with increased duration of response in individuals with CR. All responders at three months were alive at the time of evaluation, although 5/6 of the individuals with poor functional status (ECOG 2) were discontinued from the study.

Example 6.C.2

[0835] Example 6.C.2 describes the results based on the time point analysis in Example 6.A.2 and 6.B.2.

[0836] Up to the time point in Example 6.C.2, 68 individuals in the full DLBCL cohort were scored for response. For overall or objective response (OR), 3-month and 6-month objective response rates were 75% (51/68), 49% (27/55), and 40% (14/35), respectively. Complete response (CR) rates, 3-month CR rates, and 6-month CR rates were 56% (38/68), 40% (22/55), and 37% (13/35), respectively. A trend toward improved response rates at 3 months was observed in individuals treated with DL2 compared with DL1: 63% (12/19; 95% CI 38, 84) versus 40% (12/30; 95% CI 23 , 59) for ORR at p=0.148, and 58% (11/19; 95% CI 34.80) compared with 27% (8/30; 95% CI: 12.46) for CR at p=0. 0385. Among 16 individuals with double/triple rearrangement lymphoma, the ORR was 81% and the 3-month CR rate was 60%.

[0837] In the core cohort (n=49 for timepoint in Example 1.C.2), for OR, 3-month and 6-month OR rates 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. A similar trend towards improvement in long-term ORR and CR after 3 months was observed at higher doses. Specifically, for patients in the DL2-treated core cohort, the 3-month ORR 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 ORR rates and CR rates of 52% (11/21; 95% CI 30.74) and 33% (7/21; 95% CI 15.57) in individuals in the core cohort treated with DL1, with p=0.159 and p=0.0409, respectively. Among individuals in the core cohort treated with DL2 and 3-month follow-up (n=15), the 3-month ORR was 80% and the 3-month CR was 73%.

[0838] The median DOR in the full cohort and core cohort at this time point in 1.C.2 was 5.0 and 9.2 months, respectively; the median duration of CR presence was 9.2 months in the full cohort. The median duration of CR presence was not reached in the bovine cohort. Median overall survival (OS) was 13.7 months in the full cohort and was not achieved in the bovine cohort. 6-month OS was 75% in the full cohort, with a median follow-up of 5.8 months. 6-month OS was 88% in the bovine cohort, with a median follow-up of 5.6 months.

D. CAR score ⁺ T-cells in the blood

[0839] Based on data from the time point described in Example 6.A.1, 6.B.1, and 6.C.1, a pharmacokinetic analysis was performed to estimate the amounts of CAR ⁺ T cells in peripheral blood at various time points after treatment . Results were analyzed for fifty-five (55) individuals assessed at the time point in Example 6.A.1 in the DLBCL cohort and four (4) individuals (assessed at the same time point) in the mantle cell lymphoma (MCL) cohort. as described in example 7 below. Pharmacokinetic (PK) measurements were performed using validated flow cytometry to detect a marker expressed in the CAR construct and qPCR-based assays to detect integration of the CAR construct. B cell aplasia was assessed by flow cytometry using an anti-CD19 antibody. CD4 ⁺ and CD8 ⁺ expressing CAR cells, as measured by the number of cells/µl of blood (median ± quartiles) plotted on a logarithmic scale, were detected during the evaluation at both dose levels. Individuals treated with DL2 compared with DL1 had a higher median _Cmax. and median AUC _0–28 for subsets of CD3 ⁺ /CAR ⁺ , CD4 ⁺ /CAR ⁺ , and CD8 ⁺ /CAR ⁺ T cells in peripheral blood (AUC _0–28 : DL2 vs. DL1 was 1836 vs. 461,350 compared with 182 and 1628 compared with 114, for CD3 ⁺ , CD4 ⁺ and CD8 ⁺ , respectively; p<0.05 for CD8 ⁺ ; _Cmax : 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 expansion of CD3 ⁺ CAR ⁺ T cells was 15 days (range 8–29) and did not differ between dosing levels. CD4 ⁺ and CD8 ⁺ CAR-expressing T cells were homed in the bone marrow at relatively similar levels.

[0840] Increased median area under the curve (AUC) (numbers of CD8 ⁺ CAR ⁺ T cells versus time in the blood) was observed among individuals who were administered a higher dose level compared to a lower dose level, with no observed increase in toxicity . Higher peak CD8 ⁺ /CAR ⁺ T-cell exposure was observed in responders (CR/PR) than non-responders (PD); cell persistence over the evaluation time, including 3 and 6 months, was observed even in individuals whose disease progressed. Median C _max. and median AUC _0–28 CD8 ⁺ CAR ⁺ T cells were higher in responders and sustained response at month 3 (CD8 ⁺ _Cmax median=20.8 vs. 5.5; CD8 ⁺ AUC _0–28 median=235 versus 55 for CR/PR at month 3 versus PD at month 3). Among individuals assessed by CAR T cell persistence, 90% and 93% of 29 individuals had detectable CD8 ⁺ and CD4 ⁺ CAR ⁺ T cells, respectively, at month 3; Of 19 individuals, 63% and 58% 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 individuals with long-term response or relapse. CAR ⁺ T cells were detectable at the time of relapse in 89% of 11 individuals with PK, even though B cell aplasia (<1 cell/µl) was shown in almost all individuals, 97% (34/35) at month 3 and 100% (24/24) for month 6.

