Treatment of multiple sclerosis using a population of CD19 car-expressing cells

Abstract

The invention provides methods of using a population of CD19 CAR-expressing cells (for example, rapcabtagene autoleucel) for treating neuroimmune diseases or disorders. Also disclosed are methods of making a population of CD19 CAR-expressing cells.

Description

[0001] PAT059856-PCT-SEC01

[0002] TREATMENT OF MULTIPLE SCLEROSIS USING A POPULATION OF CD19 CAR¬

[0003] EXPRESSING CELLS

[0004] FIELD OF THE INVENTION

[0005] The present invention relates generally to methods of using a population of CD 19 CAR- expressing cells for treating neuroimmune diseases or disorders, compositions comprising the same, and methods of making CD 19 CAR-expressing cells.

[0006] BACKGROUND OF THE INVENTION

[0007] Multiple sclerosis (MS) is a chronic, immune-mediated disease of the central nervous system (CNS) characterized by inflammation, demyelination, and axonal / neuronal destruction, ultimately leading to severe disability. MS is the most common neuroimmune demyelinating disorder of the CNS and is one of the leading causes of nontraumatic disability in young adults, affecting approximately 2.8 million individuals worldwide. MS typically affects young adults (mean age at onset 30 years), and women are affected more often then men. Traditionally, MS is categorized into relapsing remitting MS (RRMS), secondary progressive MS (SPMS) and primary progressive MS (PPMS). In addition, if the clinical symptoms are suggestive of MS, but not all criteria met, this is called clinically isolated syndrome (CIS) as the first occurrence of MS. The term relapsing MS (RMS) includes CIS, RRMS, and active SPMS where MS disease activity (relapse and MRI lesions) is prevalent. In progressive MS (PMS) active (with relapse or MRI activity) and non-active are differentiated. Although several immune therapies are licensed for RMS, currently, ocrelizumab remains as the only approved treatment for PPMS in both US and EU and is only moderately effective in reducing disability progression and thereby does not adequately control disease progression. Thus, there is a high unmet need for a treatment that addresses the underlying MS pathophysiology to slow or completely stop disease progression, without limiting safety risks.

[0008] SUMMARY OF THE INVENTION

[0009] Disclosed herein are methods of using a population of CD 19 CAR-expressing cells for treating a disease, for example, multiple sclerosis (e.g. progressive multiple sclerosis) in a PAT059856-PCT-SEC01 subject. The present disclosure also pertains to methods of making CD 19 CAR-expressing cells, and compositions generated using such methods.

[0010] In one aspect, the disclosure provides a method of treating a subject having an multiple sclerosis (e.g. progressive multiple sclerosis) in a subject, the method comprising administering to the subject a population of cells engineered to express a CD 19 CAR (“a population of CD 19 CAR-expressing cells”), said population comprising:

[0011] (a) about the same percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells, as compared to the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ cells, in the same population of cells prior to being engineered to express the CAR;

[0012] (b) a change within about 5% to about 10% of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells, for example, as compared to the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ cells, in the same population of cells prior to being engineered to express the CAR;

[0013] (c) an increased percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells, for example, increased by at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3-fold, as compared to the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ cells, in the same population of cells prior to being engineered to express the CAR;

[0014] (d) about the same percentage of central memory cells, for example, central memory T cells, for example, CD45RA- CCR7+ CD45RO+ T cells, as compared to the percentage of central memory cells, for example, central memory T cells, for example, CD45RA- CCR7+ CD45RO+ T cells, in the same population of cells prior to being engineered to express the CAR;

[0015] (e) a change within about 5% to about 10% of central memory cells, for example, central memory T cells, for example, CD45RA- CCR7+ CD45RO+ T cells, as compared to the percentage of central memory cells, for example, central memory T cells, for example, CD45RA- CCR7+ CD45RO+ T cells, in the same population of cells prior to being engineered to express the CAR;

[0016] (f) a decreased percentage of central memory cells, for example, central memory T cells, for example, CD45RA- CCR7+ CD45RO+ T cells, for example, decreased by at least 20, 25, 30, 35, 40, 45, or 50%, as compared to the percentage of central memory cells, for example, central PAT059856-PCT-SEC01 memory T cells, for example, CD45RA- CCR7+ CD45RO+ T cells, in the same population of cells prior to being engineered to express the CAR;

[0017] (g) about the same percentage of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, as compared to the percentage of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, in the same population of cells prior to being engineered to express the CAR;

[0018] (h) a change within about 5% to about 10% of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, as compared to the percentage of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, in the same population of cells prior to being engineered to express the CAR; or

[0019] (i) an increased percentage of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, as compared to the percentage of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, in the same population of cells prior to being engineered to express the CAR.

[0020] In one aspect, the disclosure provides a method of treating a subject having a neuroimmune disease, e.g., multiple sclerosis (e.g. progressive multiple sclerosis), the method comprising administering to the subject a population of cells engineered to express a CD 19 CAR (“a population of CD 19 CAR-expressing cells”), wherein:

[0021] (a) the median GeneSetScore (Up TEM vs. Down TSCM) of the population of cells is about the same as or differs by no more than (for example, increased by no more than) about 25, 50, 75, 100, or 125% from the median GeneSetScore (Up TEM vs. Down TSCM) of the same population of cells prior to being engineered to express the CAR;

[0022] (b) the median GeneSetScore (Up Treg vs. Down Teff) of the population of cells is about the same as or differs by no more than (for example, increased by no more than) about 25, 50, 100, 150, or 200% from the median GeneSetScore (Up Treg vs. Down Teff) of the population of cells prior to being engineered to express the CAR;

[0023] (c) the median GeneSetScore (Down sternness) of the population of cells is about the same as or differs by no more than (for example, increased by no more than) about 25, 50, 100, 150, 200, or 250% from the median GeneSetScore (Down sternness) of the population of cells prior to being engineered to express the CAR; PAT059856-PCT-SEC01

[0024] (d) the median GeneSetScore (Up hypoxia) of the population of cells is about the same as or differs by no more than (for example, increased by no more than) about 125, 150, 175, or 200% from the median GeneSetScore (Up hypoxia) of the population of cells prior to being engineered to express the CAR; or

[0025] (e) the median GeneSetScore (Up autophagy) of the population of cells is about the same as or differs by no more than (for example, increased by no more than) about 180, 190, 200, or 210% from the median GeneSetScore (Up autophagy) of the population of cells prior to being engineered to express the CAR.

[0026] In one aspect, the disclosure provides a method of treating a subject having multiple sclerosis (e.g. progressive multiple sclerosis), the method comprising administering to the subject rapcabtagene autoleucel.

[0027] In one aspect, the disclosure provides a method of treating a subject having MS (e.g., progressive multiple sclerosis (PMS)), the method comprising administering to the subject a population of CD 19 CAR-expressing cells in an amount sufficient to treat the multiple sclerosis. In some embodiments, the MS is PMS. In some embodiments, the population of CD19 CAR- expressing cells is rapcabtagene autoleucel.

[0028] In some embodiments, the population of CD 19 CAR-expressing cells is administered at a dose of about 0.5 x 106to 90 x 106viable CAR-expressing cells, optionally wherein the population of CD 19 CAR-expressing cells is administered at a dose of 7.5 x 106viable CAR- expressing cells, optionally wherein the population of CAR-expressing cells is administered at a dose of 5 x 106viable CAR-expressing cells, optionally wherein the population of CAR- expressing cells is administered at a dose of 2.5 x 106viable CAR-expressing cells. In some embodiments, the population of CD 19 CAR-expressing cells is rapcabtagene autoleucel.

[0029] In some embodiments, the population of CD 19 CAR-expressing cells is administered at a dose of about 2.5 x 106to 2.5 x 108viable CAR-expressing cells, for example, about 12.5 x 106viable CAR-expressing cells. In some embodiments, the population of CD19 CAR-expressing cells is rapcabtagene autoleucel.

[0030] In some embodiments, the population of CD 19 CAR-expressing cells is administered at a dose of about 12.5 x 106to 1.25 x 109viable CAR-expressing cells, for example, about 25 x 106viable CAR-expressing cells. In some embodiments, the population of CD19 CAR-expressing cells is rapcabtagene autoleucel. PAT059856-PCT-SEC01

[0031] In some embodiments, the population of CD 19 CAR-expressing cells is administered at a dose of about 2.5 x 106to 2.5 x 108viable CAR-expressing cells, for example, about 40 x 106viable CAR-expressing cells. In some embodiments, the population of CD19 CAR-expressing cells is rapcabtagene autoleucel.

[0032] In one aspect, the disclosure provides a population of CD 19 CAR-expressing cells or a pharmaceutical composition comprising the same for use in a method of modulating an immune response in a subject having MS (e.g., progressive multiple sclerosis (PMS)), said method comprising administering to the subject an effective amount of the population of CD 19 CAR- expressing cells or an effective amount of the pharmaceutical composition. In some embodiments, the population of CD 19 CAR-expressing cells is rapcabtagene autoleucel.

[0033] In one aspect, the disclosure provides a method of treating a subject having MS (e.g., progressive multiple sclerosis (PMS)), the method comprising administering to the subject: a population of cells that express, or comprise a nucleic acid configured to express, a CD 19 chimeric antigen receptor (CD 19 CAR), optionally wherein the population of cells is rapcabtagene autoleucel, and a second therapy, wherein the second therapy and CD 19 CAR cells are present in the subject at the same time, e.g., wherein the second therapy is administered at a time when the CD 19 CAR cells are present in the subject.

[0034] In one aspect, the disclosure provides rapcabtagene autoleucel or a pharmaceutical composition comprising the same for use in a method of modulating an immune response in a subject having multiple sclerosis (e.g. progressive multiple sclerosis), said method comprising administering to the subject an effective amount of the population of rapcaptagene autoleucel or an effective amount of the pharmaceutical composition.

[0035] In one aspect, the disclosure provides rapcabtagene autoleucel or a pharmaceutical composition comprising the same for use in a method of modulating an immune response in a subject having MS (e.g., progressive multiple sclerosis (PMS)), said method comprising administering to the subject an effective amount of rapcabtagene autoleucel or an effective amount of the pharmaceutical composition.

[0036] Rapcabtagene autoleucel for use in the manufacture of a medicament for treating a subject having multiple sclerosis (e.g. progressive multiple sclerosis). PAT059856-PCT-SEC01

[0037] Rapcabtagene autoleucel for use in the manufacture of a medicament for treating a subject having MS, e.g., progressive multiple sclerosis (PMS), wherein rapcabtagene autoleucel is formulated for administration in an amount sufficient to treat the PMS.