[0841] It was not observed that higher C _max. and AUC _0–28 for DL2, compared with DL1, are associated with an increase in CRS or NT. For any NT or for CRS > grade 2, the median AUCs for CD4 ⁺ /CAR ⁺ and CD8 ⁺ /CAR ⁺ T cells were 5–10-fold and 3–5-fold higher, respectively, than the median AUC for DL2. It has been observed that higher disease burden and baseline levels of pro-inflammatory cytokines are associated with higher peak CAR ⁺ T-cell levels, higher peak cytokine levels, and a higher incidence of CRS and NT. The results were consistent with the conclusion that higher Cmax. and median AUC0–28 for DL2 did not increase CRS or NT.

Example 7 Administration of Anti-CD19 CAR Expressing Cells to Individuals with Mantle Cell Lymphoma (MCL)

[0842] CAR+ T cell therapeutic compositions containing autologous T cells expressing a chimeric antigen receptor (CAR) specific for CD19 prepared as described in Example 1 were administered to four (4) human individuals with mantle cell lymphoma (MCL) who had failed 1st line therapy. Cryopreserved cell compositions were thawed prior to intravenous administration. The therapeutic T cell composition was administered in the form of a defined cell product composition using formulations of CD4+ and CD8+ modified CAR+ T cell populations derived from the same individual administered at a target ratio of approximately 1:1. Individuals were dosed with CAR expressing T cells (in the form of a fractional dose of CD4+ and CD8+ CAR expressing T cells) in a single dose with dose level 1 (DL1) containing 5×10 ⁷ CAR expressing T cells. Beginning three (3) days prior to CAR+ T cell infusion, individuals were subjected to anti-lymphocyte chemotherapy using fludarabine (flu, 30 mg/m2) and cyclophosphamide (Cy, 300 mg/m2).

[0843] Individuals were monitored for response and toxicities as described in Example 1. No CRS or neurotoxicity was observed in any of the individuals. Of the 4 treated individuals, two (2) individuals achieved PR (non-long term) and two (2) patients had progressive disease.

Example 8 Features of a T cell therapeutic composition to be administered and method of preparing the composition

[0844] Exemplary T-cell therapeutic compositions containing autologous T-cells expressing CD19-specific chimeric antigen receptor (CAR) used for administration in Example 2 above were evaluated for more than one hundred phenotypic, functional, and health-related cell traits , using flow cytometry and in vitro assays . Therapeutic compositions of cells derived from individuals enrolled in a clinical study evaluating anti-CD19 CAR T-cell therapy for the treatment of relapsed/refractory B-cell non-Hodgkin's lymphoma (N=63; core cohort) were evaluated. Cells were evaluated before and after modification, according to various features. The features that were evaluated include those described in Example 1 and illustrative evaluated features are listed in Table E7 . Phenotypic markers associated with cell health and memory cell phenotype and other cell product phenotypes were examined using flow cytometry. T cell functionality was assessed using in vitro antigen-specific biological assays. Characterization and testing for release was performed on samples from the formulated T cell therapeutic compositions that were subjected to a representative number of freeze-thaw cycles corresponding to the cycles for the administered cell compositions.

Table E7: Representative Traits Measured in Therapeutic Compositions of Cells Containing Anti-CD19 CAR T Cells Cell composition characterization class representative trait cell health status Viability Active intracellular caspase-3 Annexin V Memory cell phenotype CCR7 (C–C chemokine type 7 receptor) cell function Inflammatory cytokines such as TNF-α (tumor necrosis factor α)

[0845] To obtain cell compositions for administration, autologous cells were isolated from individuals by leukapheresis. Samples after leukapheresis were subjected to the method of obtaining expressing CAR cells. The method involved cell washing using automated washing and immunoaffinity selection to purify CD4+ and CD8+ T cells, resulting in two compositions enriched in CD8+ (in which, at a median of 99%, an interquartile range (IQR) of 98–100%, cells were CD8+) and CD4 ⁺ (in which at a median of 99%, IQR 99-100%, cells were CD4+) cells, respectively.

[0846] As shown in FIG. 24 and summarized in Table E8 , automated T cell purification resulted in the indicated purity of CD8+ and CD4+ T cell populations. In some embodiments, such aspects of purification may reduce the likelihood of transducing non-T cells at the transduction steps in the method and/or result in high purity of T cells in the therapeutic cell composition, in a manner independent of propagation duration or other aspects. propagation or incubation with T-cell-specific reagents.

Table E8: Purity of T-cells (% of total leukocytes) by method steps Leukapheresis After cleaning In the middle of the process The composition of the medicinal product CD4+ T cell frequency 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+ T cell frequency 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

[0847] Cells from enriched CD4 ⁺ and CD8 ⁺ compositions were individually subjected to lentiviral transduction using a vector encoding an anti-CD19 CAR with a 41BB costimulatory domain. The transduced populations were then individually incubated in the presence of stimulatory reagents to expand the cells. Expanded CD8+ and CD4+ cells were composed and cryopreserved separately and stored prior to administration. To minimize variability between batches and/or compositions of cells originating from different patients, such as patients having different patient characteristics, in parameters indicative of cell health, cells were maintained at constant volumes across batches. Cell products show a narrow range of viable cells (based on cell composition assessment for one group of individuals, CD8 ⁺ : median 31×10 ⁶ cells/mL, IQR 28–40×10 ⁶ cells/mL, N=38; CD4 ⁺ : median 35×10 ⁶ cells/ml, IQR 31–40×10 ⁶ , N=36).

[0848] At the site of administration, the cells were thawed and administered, according to the target volume of each formulation corresponding to the number of CD8+CAR+ and CD4+CAR+ cells at the appropriate dose (eg, for DL1 or DL2). To minimize variability in cell health between batches, formulating, cryopreservation and storage were maintained at a constant volume, with a narrow range of viable cell concentrations (CD8 ⁺ : median 31×10 ⁶ cells/mL, IQR 28–40×10 ⁶ cells/mL , N=38, CD4 ⁺ : median 35×10 ⁶ cells/mL, IQR 31–40×10 ⁶ , N=36).

[0849] During clinical studies, there was a change in the method from a high volume of the composition to a low volume of the composition. After the change, the therapeutic composition of the cells was made at a constant low volume, with a strictly controlled range of viable cell concentrations. In some cases, a low volume formulation was used instead of a high volume formulation. Parameters indicative of the health status of CAR-expressing T cells in administration formulations were assessed, for example, by measuring, after thawing, viability, cell surface expression of annexin V, and active intracellular caspase 3 levels. compiled with low volume.

[0850] Changing the method from a high volume of the composition to a low volume of the composition led to an increase in the reliability of the method and a decrease in the variability of signs of cell health. The values for the concentration, percentage of viable cells, and percentage of active caspase-3 negative cells among CD4+ and CD8+ T cells from individual cell compositions are shown in FIG. 25 and summarized in Table E9. The median percentage of Annexin V expressing cells 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. Switching to a low volume formulation resulted in increased method reliability and reduced variability in cell health traits.

Table E9: 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

• The numbers of CAR+CD4+ and CAR+CD8+ T cells in the administration formulation were precisely controlled. It was observed that the number of cells actually administered in an exemplary group of individuals was within 8% or less of the target cell number for a given dose: 2.4–2.7×10 ⁷ (target ±8%) CD4 ⁺ CAR ⁺ T– cells and 2.4–2.7×10 ⁷ (target ±8%) CD8 ⁺ CAR ⁺ T cells for individuals injected with cells at DL1 (n=48)

• 4.6–5.1×10 ⁷ (±8% target) CD4 ⁺ CAR ⁺ T cells or 4.6–5.1×10 ⁷ (±8% target) CD8 ⁺ CAR ⁺ T cells for individuals , which were injected with cells at DL2 (n=20).

[0851] The dose range administered was found to have low variability across exemplary groups of individuals:

– 48–52 ×10 ⁶ CD3+CAR+T cells at DL1 (n=34)

– 96–101×10 ⁶ CD3+CAR+T cells at DL2 (n=29)

– 24–27×10 ⁶ CD4+CAR+ or CD8+CAR+ T cells at DL1 (n=34)

– 46–51×10 ⁶ CD4+CAR+ or CD8+CAR+ T cells at DL2 (n=29).

[0852] As shown in FIG. 26 , it was observed that CAR-expressing T-cell compositions administered to individuals showed high T-cell purity and low batch-to-batch variability. Using method and product control, therapeutic cell compositions containing CAR-T cells with low inter-batch variability in cell-specific T cell functions were obtained.