[0038] Rapcabtagene autoleucel for use in the manufacture of a medicament for treating a subject having MS, e.g., progressive multiple sclerosis (PMS), rapcabtagene autoleucel is formulated for administration at a dose of 0.5 - 50 x 106viable CAR+ T cells (e.g., 2.5-, 5-, 7.5-, 12.5-, 25-, 40 x 106viable CAR+ T cells).

[0039] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references (for example, sequence database reference numbers) mentioned herein are incorporated by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences referred to herein, for example, in any Table herein, are incorporated by reference. When one gene or protein references a plurality of sequence accession numbers, all of the sequence variants are encompassed.

[0040] In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Headings, sub-headings or numbered or lettered elements, for example, (a), (b), (i) etc., are presented merely for ease of reading. The use of headings or numbered or lettered elements in this document does not require the steps or elements be performed in alphabetical order or that the steps or elements are necessarily discrete from one another. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0041] BRIEF DESCRIPTION OF THE FIGURES

[0042] FIG. 1 is a schematic showing the clinical trial design for a phase 1 / 2 study, open-label, multi-center, to assess safety, efficacy and cellular kinetics of ARM-CD19 CAR T cells in participants with progressive MS.

[0043] FIG. 2 is a schematic showing the cohort design.

[0044] FIG. 3 is a schematic comparing the ARM process to a traditional CAR T manufacturing process. PAT059856-PCT-SEC01

[0045] DETAILED DESCRIPTION

[0046] Definitions

[0047] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

[0048] The term “a” and “an” refers to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0049] The term “about” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0050] The term “Chimeric Antigen Receptor” or alternatively a “CAR” refers to a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as “an intracellular signaling domain”) comprising a functional signaling domain derived from a stimulatory molecule as defined below. In some embodiments, the domains in the CAR polypeptide construct are in the same polypeptide chain, for example, comprise a chimeric fusion protein.

[0051] The term “autologous” refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.

[0052] The term “allogeneic” refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some embodiments, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.

[0053] The term “apheresis” as used herein refers to the art-recognized extracorporeal process by which the blood of a donor or patient is removed from the donor or patient and passed through an apparatus that separates out selected constituent(s) and returns the remainder to the circulation of PAT059856-PCT-SEC01 the donor or patient, for example, by re- transfusion. Thus, in the context of “an apheresis sample” refers to a sample obtained using apheresis.

[0054] “Immune effector cell,” as that term is used herein, refers to a cell that is involved in an immune response, for example, in the promotion of an immune effector response. Examples of immune effector cells include T cells, for example, alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

[0055] “Immune effector function or immune effector response,” as that term is used herein, refers to function or response, for example, of an immune effector cell, that enhances or promotes an immune attack of a target cell. For example, an immune effector function or response refers a property of a T or NK cell that promotes killing or the inhibition of growth or proliferation, of a target cell. In the case of a T cell, primary stimulation and costimulation are examples of immune effector function or response.

[0056] The term “effector function” refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.

[0057] The term “effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result.

[0058] The term “endogenous” refers to any material from or produced inside an organism, cell, tissue, or system.

[0059] The term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue, or system.

[0060] The term “expression” refers to the transcription and / or translation of a particular nucleotide sequence. In some embodiments, expression comprises translation of an mRNA introduced into a cell.

[0061] The term “lentivirus” refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. PAT059856-PCT-SEC01

[0062] The term “lentiviral vector” refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic, include but are not limited to, for example, the LENUVECTOR® gene delivery technology from Oxford BioMedica, the LENUMAX™ vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.

[0063] The term “parenteral” administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, intratumoral, or infusion techniques.

[0064] As used herein, “B cell antigen” refers to an antigen associated with a B cell. Nonlimiting examples of molecules associated with a B cell include proteins expressed on the surface of B cells, e.g. CD19, BCMA, CD22, CD20, CD10, CD34, CD123, FLT-3, ROR1, CD79b, CD 179b, or CD79a .

[0065] As used herein, the term “CD 19” refers to the Cluster of Differentiation 19 protein. The human and murine amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human CD19 can be found as UniProt / Swiss-Prot Accession No. Pl 5391 and the nucleic acid sequence encoding of the human CD19 can be found at Accession No. NM 001178098. It is also an early marker of B cell progenitors. See, e.g., Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). In one aspect the antigen-binding portion of the CART recognizes and binds an antigen within the extracellular domain of the CD 19 protein. In one aspect, the CD 19 protein is expressed on an autoreactive B-cell. As used herein, “CD 19” includes proteins comprising mutations, e.g., point mutations, fragments, insertions, deletions and splice variants of full length wild-type CD 19.

[0066] As used herein, the terms “treat”, “treatment” and “treating” refer to the reduction or amelioration of the progression, severity and / or duration of an neuroimmune disorder, or the amelioration of one or more symptoms (preferably, one or more discernible symptoms) of an neuroimmune disorder resulting from the administration of one or more therapies (for example, one or more therapeutic agents such as a CAR of the invention). In specific embodiments, the terms “treat,” “treatment,” and “treating” refer to the amelioration of at least one measurable PAT059856-PCT-SEC01 physical parameter of a neuroimmune disorder, such as autoantibodies or a brain lesion, not necessarily discernible by the patient. In other embodiments the terms “treat”, “treatment” and “treating” -refer to the inhibition of the progression of an neuroimmune disorder, either physically by, for example, stabilization of a discernible symptom, physiologically by, for example, stabilization of a physical parameter, or both.

[0067] The term “subject” is intended to include living organisms in which an immune response can be elicited (for example, mammals, for example, human).

[0068] The term “therapeutic” as used herein means a treatment. A therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.

[0069] The term “prophylaxis” as used herein means the prevention of or protective treatment for a disease or disease state.

[0070] “Neuroimmune disease” as used herein refers to a disease or disorder conditions characterized by the interaction between the nervous system and the immune system, leading to various symptoms and complications. These disease or disorders can result from an abnormal immune response, causing inflammation and damage to the nervous system, or from a dysfunction in the nervous system that affects immune function. An example of a neuroimmune disease is multiple sclerosis.

[0071] “Refractory” as used herein refers to a neuroimmune disease or disorder, for example, MS, which does not respond to a treatment. In embodiments, a refractory neuroimmune disease or disorder can be resistant to a treatment before or at the beginning of the treatment. In other embodiments, the refractory neuroimmune disease or disorder can become resistant during a treatment.

[0072] “Relapsed” or “relapse” as used herein refers to the return or reappearance of a disease (for example, an neuroimmune disease or disorder) or the signs and symptoms of a disease such as an neuroimmune disease or disorder after a period of improvement or responsiveness, for example, after prior treatment of a therapy, for example, standard of care therapy. The initial period of responsiveness may involve the level of autoantibodies cells falling below a certain threshold. The reappearance may involve the level of autoantibodies rising above a certain threshold.

[0073] “Remission” as used herein refers to a decrease in or disappearance of signs and symptoms of a disease (for example, a neuroimmune disease or disorder). Remission may be PAT059856-PCT-SEC01 partial or complete. In partial remission, some, but not all, signs and symptons of a disease have decreased or disappeared. In complete remission, all signs and symptoms of a disease have disappeared. Remission may be determined according to a defined set of criteria established for a particular disease (for example, an neuroimmune disease or disorder).

[0074] Ranges: throughout this disclosure, various embodiments of the invention can be presented in a 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 invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity, includes something with 95%, 96%, 97%, 98%, or 99% identity, and includes subranges such as 96-99%, 96-98%, 96- 97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the breadth of the range.

[0075] Administered “in combination”, as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, for example, the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery”. In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, for example, an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, PAT059856-PCT-SEC01 or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0076] As used herein, a “naive T cell” refers to a T cell that is antigen-inexperienced. In some embodiments, an antigen-inexperienced T cell has encountered its cognate antigen in the thymus but not in the periphery. In some embodiments, naive T cells are precursors of memory cells. In some embodiments, naive T cells express both CD45RA and CCR7, but do not express CD45RO. In some embodiments, naive T cells may be characterized by expression of CD62L, CD27, CCR7, CD45RA, CD28, and CD127, and the absence of CD95 or CD45RO isoform. In some embodiments, naive T cells express CD62L, IL-7 receptor-a, IL-6 receptor, and CD 132, but do not express CD25, CD44, CD69, or CD45RO. In some embodiments, naive T cells express CD45RA, CCR7, and CD62L and do not express CD95 or IL-2 receptor 0. In some embodiments, surface expression levels of markers are assessed using flow cytometry.

[0077] The term “central memory T cells” refers to a subset of T cells that in humans are CD45RO positive and express CCR7. In some embodiments, central memory T cells express CD95. In some embodiments, central memory T cells express IL-2R, IL-7R, and / or IL-15R. In some embodiments, central memory T cells express CD45RO, CD95, IL-2 receptor 0, CCR7, and CD62L. In some embodiments, surface expression levels of markers are assessed using flow cytometry.

[0078] The term “stem memory T cells,” “stem cell memory T cells,” “stem cell-like memory T cells,” “memory stem T cells,” “T memory stem cells,” “T stem cell memory cells,” or “TSCM cells” refers to a subset of memory T cells with stem cell-like ability, for example, the ability to self-renew and / or the multipotent capacity to reconstitute memory and / or effector T cell subsets. In some embodiments, stem memory T cells express CD45RA, CD95, IL-2 receptor 0, CCR7, and CD62L. In some embodiments, surface expression levels of markers are assessed using flow cytometry. In some embodiments, exemplary stem memory T cells are disclosed in Gattinoni et al., Nat Med. 2017 January 06; 23(1): 18-27, herein incorporated by reference in its entirety.

[0079] For clarity purposes, unless otherwise noted, classifying a cell or a population of cells as “not expressing,” or having an “absence of’ or being “negative for” a particular marker may not necessarily mean an absolute absence of the marker. The skilled artisan can readily compare the cell against a positive and / or a negative control, and / or set a predetermined threshold, and classify the cell or population of cells as not expressing or being negative for the marker when PAT059856-PCT-SEC01 the cell has an expression level below the predetermined threshold or a population of cells has an overall expression level below the predetermined threshold using conventional detection methods, e.g., using flow cytometry.

[0080] As used herein, the term “GeneSetScore (Up TEM vs. Down TSCM)” of a cell refers to a score that reflects the degree at which the cell shows an effector memory T cell (TEM) phenotype vs. a stem cell memory T cell (TSCM) phenotype. A higher GeneSetScore (Up TEM vs. Down TSCM) indicates an increasing TEM phenotype, whereas a lower GeneSetScore (Up TEM vs. Down TSCM) indicates an increasing TSCM phenotype. In some embodiments, the GeneSetScore (Up TEM vs. Down TSCM) is determined by measuring the expression of one or more genes that are up-regulated in TEM cells and / or down-regulated in TSCM cells, for example, one or more genes selected from the group consisting of MXRA7, CLIC1, NAT13, TBC1D2B, GLCCI1, DUSP10, APOBEC3D, CACNB3, ANXA2P2, TPRG1, EOMES, MATK, ARHGAP10, ADAM8, MAN1A1, SLFN12L, SH2D2A, EIF2C4, CD58, MY01F, RAB27B, ERN1, NPC1, NBEAL2, APOBEC3G, SYTL2, SLC4A4, PIK3AP1, PTGDR, MAF, PLEKHA5, ADRB2, PLXND1, GNA01, THBS1, PPP2R2B, CYTH3, KLRF1, FLJ16686, AUTS2, PTPRM, GNLY, and GFPT2. In some embodiments, the GeneSetScore (Up TEM vs. Down TSCM) is determined for each cell using RNA-seq, for example, single-cell RNA-seq (scRNA-seq), for example, as exemplified of WO / 2020 / 047452 in Example 10 with respect to FIG. 39A, hereby incorporated by reference in its entirety. In some embodiments, the GeneSetScore (Up TEM vs. Down TSCM) is calculated by taking the mean log normalized gene expression value of all of the genes in the gene set.

[0081] As used herein, the term “GeneSetScore (Up Treg vs. Down Teff)” of a cell refers to a score that reflects the degree at which the cell shows a regulatory T cell (Treg) phenotype vs. an effector T cell (Teff) phenotype. A higher GeneSetScore (Up Treg vs. Down Teff) indicates an increasing Treg phenotype, whereas a lower GeneSetScore (Up Treg vs. Down Teff) indicates an increasing Teff phenotype. In some embodiments, the GeneSetScore (Up Treg vs. Down Teff) is determined by measuring the expression of one or more genes that are up-regulated in Treg cells and / or down-regulated in Teff cells, for example, one or more genes selected from the group consisting of C12orf75, SELPLG, SWAP70, RGS1, PRR11, SPATS2L, SPATS2L, TSHR, C14orfl45, CASP8, SYT11, ACTN4, ANXA5, GLRX, HLA-DMB, PMCH, RAB11FIP1, IL32, FAM160B1, SHMT2, FRMD4B, CCR3, TNFRSF13B, NTNG2, CLDND1, BARD1, FCER1G, PAT059856-PCT-SEC01

[0082] TYMS, ATP1B1, GJB6, FGL2, TK1, SLC2A8, CDKN2A, SKAP2, GPR55, CDCA7, S100A4, GDPD5, PMAIP1, AC0T9, CEP55, SGMS1, ADPRH, AKAP2, HDAC9, IKZF4, CARD17, VAV3, 0BFC2A, ITGB1, CIITA, SETD7, HLA-DMA, CCR10, KIAA0101, SLC14A1, PTTG3P, DUSP1O, FAM164A, PYHIN1, MYO1F, SLC1A4, MYBL2, PTTG1, RRM2, TP53INP1, CCR5, ST8SIA6, TOX, BFSP2, ITPRIPL1, NCAPH, HLA-DPB2, SYT4, NINJ2, FAM46C, CCR4, GBP5, C15orf53, LMCD1, MKI67, NUSAP1, PDE4A, E2F2, CD58, ARHGEF12, LOC100188949, FAS, HLA-DPB1, SELP, WEE1, HLA-DPA1, FCRL1, ICA1, CNTNAP1, OAS1, METTL7A, CCR6, HLA-DRB4, ANXA2P3, STAM, HLA-DQB2, LGALS1, ANXA2, PI 16, DUSP4, LAYN, ANXA2P2, PTPLA, ANXA2P1, ZNF365, LAIR2, LOC541471, RASGRP4, BCAS1, UTS2, MIAT, PRDM1, SEMA3G, FAM129A, HPGD, NCF4, LGALS3, CEACAM4, JAKMIP1, TIGIT, HLA-DRA, IKZF2, HLA-DRB1, FANK1, RTKN2, TRIBI, FCRL3, and FOXP3. In some embodiments, the GeneSetScore (Up Treg vs. Down Teff) is determined using RNA-seq, for example, single-cell RNA-seq (scRNA-seq) , for example, as exemplified of WO / 2020 / 047452 in Example 10 with respect to FIG. 39B, hereby incorporated by reference in its entirety. In some embodiments, the GeneSetScore (Up Treg vs. Down Teff) is calculated by taking the mean log normalized gene expression value of all of the genes in the gene set.

[0083] As used herein, the term “GeneSetScore (Down sternness)” of a cell refers to a score that reflects the degree at which the cell shows a sternness phenotype. A lower GeneSetScore (Down sternness) indicates an increasing sternness phenotype. In some embodiments, the GeneSetScore (Down sternness) is determined by measuring the expression of one or more genes that are upregulated in a differentiating stem cell vs downregulated in a hematopoietic stem cell, for example, one or more genes selected from the group consisting of ACE, BATF, CDK6, CHD2, ERCC2, HOXB4, ME0X1, SFRP1, SP7, SRF, TALI, and XRCC5. In some embodiments, the GeneSetScore (Down sternness) is determined using RNA-seq, for example, single-cell RNA-seq (scRNA-seq) , for example, as exemplified of WO / 2020 / 047452 in Example 10 with respect to FIG. 39C, hereby incorporated by reference in its entirety. In some embodiments, the GeneSetScore (Down sternness) is calculated by taking the mean log normalized gene expression value of all of the genes in the gene set.

[0084] As used herein, the term “GeneSetScore (Up hypoxia)” of a cell refers to a score that reflects the degree at which the cell shows a hypoxia phenotype. A higher GeneSetScore (Up PAT059856-PCT-SEC01 hypoxia) indicates an increasing hypoxia phenotype. In some embodiments, the GeneSetScore (Up hypoxia) is determined by measuring the expression of one or more genes that are up- regulated in cells undergoing hypoxia, for example, one or more genes selected from the group consisting of ABCB1, ACAT1, ADM, AD0RA2B, AK2, AK3, ALDH1A1, ALDH1A3, ALDOA, ALDOC, ANGPT2, ANGPTL4, ANXA1, ANXA2, ANXA5, ARHGAP5, ARSE, ART1, BACE2, BATF3, BCL2L1, BCL2L2, BHLHE40, BHLHE41, BIK, BIRC2, BNIP3, BNIP3L, BPI, BTG1, Cllorf2, C7orf68, CA12, CA9, CALD1, CCNG2, CCT6A, CD99, CDK1, CDKN1A, CDKN1B, CITED2, CLK1, CNOT7, COL4A5, COL5A1, COL5A2, COL5A3, CP, CTSD, CXCR4, D4S234E, DDIT3, DDIT4, 1-Dec, DKC1, DR1, EDN1, EDN2, EFNA1, EGF, EGR1, EIF4A3, ELF3, ELL2, ENG, EN01, EN03, ENPEP, EPO, ERRFI1, ETS1, F3, FABP5, FGF3, FKBP4, FLT1, FN1, FOS, FTL, GAPDH, GBE1, GLRX, GPI, GPRC5A, HAP1, HBP1, HDAC1, HDAC9, HERC3, HERPUD1, HGF, HIF1A, HK1, HK2, HLA-DQB1, HM0X1, HM0X2, HSPA5, HSPD1, HSPH1, HY0U1, ICAM1, ID2, IFI27, IGF2, IGFBP1, IGFBP2, IGFBP3, IGFBP5, IL6, IL8, INSIGI, IRF6, ITGA5, JUN, KDR, KRT14, KRT18, KRT19, LDHA, LDHB, LEP, LGALS1, LONP1, LOX, LRP1, MAP4, MET, MIF, MMP13, MMP2, MMP7, MPI, MT1L, MTL3P, MUC1, MXI1, NDRG1, NFIL3, NFKB1, NFKB2, NOS1, NOS2, NOS2P1, NOS2P2, NOS3, NR3C1, NR4A1, NT5E, ODC1, P4HA1, P4HA2, PAICS, PDGFB, PDK3, PFKFB1, PFKFB3, PFKFB4, PFKL, PGAM1, PGF, PGK1, PGK2, PGM1, PIM1, PIM2, PKM2, PLAU, PLAUR, PLIN2, PLOD2, PNN, PNP, POLM, PPARA, PPAT, PR0K1, PSMA3, PSMD9, PTGS1, PTGS2, QSOX1, RBPJ, RELA, RI0K3, RNASEL, RPL36A, RRP9, SAT1, SERPINB2, SERPINE1, SGSM2, SIAH2, SIN3A, SIRPA, SLC16A1, SLC16A2, SLC20A1, SLC2A1, SLC2A3, SLC3A2, SLC6A10P, SLC6A16, SLC6A6, SLC6A8, SORL1, SPP1, SRSF6, SSSCA1, STC2, STRA13, SYT7, TBPL1, TCEAL1, TEK, TF, TFF3, TFRC, TGFA, TGFB1, TGFB3, TGFBI, TGM2, TH, THBS1, THBS2, TIMM17A, TNFAIP3, TP53, TPBG, TPD52, TPI1, TXN, TXNIP, UMPS, VEGFA, VEGFB, VEGFC, VIM, VPS11, and XRCC6. In some embodiments, the GeneSetScore (Up hypoxia) is determined using RNA-seq, for example, single-cell RNA-seq (scRNA-seq) , for example, as exemplified of WO / 2020 / 047452 in Example 10 with respect to FIG. 39D, hereby incorporated by reference in its entirety. In some embodiments, the GeneSetScore (Up hypoxia) is calculated by taking the mean log normalized gene expression value of all of the genes in the gene set. PAT059856-PCT-SEC01