[0853] Antigen-specific cytokine accumulation and intracellular cytokine (ICS) staining in vitro showed similar low variability between batches of cytokine production for a variety of cytokines (IL-2, TNF-α and IFN-γ). In an exemplary ICS experiment, cells from the formulations were stimulated with CD19, stained for cytokines including TNF-α and surface proteins including the C-C chemokine receptor type 7 (CCR7) as a memory cell 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. 27 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 presents compositions of CAR-T- cells to be injected at DL1 and DL2. These results are summarized in Table E10 . 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 narrow range was observed for the number of cells positive for TNF-α production (n=61).

Table E10: Controlled dose, T-cell phenotypes and cell-specific functions (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

[0854] The parameter indicative of tumor necrosis factor alpha (TNFα) production of CAR+ cells after stimulation with CD19 shows 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).

[0855] The results were consistent with the observation that a composition containing an accurate and uniform dose of CD4 ⁺ and CD8 ⁺ CAR-T cells, control and optimization of culture conditions for CD4 ⁺ and CD8 ⁺ T cells, low variability in cytokine production, and/or constant composition and the volume of the composition to be administered can result in a uniform health status of the cells in the composition. In the embodiments shown, aspects of such manufacturing and process control contribute to the low variability in features of such cell compositions constructed using cells from a number of different individuals and prepared for administration to a number of different individuals. Such aspects, in some aspects, include the use of an accurate, uniform fixed dose of administered CD4+ and CD8+ cells among individuals; control and optimization of culture conditions for CD4+ and CD8+ T cells, such as those resulting in low batch-to-batch variability in drug product phenotypes (eg, CCR7) and in vitro function (eg, production of IL-2, TNF-α, and IFN-γ after stimulation with antigen) for example, among different individuals; and the use of a constant composition and volume of the drug product, which can result in or contribute to uniformity among therapeutic compositions of cells obtained by this method for features indicative of cell health.

Example 9: Additional background factors associated with the development of neurotoxicity after CAR+ T-cell therapy

[0856] Various background factors measured prior to treatment with CAR-T cell compositions in a number of different clinical studies for B-cell malignancies such as ALL and NHL were evaluated and compared among individuals who developed and who did not Grade 3 neurotoxicity or higher has occurred. Data from 3 clinical trials were analyzed (including the trial described in Example 1 involving treatment with anti-CD19 CAR T cells in individuals with ALL (Study 1), the clinical trial described in Example 2 including similar treatment using CAR against CD19 in individuals with relapsed or refractory adult ALL (study 2) and additional clinical studies in which individuals with B-cell malignancies, including ALL and non-Hodgkin's lymphoma (NHL), were treated with cells expressing various CARs against CD19 (study 3)). Individuals were administered anti-CD19 CAR T cell compositions and monitored for clinical response and adverse outcomes. Neurotoxicity was graded on a scale of 0-5 as described in Example 1.

[0857] Background factors were analyzed post-treatment and compared among individuals using a series of statistical analyses. Exemplary background factors observed to be associated with grade 3 or higher neurotoxicity in individuals are listed in Tables E11-A and E11-B below, including serum IL-15 levels and platelet cell count before CAR-T cell administration, disease type (non-Hodgkin's lymphoma (NHL) versus ALL) and disease burden, as determined by the percentage of blast cells in the bone marrow. Gene expression profiles were also present among the parameters identified: individuals who were Philadelphia chromosome positive (Ph+) or who had a gene expression profile resembling a Ph+ gene expression profile (Ph-like) had significantly fewer cases of grade 3 or higher neurotoxicity than non-positive individuals. (non-Ph). Exemplary background factors associated with grade 3 or higher neurotoxicity in Study 1 are shown in Table E11-A. Exemplary background factors associated with grade 3 or greater neurotoxicity among three clinical studies are shown in Table E11-B.

Table E11–A: Exemplary Baseline Parameters Associated with Grade 3 or Higher Neurotoxicity in Study 1

background factor sign Criterion NTX degree > 3 p ^a value Marker of inflammation: IL-15 in plasma B-cell malignancies High IL- ^15b 10/11 (91%) 0.01 Low IL-15 10/24(42%) Gene Expression Profile ALL non-Ph 12/15 (80%) grade 4 or 5: 7 events 0.03 Ph+/Ph-like 6/16 (38%) grade 4 or 5: 0 events

Table E11-B: Exemplary Background Parameters Associated with Grade 3 or Higher Neurotoxicity

background factor sign Criterion NTX degree > 3 p ^a value Source Marker of inflammation: Serum IL-15 B-cell malignancies High IL- ^15b 26/45 (58%) <0.0001 Study 1 Low IL-15 19/109(17%) Study 3 Platelet count ALL <120K 60/162(37%) 0.002 Study 1 Study 2 >120K 9/60(15%) Study 3 Type of disease B-cell malignancies ALL 14/47(30%) 0.003 Study 3 NHL 8/62(13%) Disease load ALL ≥5% BM of blast cells 40/94(43%) 0.02 Study 1 Study 2 < 5% BM of blast cells 9/42(21%) Study 3 Gene Expression Profile ALL non-Ph 12/15 (80%) grade 5: 5 events 0.03 Study 1 Ph+/Ph-like 6/16 (38%) grade 5: no

^a Two-tailed Fisher exact test

^b High IL–15 > 30 pg/mL.