[0085] As used herein, the term “GeneSetScore (Up autophagy)” of a cell refers to a score that reflects the degree at which the cell shows an autophagy phenotype. A higher GeneSetScore (Up autophagy) indicates an increasing autophagy phenotype. In some embodiments, the GeneSetScore (Up autophagy) is determined by measuring the expression of one or more genes that are up-regulated in cells undergoing autophagy, for example, one or more genes selected from the group consisting of ABL1, ACBD5, ACINI, ACTRT1, ADAMTS7, AKR1E2, ALKBH5, ALPK1, AMBRA1, ANXA5, ANXA7, ARSB, ASB2, ATG10, ATG12, ATG13, ATG14, ATG16L1, ATG16L2, ATG2A, ATG2B, ATG3, ATG4A, ATG4B, ATG4C, ATG4D, ATG5, ATG7, ATG9A, ATG9B, ATP13A2, ATP1B1, ATPAF1-AS1, ATPIF1, BECN1, BECN1P1, BLOC1S1, BMP2KL, BNIP1, BNIP3, BOC, Cllorf2, Cl lorf41, C12orf44, C12orf5, C14orfl33, Clorf210, C5, C6orfl06, C7orf59, C7orf68, C8orf59, C9orf72, CA7, CALCB, CALCOCO2, CAPS, CCDC36, CD163L1, CD93, CDC37, CDKN2A, CHAF1B, CHMP2A, CHMP2B, CHMP3, CHMP4A, CHMP4B, CHMP4C, CHMP6, CHST3, CISD2, CLDN7, CLEC16A, CLN3, CLVS1, C0X8A, CPA3, CRNKL1, CSPG5, CTSA, CTSB, CTSD, CXCR7, DAP, DKKL1, DNAAF2, DPF3, DRAM1, DRAM2, DYNLL1, DYNLL2, DZANK1, EI24, EIF2S1, EPG5, EPM2A, FABP1, FAM125A, FAM131B, FAM134B, FAM13B, F AMI 76 A, FAM176B, FAM48A, FANCC, FANCF, FANCL, FBX07, FCGR3B, FGF14, FGF7, FGFBP1, FIS1, FNBP1L, F0X01, FUNDCI, FUNDC2, FXR2, GAB ARAP, GABARAPL1, GABARAPL2, GABARAPL3, GABRA5, GDF5, GMIP, HAP1, HAPLN1, HBXIP, HCAR1, HDAC6, HGS, HIST1H3A, HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E, HIST1H3F, HIST1H3G, HIST1H3H, HIST1H3I, HIST1H3J, HK2, HMGB1, HPR, HSF2BP, HSP90AA1, HSPA8, IFI16, IPPK, IRGM, IST1, ITGB4, ITPKC, KCNK3, KCNQ1, KIAA0226, KIAA1324, KRCC1, KRT15, KRT73, LAMP1, LAMP2, LAMT0R1, LAMT0R2, LAMT0R3, LARP1B, LENG9, LGALS8, LIX1, LIX1L, LMCD1, LRRK2, LRSAM1, LSM4, MAP1A, MAP1LC3A, MAP1LC3B, MAP1LC3B2, MAP1LC3C, MAP1S, MAP2K1, MAP3K12, MARK2, MBD5, MDH1, MEX3C, MFN1, MFN2, MLST8, MRPS10, MRPS2, MSTN, MTERFD1, MTMR14, MTMR3, MTOR, MTSS1, MYH11, MYLK, MY0M1, NBR1, NDUFB9, NEFM, NHLRC1, NME2, NPC1, NR2C2, NRBF2, NTHL1, NUP93, OBSCN, OPTN, P2RX5, PACS2, PARK2, PARK7, PDK1, PDK4, PEX13, PEX3, PFKP, PGK2, PHF23, PHYHIP, PI4K2A, PIK3C3, PIK3CA, PIK3CB, PIK3R4, PINK1, PLEKHM1, PLOD2, PNPO, PPARGC1A, PPY, PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, PRKAG3, PAT059856-PCT-SEC01

[0086] PRKD2, PRKG1, PSEN1, PTPN22, RAB12, RAB1A, RAB1B, RAB23, RAB24, RAB33B, RAB39, RAB7A, RB1CC1, RBM18, REEP2, REP15, RFWD3, RGS19, RHEB, RIMS3, RNF185, RNF41, RPS27A, RPTOR, RRAGA, RRAGB, RRAGC, RRAGD, S100A8, S100A9, SCN1A, SERPINB10, SESN2, SFRP4, SH3GLB1, SIRT2, SLC1A3, SLC1A4, SLC22A3, SLC25A19, SLC35B3, SLC35C1, SLC37A4, SLC6A1, SLCO1A2, SMURF1, SNAP29, SNAPIN, SNF8, SNRPB, SNRPB2, SNRPD1, SNRPF, SNTG1, SNX14, SPATAI 8, SQSTM1, SRPX, STAM, STAM2, STAT2, STBD1, STK11, STK32A, STOM, STX12, STX17, SUPT3H, TBC1D17, TBC1D25, TBC1D5, TCIRG1, TEAD4, TECPR1, TECPR2, TFEB, TM9SF1, TMBIM6, TMEM203, TMEM208, TMEM39A, TMEM39B, TMEM59, TMEM74, TMEM93, TNIK, TOLLIP, TOMM20, TOMM22, TOMM40, T0MM5, T0MM6, T0MM7, TOMM70A, TP53INP1, TP53INP2, TRAPPC8, TREM1, TRIM17, TRIM5, TSG101, TXLNA, UBA52, UBB, UBC, UBQLN1, UBQLN2, UBQLN4, ULK1, ULK2, ULK3, USP10, USP13, USP30, UVRAG, VAMP7, VAMP8, VDAC1, VMP1, VPS11, VPS16, VPS18, VPS25, VPS28, VPS33A, VPS33B, VPS36, VPS37A, VPS37B, VPS37C, VPS37D, VPS39, VPS41, VPS4A, VPS4B, VTA1, VTT1A, VTI1B, WDFY3, WDR45, WDR45L, WIPI1, WIPI2, XBP1, YIPF1, ZCCHC17, ZFYVE1, ZKSCAN3, ZNF189, ZNF593, and ZNF681. In some embodiments, the GeneSetScore (Up autophagy) is determined using RNA-seq, for example, single-cell RNA-seq (scRNA-seq) , for example, as exemplified of WO / 2020 / 047452 in Example 10 with respect to FIG. 39E, hereby incorporated by reference in its entirety. In some embodiments, the GeneSetScore (Up autophagy) is calculated by taking the mean log normalized gene expression value of all of the genes in the gene set.

[0087] As used herein, the term “GeneSetScore (Up resting vs. Down activated)” of a cell refers to a score that reflects the degree at which the cell shows a resting T cell phenotype vs. an activated T cell phenotype. A higher GeneSetScore (Up resting vs. Down activated) indicates an increasing resting T cell phenotype, whereas a lower GeneSetScore (Up resting vs. Down activated) indicates an increasing activated T cell phenotype. In some embodiments, the GeneSetScore (Up resting vs. Down activated) is determined by measuring the expression of one or more genes that are up-regulated in resting T cells and / or down- regulated in activated T cells, for example, one or more genes selected from the group consisting of ABCA7, ABCF3, ACAP2, AMT, ANKH, ATF7IP2, ATG14, ATP1A1, ATXN7, ATXN7L3B, BCL7A, BEX4, BSDC1, BTG1, BTG2, BTN3A1, Cllorf21, C19orf22, C21orf2, CAMK2G, CARS2, CCNL2, CD248, PAT059856-PCT-SEC01

[0088] CD5, CD55, CEP164, CHKB, CLK1, CLK4, CTSL1, DBP, DCUN1D2, DENND1C, DGKD, DLG1, DUSP1, EAPP, ECE1, ECHDC2, ERBB2IP, FAM117A, FAM134B, FAM134C, F AMI 69 A, FAM190B, FAU, FLJ10038, F0XJ2, F0XJ3, F0XL1, FOXO1, FXYD5, FYB, HLA-E, HSPA1L, HYAL2, ICAM2, IFIT5, IFITM1, IKBKB, IQSEC1, IRS4, KIAA0664L3, KIAA0748, KLF3, KLF9, KRT18, LEF1, LINC00342, LIPA, LIPT1, LLGL2, LMBR1L, LPAR2, LTBP3, LYPD3, LZTFL1, MANBA, MAP2K6, MAP3K1, MARCH8, MAU2, MGEA5, MMP8, MPO, MSL1, MSL3, MYH3, MYLIP, NAGPA, NDST2, NISCH, NICER, NLRP1, NOSIP, NPIP, NUMA1, PAIP2B, PAPD7, PBXIP1, PCIF1, PI4KA, PLCL2, PLEKHA1, PLEKHF2, PNISR, PPFIBP2, PRKCA, PRKCZ, PRKD3, PRMT2, PTP4A3, PXN, RASA2, RASA3, RASGRP2, RBM38, REPIN1, RNF38, RNF44, R0R1, RPL30, RPL32, RPLP1, RPS20, RPS24, RPS27, RPS6, RPS9, RXRA, RYK, SCAND2, SEMA4C, SETD1B, SETD6, SETX, SF3B1, SH2B1, SLC2A4RG, SLC35E2B, SLC46A3, SMAGP, SMARCE1, SMPD1, SNPH, SP140L, SPATA6, SPG7, SREK1IP1, SRSF5, STAT5B, SVIL, SYF2, SYNJ2BP, TAF1C, TBC1D4, TCF20, TECTA, TES, TMEM127, TMEM159, TMEM30B, TMEM66, TMEM8B, TP53TG1, TPCN1, TRIM22, TRIM44, TSC1, TSC22D1, TSC22D3, TSPYL2, TTC9, TTN, UBE2G2, USP33, USP34, VAMP1, VILL, VIPR1, VPS13C, ZBED5, ZBTB25, ZBTB40, ZC3H3, ZFP161, ZFP36L1, ZFP36L2, ZHX2, ZMYM5, ZNF136, ZNF148, ZNF318, ZNF350, ZNF512B, ZNF609, ZNF652, ZNF83, ZNF862, and ZNF91. In some embodiments, the GeneSetScore (Up resting vs. Down activated) is determined using RNA-seq, for example, single-cell RNA-seq (scRNA-seq) , for example, as exemplified of WO / 2020 / 047452 in Example 10 with respect to FIG. 38D, hereby incorporated by reference in its entirety. In some embodiments, the GeneSetScore (Up resting vs. Down activated) is calculated by taking the mean log normalized gene expression value of all of the genes in the gene set.