[0858] Blood samples collected from individuals at various time points after administration of CAR-T cells in Study 1 were analyzed for the number of CAR+-T cells. Cytokine levels, eg IL-15, in serum from individuals were assessed before treatment (screening) and at various time points after treatment (day 2, day 4 and day 7). The results showed that long-term and lethal neurotoxicity correlated with early and rapid increases in levels of IL-15, a T-cell stimulating cytokine, and with CAR-T cell proliferation.

[0859] As discussed in Example 1, faster proliferation of CAR T cells and earlier maximization of CAR expressing T cells were observed in the blood of individuals who continued to develop more severe neurotoxicity, and in particular those who developed neurotoxicity. grade 5 and cerebral edema. For example, the results showed that these individuals had a detectable level of CAR+ cells per µl of blood within 4-7 days after the first dose of cells, with more than 20 CAR+ cells per µl of blood observed in the blood of all such individuals within seven days after administration. cells (see FIG. 1 and 2). In individuals with less severe neurotoxicity, maximum proliferation of CAR+ T cells was generally observed later and with a lower maximum number of CAR+ T cells per microliter (FIG. 2, 28).

[0860] Among the cytokines, the median levels of which increased several times, compared with background levels, after infusion of CAR-T cells, there were IL-2 (14 times), IL-6 (2.7 times), IL-10 (3.5 times), IL-15 (2.5 times) and IFN-gamma (3.3 times). For the group of individuals who experienced earlier maximum CAR-T cell expansion (maximum CAR+-T cell counts by day 7), higher mean baseline serum IL-15 levels and an increased increase in serum 4 days after administration were observed, according to compared with individuals with a later maximum reproduction of CAR-T-cells (FIG. 28). By the next time point (Day 7) at which serum IL-15 levels were measured, the serum levels of IL-15 in these fast maximizing individuals had decreased and were approximately comparable to or lower than levels in individuals with later maxima. reproduction. The results were consistent with the conclusion that baseline serum IL-15 levels and/or a higher or faster increase in IL-15 levels may indicate or correlate with the potential risk of neurotoxicity or cerebral edema, or their severity, after treatment with CAR- T cells. It was also observed that cycles of antilymphocyte and transitional chemotherapy were also associated with increased levels of cytokines stimulating T cell proliferation, such as IL-15.

[0861] The present invention is not limited in scope to the specific embodiments described, which are presented, for example, to illustrate various aspects of the invention. Various modifications of the described compositions and methods are obvious from the description and explanations in the present description. Such variations may be practiced without departing from the scope and scope of the description, and are included within the scope of the present description.