[0089] As used herein, the term “GeneSetScore (Progressively up in memory differentiation)” of a cell refers to a score that reflects the stage of the cell in memory differentiation. A higher GeneSetScore (Progressively up in memory differentiation) indicates an increasing late memory T cell phenotype, whereas a lower GeneSetScore (Progressively up in memory differentiation) indicates an increasing early memory T cell phenotype. In some embodiments, the GeneSetScore (Up autophagy) is determined by measuring the expression of one or more genes that are up-regulated during memory differentiation, for example, one or more genes selected PAT059856-PCT-SEC01 from the group consisting of MTCH2, RAB6C, KIAA0195, SETD2, C2orf24, NRD1, GNA13, COP A, SELT, TNIP1, CBFA2T2, LRP10, PRKCI, BRE, ANKS1A, PNPLA6, ARL6IP1, WDFY1, MAPK1, GPR153, SHKBP1, MAP1LC3B2, PIP4K2A, HCN3, GTPBP1, TLN1, C4orf34, KIF3B, TCIRG1, PPP3CA, ATG4D, TYMP, TRAF6, C17orf76, WIPF1, FAM108A1, MYL6, NRM, SPCS2, GGT3P, GALK1, CLIP4, ARL4C, YWHAQ, LPCAT4, ATG2A, IDS, TBC1D5, DMPK, ST6GALNAC6, REEP5, ABHD6, KIAA0247, EMB, TSEN54, SPIRE2, PIWIL4, ZSCAN22, ICAM1, CHD9, LPIN2, SETD8, ZC3H12A, ULBP3, IL15RA, HLA- DQB2, LCP1, CHP, RUNX3, TMEM43, REEP4, MEF2D, ABL1, TMEM39A, PCBP4, PLCD1, CHST12, RASGRP1, Clorf58, Cl lorf63, C6orfl29, FH0D1, DKFZp434F142, PIK3CG, ITPR3, BTG3, C4orf50, CNNM3, IFI16, AK1, CDK2AP1, REL, BCL2L1, MVD, TTC39C, PLEKHA2, FKBP11, EML4, FANCA, CDCA4, FUCA2, MFSD10, TBCD, CAPN2, IQGAP1, CHST11, PIK3R1, MY05A, KIR2DL3, DLG3, MXD4, RALGDS, S1PR5, WSB2, CCR3, TIP ARP, SP140, CD151, SOX13, KRTAP5-2, NF1, PEA15, PARP8, RNF166, UEVLD, LIMK1, CACNB1, TMX4, SLC6A6, LBA1, SV2A, LLGL2, IRF1, PPP2R5C, CD99, RAPGEF1, PPP4R1, OSBPL7, FOXP4, SLA2, TBC1D2B, ST7, JAZF1, GGA2, PI4K2A, CD68, LPGAT1, STX11, ZAK, FAM160B1, RORA, C8orf80, APOBEC3F, TGFBI, DNAJC1, GPR114, LRP8, CD69, CMIP, NAT13, TGFBI, FLJ00049, ANTXR2, NR4A3, IL12RB1, NTNG2, RDX, MLLT4, GPRIN3, ADCY9, CD300A, SCD5, ABB, PTPN22, LGALS1, SYTL3, BMPR1A, TBK1, PMAIP1, RASGEF1A, GCNT1, GABARAPL1, STOM, CALHM2, ABCA2, PPP1R16B, SYNE2, PAM, C12orf75, CLCF1, MXRA7, APOBEC3C, CLSTN3, ACOT9, HIP1, LAG3, TNFAIP3, DCBLD1, KLF6, CACNB3, RNF19A, RAB27A, FADS3, DLG5, APOBEC3D, TNFRSF1B, ACTN4, TBKBP1, ATXN1, ARAP2, ARHGEF12, FAM53B, MAN1A1, FAM38A, PLXNC1, GRLF1, SRGN, HLA-DRB5, B4GALT5, WIPI1, PTPRJ, SLFN11, DUSP2, ANXA5, AHNAK, NEO1, CLIC1, EIF2C4, MAP3K5, IL2RB, PLEKHG1, MY06, GTDC1, EDARADD, GALM, TARP, ADAM8, MSC, HNRPLL, SYT11, ATP2B4, NHSL2, MATK, ARHGAP18, SLFN12L, SPATS2L, RAB27B, PIK3R3, TP53INP1, MB0AT1, GYG1, KATNAL1, FAM46C, ZC3HAV1L, ANXA2P2, CTNNA1, NPC1, C3AR1, CRIM1, SH2D2A, ERN1, YPEL1, TBX21, SLC1A4, FASLG, PHACTR2, GALNT3, ADRB2, PIK3AP1, TLR3, PLEKHA5, DUSP10, GNAO1, PTGDR, FRMD4B, ANXA2, EOMES, CADM1, MAF, TPRG1, NBEAL2, PPP2R2B, PELO, SLC4A4, KLRF1, FOSL2, RGS2, TGFBR3, PRF1, MY01F, GAB3, C17orf66, MICAL2, CYTH3, TOX, HLA-DRA, SYNE1, PAT059856-PCT-SEC01

[0090] WEE1, PYHIN1, F2R, PLD1, THBS1, CD58, FAS, NETO2, CXCR6, ST6GALNAC2, DUSP4, AUTS2, Clorf21, KLRG1, TNIP3, GZMA, PRR5L, PRDM1, ST8SIA6, PLXND1, PTPRM, GFPT2, MYBL1, SLAMF7, FLJ16686, GNLY, ZEB2, CST7, IL18RAP, CCL5, KLRD1, and KLRBE In some embodiments, the GeneSetScore (Progressively up in memory differentiation) is determined using RNA-seq, for example, as exemplified of WO / 2020 / 047452 in Example 10 with respect to FIG. 40B, hereby incorporated by reference in its entirety. In some embodiments, the GeneSetScore (Progressively up in memory differentiation) is calculated by taking the mean log normalized gene expression value of all of the genes in the gene set.

[0091] As used herein, the term “GeneSetScore (Up TEM vs. Down TN)” of a cell refers to a score that reflects the degree at which the cell shows an effector memory T cell (TEM) phenotype vs. a naive T cell (TN) phenotype. A higher GeneSetScore (Up TEM vs. Down TN) indicates an increasing TEM phenotype, whereas a lower GeneSetScore (Up TEM vs. Down TN) indicates an increasing TN phenotype. In some embodiments, the GeneSetScore (Up TEM vs. Down TN) is determined by measuring the expression of one or more genes that are up-regulated in TEM cells and / or down-regulated in TN cells, for example, one or more genes selected from the group consisting of MY05A, MXD4, STK3, S1PR5, GLCCI1, CCR3, SOX13, KRTAP5-2, PEA15, PARP8, RNF166, UEVLD, LIMK1, SLC6A6, SV2A, KPNA2, OSBPL7, ST7, GGA2, PI4K2A, CD68, ZAK, RORA, TGFBI, DNAJC1, JOSD1, ZFYVE28, LRP8, OSBPL3, CMIP, NAT13, TGFBI, ANTXR2, NR4A3, RDX, ADCY9, CHN1, CD300A, SCD5, PTPN22, LGALS1, RASGEF1A, GCNT1, GLUL, ABCA2, CLDND1, PAM, CLCF1, MXRA7, CLSTN3, ACOT9, METRNL, BMPR1A, LRIG1, APOBEC3G, CACNB3, RNF19A, RAB27A, FADS3, ACTN4, TBKBP1, FAM53B, MAN1A1, FAM38A, GRLF1, B4GALT5, WIPI1, DUSP2, ANXA5, AHNAK, CLIC1, MAP3K5, ST8SIA1, TARP, ADAM8, MATK, SLFN12L, PIK3R3, FAM46C, ANXA2P2, CTNNA1, NPC1, SH2D2A, ERN1, YPEL1, TBX21, STOM, PHACTR2, GBP5, ADRB2, PIK3AP1, DUSP10, PTGDR, EOMES, MAF, TPRG1, NBEAL2, NCAPH, SLC4A4, FOSL2, RGS2, TGFBR3, MYO1F, C17orf66, CYTH3, WEE1, PYHIN1, F2R, THBS1, CD58, AUTS2, FAM129A, TNIP3, GZMA, PRR5L, PRDM1, PLXND1, PTPRM, GFPT2, MYBL1, SLAMF7, ZEB2, CST7, CCL5, GZMK, and KLRB1. In some embodiments, the GeneSetScore (Up TEM vs. Down TN) is determined using RNA-seq, for example, singlecell RNA-seq (scRNA-seq) , for example, as exemplified of WO / 2020 / 047452 in Example 10 with respect to FIG. 40C, hereby incorporated by reference in its entirety. In some PAT059856-PCT-SEC01 embodiments, the GeneSetScore (Up TEM vs. Down TN) is calculated by taking the mean log normalized gene expression value of all of the genes in the gene set.

[0092] In the context of GeneSetScore values (e.g., median GeneSetScore values), when a positive GeneSetScore is reduced by 100%, the value becomes 0. When a negative GeneSetScore is increased by 100%, the value becomes 0. For example, as disclosed in WO / 2020 / 047452, the median GeneSetScore of the Dayl sample is -0.084; the median GeneSetScore of the Day9 sample is 0.035; and the median GeneSetScore of the input sample is -0.1. In WO / 2020 / 047452 in FIG. 39A, increasing the median GeneSetScore of the input sample by 100% leads to a GeneSetScore value of 0; and increasing the median GeneSetScore of the input sample by 200% leads to a GeneSetScore value of 0.1. In WO / 2020 / 047452 in FIG. 39A, decreasing the median GeneSetScore of the Day9 sample by 100% leads to a GeneSetScore value of 0; and decreasing the median GeneSetScore of the Day9 sample by 200% leads to a GeneSetScore value of -0.035.

[0093] Various embodiments of the compositions and methods herein are described in further detail below. Additional definitions are set out throughout the specification.

[0094] CD19 CAR-Expressing Cells

[0095] The present disclosure provides CAR-expressing cell compositions and their use in medicaments or methods for treating, among other diseases, multiple sclerosis (e.g. progressive multiple sclerosis) involving cells or tissues which express an antigen as described herein. In some embodiments, provided herein are pharmaceutical compositions comprising a CAR- expressing cell, for example, a plurality of CD 19 CAR-expressing cells, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. In some embodiments, the CD 19 CAR-expressing cells is rapcabtagene autoleucel.

[0096] In some embodiments, the antigen-binding domain of the CD 19 CAR has the same or a similar binding specificity as the FMC63 scFv fragment described in Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). In some embodiments, the antigen-binding domain of the CD19 CAR includes the scFv fragment described in Nicholson et al. Mol. Immun. 34 (16- 17): 1157-1165 (1997). In some embodiments, the CD19 CAR includes an antigen-binding domain (for example, a humanized antigen-binding domain) according to Table 3 of PAT059856-PCT-SEC01

[0097] WO2014 / 153270, incorporated herein by reference. In some embodiments, the CD19 CAR comprises an amino acid sequence provided as SEQ ID NO: 12 in PCT publication WO2012 / 079000, incorporated herein by reference.