SEQUENCES

SEQID NO. SUBSEQUENCE DESCRIPTION one ESKYGPPCPPCP spacer (IgG4 hinge) (AK), Homo sapiens 2 GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCT spacer (IgG4 hinge) (n.), homo sapiens 3 ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Spacer hinge–CH3, Homo sapiens four ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Hinge spacer–CH2–CH3, Homo sapiens 5 RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH IgD-hinge-Fc, Homo sapiens 6 LEGGGEGRGSLTCGDVEENPGPR T2A, artificial 7 RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM tEGFR, artificial eight FWVLVVVGGVLACYSLLVTVAFIFWV CD28 (amino acids 153-179 from Accession No. P10747), Homo sapiens 9 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV CD28 (amino acids 114-179 from Accession No. P10747), Homo sapiens ten RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 (amino acids 180–220 from P10747), Homo sapiens eleven RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 (LL to GG), Homo sapiens 12 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 4–1BB (amino acids 214–255 from Q07011.1), Homo sapiens 13 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3-zeta, Homo sapiens fourteen RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3-zeta, Homo sapiens fifteen RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN ELQKDKMAEA YSEIGMKGER RRGKGHDGLY QGLSTATKDTYDALHMQALP PR CD3-zeta, Homo sapiens 16 PGGG–(SGGGG)5–P– linker 17 GSADDAKKDAAKKDGKS linker eighteen EGRGSLLTCGDVEENPGP T2A 19 GSGATNFSLLKQAGDVEENPGP P2A twenty ATNFSLLKQAGDVEENPGP P2A 21 QCTNYALLKLAGDVESNPGP E2A 22 VKQTLNFDLLKLAGDVESNPGP F2A 23 MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM EGFRt, artificial 24 atgcttctcctggtgacaagccttctgctctgtgagtttaccacacccagcattcctcctgatccca GMCSFR alpha chain signal sequence 25 MLLLVTSLLLCELPHPAFLLIP GMCSFR alpha chain signal sequence 26 MALPVTALLLPLALLLHA signal peptide CD8-alpha 27 EPKSCDKTHTCPPCP Hinge 28 ERKCCVECPPCP Hinge 29 ELKTPLGDTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP Hinge thirty ESKYGPPCPSCP Hinge 31 _{X1PPX2P _}
X1 is glycine, cysteine or arginine
X2 is cysteine or threonine 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 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS 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 fifty EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTVTVSS SJ25C1 VH 51 DIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKR SJ25C1 VL 52 GGGGSGGGGSGGGGS Linker 53 EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKR SJ25C1 scFv 54 HYYYGGSYAMDY FMC63HC-CDR3 55 HTSRLHS FMC63LC-CDR2 56 QQGNTLPYT FMC63LC-CDR3 57 gacatccagatgacccagaccacctccagcctgagcgccagcctgggcgaccgggtgaccatcagctgccgggccagccaggacatcagcaagtacctgaactggtatcagcagaagcccgacggcaccgtcaagctgctgatctaccacaccagccggctgcacagcggcgtgcccagccggttметкуcggcagcggctccggcaccgactacagcctgaccatctccaacctggaacaggaagatatcgccacctacttttgccagcagggcaacacactgccctacacctttggcggcggaacaaagctggaaatcaccggcagcacctccggcagcggcaagcctggcagcggcgagggcagcaccaagggcgaggtgaagctgcaggaaagcggccctggcctggtggcccccagccagagcctgagcgtgacctgcaccgtgagcggcgtgagcctgcccgactacggcgtgagctggatccggcagccccccaggaagggcctggaatggctgggcgtgatctggggcagcgagaccacctactacaacagcgccctgaagagccggctgaccatcatcaaggacaacagcaagagccaggtgttcctgaagatgaacagcctgcagaccgacgacaccgccatctactactgcgccaagcactactactacggcggcagctacgccatggactactggggccagggcaccagcgtgaccgtgagcagc scFv encoding sequence 58 GSTSGSGKPGSGEGSTKG Linker

--->

SEQUENCE LIST

<110> JUNO THERAPUTICS, INC.

COVINGTON, Michael, Gerard

DAVE, Kedar, Himanshu

GHETTO, Richard, James, Jr.

KOWSKI, Tom

LARSON, Ryan, P.

RAMSBOG, Christopher, Glen

TREDE, Nikolaus, Sebastian

WEBER, Clinton

Whitmore, James, Boyd

YI, Nathan

BOSCHEN, Pascal

Beckett, Travis

BLACKMAN, Samuel, Charles

CHARTRAN, Nathaniel

DAVIS-PICKETT, Mel

Gilbert, Mark

LAMBERT, Nathaniel

LEE, He

MALLANY, Mary

Pollock, Katherine, Lindsey

ODEGARD, Valerie

SMITH, Jeff

SUZERLAND, Claire

WALKER, Andrew W.

<120> COMPOSITIONS, PRODUCTS AND METHODS RELATED TO DOSING

IN CELL THERAPY

<130> 735042009340

<150> 62/464371

<151> 2017-02-27

<150> 62/465817

<151> 2017-03-01

<150> 62/470180

<151> 2017-03-10

<150> 62/527002

<151> 2017-06-29

<150> 62/580416

<151> 2017-11-01

<150> 62/584740

<151> 2017-11-10

<150> 62/596703

<151> 2017-12-08

<160> 58

<170> PatentIn version 3.5

<210> 1

<211> 12

<212> Protein

<213> Homo sapiens

<220>

<221> undefined attribute

<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>

<221> undefined attribute

<223> spacer (IgG4 hinge)