[0098] In some embodiments, the population of CAR T cells that specifically bind to CD 19 comprises rapcabtagene autoleucel. The rapcabtagene autoleucel is made using autologous T cells obtained from peripheral blood mononuclear cells from a subject (e.g., from a subject having an neuroimmune disease or disorder) by leukapheresis and subsequently transduced with a self-inactivating, non-replicating lentiviral vector encoding a T cell chimeric antigen receptor targeting CD 19. The expressed transgene comprises a CD8a leader sequence, a murine anti- CD19 single chain variable fragment (scFv) derived from the mouse hybridoma FMC63, a CD8a hinge and transmembrane region, and a 4- IBB (CD 137) and CD3(j (TCRQ signaling domain, and is under control of the elongation factor 1 alpha (EFla) promoter. The construct is flanked by 5' and 3' long terminal repeats (LTRs) and also contains a \| / packaging signal, a Rev response element (RRE), a central polypurine tract (cPPT) sequence, and an optimized Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). The leukapheresis material is enriched for CD4 / CD8 T cells by positive immunoselection, activated by CD3 and CD28 agonists and transduced with the vector. Without further cell propagation, the T cells are washed, formulated for infusion, and cryopreserved. Rapcabtagene autoleucel is composed of >80% T cells and <1% B cells, with a mixture of transgene positive (>3.4%) and negative T cells. The CD4+ and CD8+ naive T cell subsets (CD45RA+CCR7+) present in the leukapheresis material are largely retained. In some embodiments, the rapcabtagene autoleucel is made by the ARM manufacturing process described herein.

[0099] In some embodiments, the CD19 CAR-expressing cell population has:

[0100] (a) about the same percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells, as compared to the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ cells, in the same population of cells prior to being engineered to express the CAR;

[0101] (b) a change within about 5% to about 10% of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells, for example, as compared to the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ cells, in the same population of cells prior to being engineered to express the CAR; PAT059856-PCT-SEC01

[0102] (c) an increased percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells, for example, increased by at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3-fold, as compared to the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ cells, in the same population of cells prior to being engineered to express the CAR;

[0103] (d) about the same percentage of central memory cells, for example, central memory T cells, for example, CD45RA- CCR7+ CD45RO+ T cells, as compared to the percentage of central memory cells, for example, central memory T cells, for example, CD45RA- CCR7+ CD45RO+ T cells, in the same population of cells prior to being engineered to express the CAR;

[0104] (e) a change within about 5% to about 10% of central memory cells, for example, central memory T cells, for example, CD45RA- CCR7+ CD45RO+ T cells, as compared to the percentage of central memory cells, for example, central memory T cells, for example, CD45RA- CCR7+ CD45RO+ T cells, in the same population of cells prior to being engineered to express the CAR;

[0105] (f) a decreased percentage of central memory cells, for example, central memory T cells, for example, CD45RA- CCR7+ CD45RO+ T cells, for example, decreased by at least 20, 25, 30, 35, 40, 45, or 50%, as compared to the percentage of central memory cells, for example, central memory T cells, for example, CD45RA- CCR7+ CD45RO+ T cells, in the same population of cells prior to being engineered to express the CAR;

[0106] (g) about the same percentage of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, as compared to the percentage of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, in the same population of cells prior to being engineered to express the CAR;

[0107] (h) a change within about 5% to about 10% of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, as compared to the percentage of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, in the same population of cells prior to being engineered to express the CAR; or

[0108] (i) an increased percentage of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, as compared to the percentage of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, in the same population of cells prior to being engineered to express the CAR. PAT059856-PCT-SEC01

[0109] In some embodiments, the CD 19 CAR-expressing population of cells made via the ARM process described herein shows a higher percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells (for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50% higher), compared with cells made by an otherwise similar method which lasts, for example, more than 26 hours (for example, which lasts more than 5, 6, 7, 8, 9, 10, 11, or 12 days) or which involves expanding the population of cells in vitro for, for example, more than 3 days (for example, expanding the population of cells in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days).

[0110] In some embodiments, the CD 19 CAR-expressing population of cells made via the ARM process described herein shows a lower percentage of central memory cells, for example, central memory T cells, for example, CD95+ central memory T cells (for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50% lower), compared with cells made by an otherwise similar method which lasts, for example, more than 26 hours (for example, which lasts more than 5, 6, 7, 8, 9, 10, 11, or 12 days) or which involves expanding the population of cells in vitro for, for example, more than 3 days (for example, expanding the population of cells in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days).

[0111] In some embodiments, the CD 19 CAR-expressing population of cells made via the ARM process described herein after being administered in vivo, persists longer or expands at a higher level (for example, at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% higher), compared with cells made by an otherwise similar method which lasts, for example, more than 26 hours (for example, which lasts more than 5, 6, 7, 8, 9, 10, 11, or 12 days) or which involves expanding the population of cells in vitro for, for example, more than 3 days (for example, expanding the population of cells in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days).

[0112] Any known CD 19 CAR, for example, the CD 19 antigen-binding domain of any known CD 19 CAR, in the art can be used in accordance with the present disclosure. Examples include tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, lisocabtagene maraleucel, relmacabtagene autoleucel, CC-97540, AT101, CABA-201, KYV-101, IMPT-514, or MB- CART19.1. Further exemplary CD 19 CARs include CD 19 CARs described herein or an antiCD F CAR described in Xu et al. Blood 123.24(2014):3750-9; Kochenderfer et al. Blood 122.25(2013):4129-39, Cruz et al. Blood 122.17(2013):2965-73, NCT00586391, NCT01087294, NCT02456350, NCT00840853, NCT02659943, NCT02650999, NCT02640209, NCT01747486, PAT059856-PCT-SEC01

[0113] NCT02546739, NCT02656147, NCT02772198, NCT00709033, NCT02081937, NCT00924326, NCT02735083, NCT02794246, NCT02746952, NCT01593696, NCT02134262, NCT01853631, NCT02443831, NCT02277522, NCT02348216, NCT02614066, NCT02030834, NCT02624258, NCT02625480, NCT02030847, NCT02644655, NCT02349698, NCT02813837, NCT02050347, NCT01683279, NCT02529813, NCT02537977, NCT02799550, NCT02672501, NCT02819583, NCT02028455, NCT01840566, NCT01318317, NCT01864889, NCT02706405, NCT01475058, NCT01430390, NCT02146924, NCT02051257, NCT02431988, NCT01815749, NCT02153580, NCT01865617, NCT02208362, NCT02685670, NCT02535364, NCT02631044, NCT02728882, NCT02735291, NCT01860937, NCT02822326, NCT02737085, NCT02465983, NCT02132624, NCT02782351, NCT01493453, NCT02652910, NCT02247609, NCT01029366, NCT01626495, NCT02721407, NCT01044069, NCT00422383, NCT01680991, NCT02794961, or NCT02456207, NCT05338931, NCT05869955, NCT06056921, NCT06106906, NCT06121297, NCT06152172, NCT06189157, NCT05459870, NCT06153095, each of which is incorporated herein by reference in its entirety.

[0114] Methods of Treating

[0115] The present application discloses methods of treating method of treating a subject having multiple sclerosis (e.g. progressive multiple sclerosis) the method comprising administering to the subject a population of cells engineered to express a CD 19 CAR (“a population of CD 19 CAR-expressing cells”). In some embodiments, the method further comprises identifying the subject as a responder or a non-responder, and / or one who has achieved remission, based on a measure of one or more of the parameters of efficacy disclosed herein. In some embodiments, the measure of one or more of the parameters disclosed herein is obtained from a sample acquired from the subject. In some embodiments of any of the methods disclosed herein, the subject is evaluated prior to receiving, during, or after receiving, the CD 19 CAR-expressing cell therapy. In some embodiments, the CD 19 CAR-expressing cell therapy is rapcabtagene autoleucel.

[0116] In some embodiments, the identification of a subject as a responder, non-responder and / or one who has achieved remission, following administration of the CD 19 CAR-expressing cells according to the methods described herein can be determined by evaluating the subject according to clinical criteria. Efficacy may be evaluated, for example, using the Expanded PAT059856-PCT-SEC01

[0117] Disability Status Scale (EDSS), magnetic resonance imaging (MRI), Symbol Digit Modalities Test (SDMT), Modified Fatigue Impact Scale (MFIS), Short Form Health Survey (SF-36 v2), 9 Hole Peg Test (9HPT), or Timed 25 Foot Walk (T25FW). The above criteria are explained in further detail below. EDSS provides a total score on a scale that ranges from 0 to 10. The first levels 1.0 to 4.5 refer to people with a high degree of ambulatory ability and the subsequent levels 5.0 to 9.5 refer to the progressive loss of ambulatory ability.

[0118] Table 1: Expanded Disability Status Scale Scores PAT059856-PCT-SEC01

[0119] In addition, the EDSS also provides eight subscale measurements called Functional System (FS) scores. The levels of function within each category refer to the eight functional systems affected by MS. PAT059856-PCT-SEC01

[0120] 1 . Pyramidal (motor function) (P)

[0121] 2. Cerebellar (C11)

[0122] 3. Brainstem (BS)

[0123] 4. Sensory (S)

[0124] 5 Bowel and Bladder (BB)

[0125] 6. Visual (V)

[0126] 7. Cerebral or Mental (Cb)

[0127] 3. Other (O)

[0128] The FS are scored on a scale of 0 (low level of problems) to 5 or 6 (high level of problems) to best reflect the level of disability observed clinically. The “Other” category consists of any other neurologic findings attributed to MS and is dichotomous, with 0 as none and 1 as any present.

[0129] In contrast, the total EDSS score is determined by two factors: gait and FS scores. EDSS scores below 4.0 are determined by the FS scores alone. People with EDSS scores of 4.0 and above may have some degree of gait impairment. Scores between 4.0 and 9.5 are determined by both gait abilities and the FS scores. For simplicity, many experts gauge the EDSS scores between 4.0 and 9.5 entirely by gait, without considering the FS scores. The EDSS is widely used and accepted as a valid tool to clinically measure and evaluate MS patients’ level of functioning. An improvement can be defined as a decrease in the EDSS score of at least 1 point for a baseline EDSS less than 5.5, or of at least 0.5 points for baseline EDSS greater than or equal to 5.5.