<400> 2

gaatctaagt acggaccgcc ctgcccccct tgccct 36

<210> 3

<211> 119

<212> Protein

<213> Homo sapiens

<220>

<221> undefined attribute

<223> Spacer Hinge-CH3

<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>

<221> undefined attribute

<223> Spacer Hinge-CH2-CH3

<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>

<221> undefined attribute

<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

twenty

<210> 7

<211> 335

<212> Protein

<213> Artificial sequence

<220>

<223>tEGFR

<400> 7

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> 8

<211> 27

<212> Protein

<213> Homo sapiens

<220>

<221> undefined attribute

<223>CD28

<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>

<221> undefined attribute

<223>CD28

<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>

<221> undefined attribute

<223>CD28

<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>

<221> undefined attribute

<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>

<221> undefined attribute

<223> 4-1BB

<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>

<221> undefined attribute

<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>

<221> undefined attribute

<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>

<221> undefined attribute

<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> 30

<212> Protein

<213> Artificial sequence

<220>

<223> Linker sequence

<400> 16

Pro Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Pro

20 25 30

<210> 17

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> linker sequence

<400> 17

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

1 5 10 15

Ser

<210> 18

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> T2A

<400> 18

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

1 5 10 15

GlyPro

<210> 19

<211> 22

<212> Protein

<213> Artificial sequence

<220>

<223> P2A

<400> 19

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

twenty

<210> 20

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> P2A

<400> 20

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

1 5 10 15

Pro Gly Pro

<210> 21

<211> 20

<212> Protein

<213> Artificial sequence

<220>

<223>E2A

<400> 21

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

1 5 10 15

Asn Pro Gly Pro

twenty

<210> 22

<211> 22

<212> Protein

<213> Artificial sequence

<220>

<223> F2A

<400> 22

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

twenty

<210> 23

<211> 357

<212> Protein

<213> Artificial sequence

<220>

<223>tEGFR

<400> 23

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> 24

<211> 66

<212> DNA

<213> Homo sapiens

<220>

<221> undefined attribute

<223> GMCSFR alpha chain signal sequence

<400> 24

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcca 66

<210> 25

<211> 22

<212> Protein

<213> Homo sapiens

<220>

<221> undefined attribute

<223> GMCSFR alpha chain signal sequence

<400> 25

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

twenty

<210> 26

<211> 18

<212> Protein

<213> Homo sapiens

<220>

<221> undefined attribute

<223> signal peptide CD8-alpha

<400> 26

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

1 5 10 15

His Ala

<210> 27

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Hinge sequence

<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 sequence

<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 sequence

<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 sequence

<400> 30

Glu Ser Lys Tyr Gly Pro Cys Pro Ser Cys Pro

1 5 10

<210> 31

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Hinge sequence

<220>

<221> undefined attribute

<222> (1)..(1)

<223> glycine, cysteine or arginine

<220>

<221> undefined attribute

<222> (4)..(4)

<223> Xaa is cysteine or threonine

<400> 31

Xaa Pro Pro Xaa Pro

fifteen

<210> 32

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Hinge sequence

<400> 32

Tyr Gly Pro Pro Cys Pro Pro Cys Pro

fifteen

<210> 33

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Hinge sequence

<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 sequence

<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

fifteen

<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

fifteen

<210> 38

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> FMC63 CDR H1

<400> 38

Asp Tyr Gly Val Ser

fifteen

<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

fifteen

<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

fifteen

<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

fifteen

<210> 47

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> SJ25C1 CDR H1

<400> 47

Ser Tyr Trp Met Asn

fifteen

<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 sequence

<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 HC-CDR3

<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 LC-CDR2

<400> 55

His Thr Ser Arg Leu His Ser

fifteen

<210> 56

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> FMC63 LC-CDR3

<400> 56

Gln Gln Gly Asn Thr Leu Pro Tyr Thr

fifteen

<210> 57

<211> 735

<212> DNA

<213> Artificial sequence

<220>

<223> sequence encoding scFv

<400> 57

gacatccaga tgacccagac cacctccagc ctgagcgcca gcctgggcga ccgggtgacc 60

atcagctgcc gggccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

gacggcaccg tcaagctgct gatctaccac accagccggc tgcacagcgg cgtgcccagc 180

cggtttagcg gcagcggctc cggcaccgac tacagcctga ccatctccaa cctggaacag 240

gaagatatcg ccacctactt ttgccagcag ggcaacacac tgccctacac ctttggcggc 300

ggaacaaagc tggaaatcac cggcagcacc tccggcagcg gcaagcctgg cagcggcgag 360

ggcagcacca agggcgaggt gaagctgcag gaaagcggcc ctggcctggt ggcccccagc 420

cagagcctga gcgtgacctg caccgtgagc ggcgtgagcc tgcccgacta cggcgtgagc 480

tggatccggc agccccccag gaagggcctg gaatggctgg gcgtgatctg gggcagcgag 540

accacctact acaacagcgc cctgaagagc cggctgacca tcatcaagga caacagcaag 600

agccaggtgt tcctgaagat gaacagcctg cagaccgacg acaccgccat ctactactgc 660

gccaagcact actactacgg cggcagctac gccatggact actggggcca gggcaccagc 720

gtgaccgtga gcagc 735

<210> 58

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> Linker sequence

<400> 58

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr

1 5 10 15

Lys Gly

<---

Claims (35)