[0130] In some embodiments, a subject is identified as showing improvement as measured by a lowering of the EDSS score from baseline following administration of the CD19 CAR- expressing cells (e.g., rapcabtag ene autoleucel) to the subject according to the methods described herein. In some embodiments, the baseline EDSS score of the patient is the EDSS score measured prior to the first administration of the CD 19 CAR-expressing cells. In some embodiments, the baseline EDSS score of the patient is the first EDSS score measured after administration of the CD 19 CAR-expressing cells.

[0131] In some embodiments, the subject’s EDSS score dose not worsen. In some embodiments, the subject’s EDSS score is lowered by at least 0.5 points. In another embodiment, the subject’s EDSS score is lowered by at least 1.0 points. In another embodiment, the subject’s EDSS score is lowered by at least 1.5 points. In another embodiment, the subject’s EDSS score is lowered by PAT059856-PCT-SEC01 at least 2.0 points. In some embodiments, the subject’s EDSS score is lowered by at least 2.5 points. In another embodiment, the subject’s EDSS score is lowered by at least 3.0 points. In another embodiment, the subject’s EDSS score is lowered by at least 3.5 points. In another embodiment, the subject’s EDSS score is lowered by at least 4.0 points. In some embodiments, the subject’s EDSS score is lowered by at least 4.5 points. In another embodiment, the subject’s EDSS score is lowered by at least 5.0 points. In another embodiment, the subject’s EDSS score is lowered by at least 5.5 points. In another embodiment, the subject’s EDSS score is lowered by at least 6.0 points. In some embodiments, the subject’s EDSS score is lowered by at least 6.5 points. In another embodiment, the subject’s EDSS score is lowered by at least 7.0 points. In another embodiment, the subject’s EDSS score is lowered by at least 7.5 points. In another embodiment, the subject’s EDSS score is lowered by at least 8.0 points. In some embodiments, the subject’s EDSS score is lowered by at least 8.5 points. In another embodiment, the subject’s EDSS score is lowered by at least 9.0 points. In another embodiment, the subject’s EDSS score is lowered by at least 9.5 points.

[0132] In some embodiments, a subject is identified as showing improvement as measured by a lowering of the EDSS score from baseline following administration of the CD19 CAR- expressing cells (e.g., rapcabtag ene autoleucel) to the subject according to the methods described herein. In some embodiments, the lowering of the EDSS score is achieved within 3 months of administration. In some embodiments, lowering of the EDSS score is achieved within 6 months of administration. In some embodiments, the lowering of the EDSS score is achieved within 9 months of administration. In some embodiments, lowering of the EDSS score is achieved within 12 months of administration. In some embodiments, the lowering of the EDSS score is achieved within 18 months of administration. In some embodiments, lowering of the EDSS score is achieved within 24 months of administration.

[0133] In some embodiments, the lowering of the EDSS score from baseline achieved by a subject following administration of the CD 19 CAR-expressing cells (e.g., rapcabtag ene autoleucel) is sustained for 3 months or more. In some embodiments, lowering of the EDSS score is sustained for 6 months or more. In some embodiments lowering of the EDSS score is sustained for 9 months or more. In some embodiments, the lowering of the EDSS score is sustained for 12 months or more. In some embodiments, the lowering of the EDSS score is sustained for 18 months or more. In some embodiments, the lowering of the EDSS score is PAT059856-PCT-SEC01 sustained for 24 months or more. In some embodiments, the lowering of the EDSS score is sustained indefinitely.

[0134] In some embodiments, a subject is identified as achieving a stable disease state as measured by no worsening of the EDSS score from baseline following administration of the CD19 CAR-expressing cells (e.g., rapcabtagene autoleucel) to the subject according to the methods described herein. In some embodiments, the EDSS score does not increase from the baseline EDSS score following administration of the CD19 CAR-expressing cells. In some embodiments, the baseline EDSS score of the patient is the EDSS score measured prior to the first administration of the CD 19 CAR-expressing cells. In some embodiments, the baseline EDSS score of the patient is the first EDSS score measured after administration of the CD19 CAR-expressing cells.

[0135] In some embodiments, a subject is identified as achieving a stable disease state as measured by no worsening of the EDSS score from baseline following administration of the CD19 CAR-expressing cells (e.g., rapcabtagene autoleucel) to the subject according to the methods described herein. In some embodiments, the EDSS score does not increase during the first 3 months following administration. In some embodiments, the EDSS score does not increase during the first 6 months following administration. In some embodiments, the EDSS score does not increase during the first 9 months following administration. In some embodiments, the EDSS score does not increase during the first 12 months following administration. In some embodiments, the EDSS score does not increase during the first 18 months following administration. In some embodiments, the EDSS score does not increase during the first 24 months following administration.

[0136] In some embodiments, the EDSS score of the patient does not increase from baseline following administration of the CD 19 CAR-expressing cells (e.g., rapcabtagene autoleucel) for at least 3 months. In some embodiments, the EDSS score does not increase for at least 6 months. In some embodiments, the EDSS score does not increase for at least 9 months. In some embodiments, the EDSS score does not increase for at least 12 months. In some embodiments, the EDSS score does not increase for at least 15 months. In some embodiments, the EDSS score does not increase for at least 18 months. In some embodiments, the EDSS score does not increase for at least 24 months. In some embodiments, the EDSS score of the patient remains stable indefinitely. PAT059856-PCT-SEC01

[0137] Magnetic resonance imaging (MRI) is currently the most sensitive, noninvasive way of imaging the brain, spinal cord or other areas of the body. It is the preferred imaging method to help establish an MS diagnosis and to monitor disease progression. MRI uses magnetic fields and radio waves to measure the relative water content in the body’s tissues. Because the layer of myelin that protects nerve cell fibers is fatty, it repels water. In the areas where the myelin has been damaged by MS, the fat is stripped away. With the fat gone, the area holds more water and shows up on an MRI scan as either a bright white spot or a darkened area depending on the type of scan used.

[0138] Various types of MRI scans are used to test for and track MS. Sometimes gadolinium, a contrast agent, is injected into the vein during an MRI to help detect areas of new inflammation. Because gadolinium is a large molecule, it normally cannot pass through the blood-brain barrier, which prevents substances from passing from the bloodstream into the central nervous system. However, active inflammation can disrupt the blood-brain barrier. Then gadolinium can enter and highlight the inflamed areas.

[0139] Common MRI sequences used in MS include:

[0140] • T-l weighted without gadolinium — may show dark areas (hypointensities) thought to indicate areas of permanent nerve damage

[0141] • T-l weighted with gadolinium — may show bright areas (enhancing lesions) that indicate areas of active inflammation

[0142] • T-2 weighted — shows overall disease burden or lesion load (meaning the total number of lesions, both old and new)

[0143] • Fluid attenuated inversion recovery (FLAIR) — shows MS activity by reducing interference from the spinal fluid

[0144] In some embodiments, the disease activity of a subject following administration of the CD 19 CAR-expressing cells (e.g., rapcabtagene autoleucel) is improved as determined by a reduction in the size and number of brain lesions as detected by MRI. In some embodiments, there are no new or enlarged T2 lesions. In some embodiments, there are no new or enlarged gadolinium-enhanced lesions. In some embodiments, the presence of new lesions and / or the size of existing lesions is detected by comparing an MRI image taken prior to administration of the CD 19 CAR-expression cells to an MRI taken after administration of the CD 19 CAR-expression cells. PAT059856-PCT-SEC01

[0145] In some embodiments, the improved disease activity of a subject following administration of the CD 19 CAR-expressing cells (e.g., rapcabtagene autoleucel) as determined by a reduction in the size and number of brain lesions as detected by an MRI is sustained for 3 months or more. In some embodiments, improved disease activity is sustained for 6 months or more. In some embodiments improved disease activity is sustained for 9 months or more. In some embodiments, the improved disease activity is sustained for 12 months or more. In some embodiments, the improved disease activity is sustained for 18 months or more. In some embodiments, the improved disease activity is sustained for 24 months or more. In some embodiments, the improved disease activity is sustained indefinitely.

[0146] In some embodiments, the patient exhibits a stable disease state following administration of the CD19 CAR-expressing cells (e.g., rapcabtagene autoleucel) as determined by no new or enlarged T2 lesions as detected by MRI. In some embodiments, no new or enlarged T2 lesions are detected for at least 3 months. In some embodiments, no new or enlarged T2 lesions are detected for at least 6 months. In some embodiments, no new or enlarged T2 lesions are detected for at least 9 months. In some embodiments, no new or enlarged T2 lesions are detected for at least 12 months. In some embodiments, no new or enlarged T2 lesions are detected for at least 15 months. In some embodiments, no new or enlarged T2 lesions are detected for at least 18 months. In some embodiments, no new or enlarged T2 lesions are detected for at least 24 months.

[0147] In some embodiments, the patient exhibits a stable disease state following administration of the CD19 CAR-expressing cells (e.g., rapcabtagene autoleucel) as determined by no new or enlarged gadolinium-enhanced lesions as detected by MRI. In some embodiments, no new or enlarged gadolinium-enhanced lesions are detected for at least 3 months. In some embodiments, no new or enlarged gadolinium-enhanced lesions are detected for at least 6 months. In some embodiments, no new or enlarged gadolinium-enhanced lesions are detected for at least 9 months. In some embodiments, no new or enlarged gadolinium-enhanced lesions are detected for at least 12 months. In some embodiments, no new or enlarged gadolinium-enhanced lesions are detected for at least 15 months. In some embodiments, no new or enlarged gadolinium- enhanced lesions are detected for at least 18 months. In some embodiments, no new or enlarged gadolinium-enhanced lesions are detected for at least 24 months. PAT059856-PCT-SEC01

[0148] In some embodiments, the patient exhibits a stable disease state following administration of the CD19 CAR-expressing cells (e.g., rapcabtagene autoleucel) as determined by no new or additional brain atrophy as detected by MRI. In some embodiments, no new or additional brain atrophy is detected for at least 3 months. In some embodiments, no new or additional brain atrophy is detected for at least 6 months. In some embodiments, no new or additional brain atrophy detected for at least 9 months. In some embodiments, no new or additional brain atrophy detected for at least 12 months. In some embodiments, no new or additional brain atrophy is detected for at least 15 months. In some embodiments, no new or additional brain atrophy is detected for at least 18 months. In some embodiments, no new or additional brain atrophy is detected for at least 24 months.