1. A method for determining the risk of developing toxicity in an individual, including an analysis of the number of cells expressing a recombinant receptor in a blood sample of an individual, wherein said individual has previously been dosed with cells expressing the recombinant receptor, where an individual is at risk of developing toxicity if: (i) no more than four days after the start of administration, the number of recombinant receptor-expressing cells in the individual's blood sample is at least about 2 recombinant receptor-expressing cells per microliter; (ii) no more than five or six days after the start of administration, the number of recombinant receptor-expressing cells in the individual's blood sample is at least about 5 recombinant receptor-expressing cells per microliter, or at least 10 recombinant receptor-expressing cells per microliter; or (iii) no more than seven days after the start of administration, the number of recombinant receptor-expressing cells in the individual's blood sample is at least about 15 recombinant receptor-expressing cells per microliter. 2. The method of claim 1, wherein the individual has previously been dosed with recombinant receptor-expressing cells in connection with the method of treating a disease or condition. 3. A method for determining the risk of developing toxicity in an individual, including: (a) administering to an individual suffering from the disease or condition a dose of cells expressing the recombinant receptor; and (b) after the administration of the cells, assaying the number of cells expressing the recombinant receptor in the individual's blood sample, where an individual is at risk of developing toxicity if: (i) no more than four days after the start of administration, the number of recombinant receptor-expressing cells in the individual's blood sample is at least about 2 recombinant receptor-expressing cells per microliter; (ii) no more than five or six days after the start of administration, the number of recombinant receptor-expressing cells in the individual's blood sample is at least about 5 recombinant receptor-expressing cells per microliter, or at least about 10 recombinant receptor-expressing cells per microliter; or (iii) no more than seven days after the start of administration, the number of recombinant receptor-expressing cells in the individual's blood sample is at least about 15 recombinant receptor-expressing cells per microliter. 4. The method according to any one of claims 1 to 3, wherein the assay comprises the use of a nucleic acid detection method or flow cytometry. 5. The method according to any one of claims 1 to 4, wherein the assay comprises using a detection method based on a quantitative polymerase chain reaction (qPCR). 6. The method according to any one of claims 1 to 5, wherein the individual is at risk of developing toxicity if, in addition, the level, amount or concentration of interleukin-15 (IL-15) detected in the individual's blood sample or serum is or exceeds 30 pg/ml. 7. The method of claim 6, wherein the sample is taken from an individual no more than about 1 day, 2 days, 3 days, 4 days, or 5 days after the start of the dose of recombinant receptor-expressing cells. 8. The method according to any one of claims 1 to 7, wherein the toxicity is neurotoxicity equal to or greater than grade 4 or grade 5, or at least long-term neurotoxicity grade 3. 9. The method according to any one of claims 1 to 7, wherein the toxicity is caused by or associated with cerebral edema. 10. The method of any one of claims 1 to 9, wherein the recombinant receptor is a chimeric antigen receptor (CAR). 11. The method of any one of claims 2-10, wherein the disease or condition is cancer. 12. The method of any one of claims 2-11, wherein the disease or condition is myeloma, lymphoma, or leukemia. 13. The method of any one of claims 2-12, wherein the disease or condition is a B cell cancer. 14. The method of claim 13, wherein the B-cell cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin's lymphoma (NHL), and diffuse large B-cell lymphoma ( DLBCL). 15. The method of any one of claims 1 to 14, wherein the recombinant receptor targets an antigen associated with a B cell cancer. 16. The method according to any one of claims 1 to 15, wherein the recombinant receptor targets an antigen that is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, kappa Ig, lambda Ig, CD79a, CD79b, or CD30. 17. The method according to any one of claims 1 to 16, wherein if it is determined that the individual is at risk of toxicity, the method further comprises stopping the administration of cells expressing the recombinant receptor, administering to the individual a lower dose of cells expressing the recombinant receptor, administering to the individual cells expressing another recombinant receptor; and/or administering to the individual an agent capable of treating, preventing, slowing down, or attenuating the development of toxicity. 18. The method of claim 17 wherein the agent is a steroid, a cytokine receptor antagonist or inhibitor, or a cytokine inhibitor. 19. The method of claim 18, wherein the cytokine receptor is an IL-6 receptor, a CD122 receptor (IL-2Rbeta receptor), or a CCR2. 20. The method according to claim 18 or 19, wherein the cytokine is IL-6, CP-1, IL-10, IFN-γ, IL-8, or IL-18. 21. The method of claim 18 wherein the steroid is a corticosteroid. 22. The method of claim 21 wherein the corticosteroid is a glucocorticoid. 23. The method of claim 22 wherein the glucocorticoid is dexamethasone. 24. The method according to any one of claims 17-20, wherein the agent is tocilizumab, siltuximab, sarilumab, olokizumab (CDP6038), elsilimoab, ALD518/BMS-945429, sirukumab (CNTO 136), CPSI-2634, ARGX-109, FE301 or FM101.

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