[0149] In some embodiments, the patient exhibits a stable disease state following administration of the CD19 CAR-expressing cells (e.g., rapcabtagene autoleucel) as determined by no new or enlarged paramagnetic rim lesions as detected by MRI. In some embodiments, no new or enlarged paramagnetic rim lesions are detected for at least 3 months. In some embodiments, no new or enlarged paramagnetic rim lesions are detected for at least 6 months. In some embodiments, no new or enlarged paramagnetic rim lesions are detected for at least 9 months. In some embodiments, no new or enlarged paramagnetic rim lesions are detected for at least 12 months. In some embodiments, no new or enlarged paramagnetic rim lesions are detected for at least 15 months. In some embodiments, no new or enlarged paramagnetic rim lesions are detected for at least 18 months. In some embodiments, no new or enlarged paramagnetic rim lesions are detected for at least 24 months.

[0150] In some embodiments, the patient exhibits a stable disease state following administration of the CD19 CAR-expressing cells (e.g., rapcabtagene autoleucel) as determined by no new or enlarged slowly expanding lesions as detected by MRI. In some embodiments, no new or enlarged slowly expanding lesions are detected for at least 3 months. In some embodiments, no new or enlarged slowly expanding lesions are detected for at least 6 months. In some embodiments, no new or enlarged slowly expanding lesions are detected for at least 9 months. In some embodiments, no new or enlarged slowly expanding lesions are detected for at least 12 months. In some embodiments, no new or enlarged slowly expanding lesions are detected for at least 15 months. In some embodiments, no new or enlarged slowly expanding lesions are PAT059856-PCT-SEC01 detected for at least 18 months. In some embodiments, no new or enlarged slowly expanding lesions are detected for at least 24 months.

[0151] The Symbol Digit Modalities Test (SDMT) (Smith, 1982) is a measure of speeded information processing. Participants are provided an 812x11 inch sheet with nine symbols, each paired with a number on top of the page. The remainder of the page consists of a randomized, sequential assortment of these symbols. Participants are asked to verbally respond with the number that corresponds with each symbol. The dependent variable is the total number correct in 90 seconds.

[0152] In some embodiments, a subject is identified as showing improvement as measured by a lowering of the SDMT score from baseline following administration of the CD 19 CAR- expressing cells (e.g., rapcabtag ene autoleucel) to the subject according to the methods described herein. In some embodiments, the baseline SDMT score of the patient is the SDMT score measured prior to the first administration of the CD 19 CAR-expressing cells. In some embodiments, the baseline SDMT score of the patient is the first SDMT score measured after administration of the CD 19 CAR-expressing cells.

[0153] In some embodiments, the SDMT score is lowered by at least 4 points. In some embodiments, the SDMT score is lowered by at least 5 points. In some embodiments, the SDMT score is lowered by at least 6 points. In some embodiments, the SDMT score is lowered by at least 7 points. In some embodiments, the SDMT score is lowered by at least 8 points. In some embodiments, the SDMT score is lowered by at least 9 points. In some embodiments, the SDMT score is lowered by at least 10 points. In some embodiments, the SDMT score is lowered by at least 11 points. In some embodiments, the SDMT score is lowered by at least 12 points. In some embodiments, the SDMT score is lowered by at least 13 points. In some embodiments, the SDMT score is lowered by at least 14 points.

[0154] In some embodiments, a subject is identified as showing improvement as measured by a lowering of the SDMT score from baseline following administration of the CD 19 CAR- expressing cells (e.g., rapcabtag ene autoleucel) to the subject according to the methods described herein. In some embodiments, the baseline SDMT score of the patient is the SMDT score measured prior to the first administration of the CD 19 CAR-expressing cells. In some embodiments, the baseline SDMT score of the patient is the first SMDT score measured after administration of the CD 19 CAR-expressing cells. In some embodiments, the SDMT score is PAT059856-PCT-SEC01 lowered by at least 10%. In some embodiments, the SDMT score is lowered by at least 15%. In some embodiments, the SDMT score is lowered by at least 20%.

[0155] In some embodiments, a subject is identified as showing improvement as measured by a lowering of the SDMT score from baseline following administration of the CD 19 CAR- expressing cells (e.g., rapcabtag ene autoleucel) to the subject according to the methods described herein. In some embodiments, the lowering of the SDMT score is achieved within 3 months of administration. In some embodiments, lowering of the SDMT score is achieved within 6 months of administration. In some embodiments, the lowering of the SDMT score is achieved within 9 months of administration. In some embodiments, lowering of the SDMT score is achieved within 12 months of administration. In some embodiments, the lowering of the SDMT score is achieved within 18 months of administration. In some embodiments, lowering of the SDMT score is achieved within 24 months of administration.

[0156] In some embodiments, the lowering of the SDMT score from baseline achieved by a subject following administration of the CD 19 CAR-expressing cells (e.g., rapcabtag ene autoleucel) is sustained for 3 months or more. In some embodiments, lowering of the SDMT score is sustained for 6 months or more. In some embodiments lowering of the SDMT score is sustained for 9 months or more. In some embodiments, the lowering of the SDMT score is sustained for 12 months or more. In some embodiments, the lowering of the SDMT score is sustained for 18 months or more. In some embodiments, the lowering of the SDMT score is sustained for 24 months or more. In some embodiments, the lowering of the SDMT score is sustained indefinitely.

[0157] In some embodiments, a subject is identified as achieving a stable disease state as measured by no worsening of the SDMT score from baseline following administration of the CD19 CAR-expressing cells (e.g., rapcabtagene autoleucel) to the subject according to the methods described herein. In some embodiments, the SMDT score does not increase from the baseline SDMT score following administration of the CD 19 CAR-expressing cells. In some embodiments, the baseline SDMT score of the patient is the SDMT score measured prior to the first administration of the CD 19 CAR-expressing cells. In some embodiments, the baseline SDMT score of the patient is the first SMDT score measured after administration of the CD 19 CAR-expressing cells. PAT059856-PCT-SEC01

[0158] In some embodiments, a subject is identified as achieving a stable disease state as measured by no worsening of the SMDT score from baseline following administration of the CD19 CAR-expressing cells (e.g., rapcabtagene autoleucel) to the subject according to the methods described herein. In some embodiments, the SMDT score does not increase during the first 3 months following administration. In some embodiments, the SMDT score does not increase during the first 6 months following administration. In some embodiments, the SMDT score does not increase during the first 9 months following administration. In some embodiments, the SMDT score does not increase during the first 12 months following administration. In some embodiments, the SMDT score does not increase during the first 18 months following administration. In some embodiments, the SMDT score does not increase during the first 24 months following administration.

[0159] In some embodiments, the SMDT score of the patient does not increase following administration of the CD19 CAR-expressing cells (e.g., rapcabtagene autoleucel) for at least 3. In some embodiments, the SMDT score does not increase for at least 6 months. In some embodiments, the SMDT score does not increase for at least 9 months. In some embodiments, the SMDT score does not increase for at least 12 months. In some embodiments, the SMDT score does not increase for at least 15 months. In some embodiments, the SMDT score does not increase for at least 18 months. In some embodiments, the SMDT score does not increase for at least 24 months. In some embodiments, the SMDT score of the patient remains stable indefinitely.

[0160] The Modified Fatigue Impact Scale (MFIS) is a modified version of the 40-item Fatigue Impact Scale (FIS), which was originally developed to assess the effects of fatigue on quality of life in patients with chronic diseases, specifically MS. The MFIS contains 9 “physical” items, 10 “cognitive” items, and 2 “psychosocial” items. Participants are asked to rate the extent to which their fatigue has affected their life in the past 4 weeks, with “0” indicating “no problem” and “4” indicating “extreme problem.” The maximum possible score is 84, with higher scores indicating a greater impact on quality of life. The ranges of scores for each subscale are as follows: physical, 0 to 36; cognitive, 0 to 40; and psychosocial, 0 to 8.

[0161] In some embodiments, a subject is identified as showing improvement as measured by a lowering of the MFIS score from baseline following administration of the CD 19 CAR- expressing cells (e.g., rapcabtagene autoleucel) to the subject according to the methods described PAT059856-PCT-SEC01 herein. In some embodiments, the baseline MFIS score of the patient is the MFIS score measured prior to the first administration of the CD 19 CAR-expressing cells. In some embodiments, the baseline MFIS score of the patient is the first MFIS score measured after administration of the CD 19 CAR-expressing cells.

[0162] In some embodiments, the MFIS score is lowered by 4 or more points. In some embodiments, the MFIS score is lowered by 6 or more points, embodiments, the MFIS score is lowered by 8 or more points. In some embodiments, the MFIS score is lowered by 10 or more points, embodiments, the MFIS score is lowered by 12 or more points. In some embodiments, the MFIS score is lowered by 14 or more points. In some embodiments, the MFIS score is lowered by 16 or more points. In some embodiments, the MFIS score is lowered by 18 or more points. In some embodiments, the MFIS score is lowered by 20 or more points. In some embodiments, the MFIS score is lowered by 22 or more points. In some embodiments, the MFIS score is lowered by 24 or more points. In some embodiments, the MFIS score is lowered by 26 or more points. In some embodiments, the MFIS score is lowered by 28 or more points. In some embodiments, the MFIS score is lowered by 30 or more points.

[0163] In some embodiments, a subject is identified as showing improvement as measured by a lowering of the MFIS score from baseline following administration of the CD 19 CAR- expressing cells (e.g., rapcabtag ene autoleucel) to the subject according to the methods described herein. In some embodiments, the lowering of the MFIS score is achieved within 3 months of administration. In some embodiments, lowering of the MFIS score is achieved within 6 months of administration. In some embodiments, the lowering of the MFIS score is achieved within 9 months of administration. In some embodiments, lowering of the MFIS score is achieved within 12 months of administration.

[0164] In some embodiments, the lowering of the MFIS score from baseline achieved by a subject following administration of the CD 19 CAR-expressing cells (e.g., rapcabtag ene autoleucel) is sustained for 3 months or more. In some embodiments, lowering of the MFIS score is sustained for 6 months or more. In some embodiments lowering of the MFIS score is sustained for 9 months or more. In some embodiments, the lowering of the MFIS score is sustained for 12 months or more. In some embodiments, the lowering of the MFIS score is sustained for 18 months or more. In some embodiments, the lowering of the MFIS score is PAT059856-PCT-SEC01 sustained for 24 months or more. In some embodiments, the lowering of the MFIS score is sustained indefinitely.

[0165] In some embodiments, a subject is identified as achieving a stable disease state as measured by no worsening of the MFIS score from baseline following administration of the CD19 CAR-expressing cells (e.g., rapcabtagene autoleucel) to the subject according to the methods described herein. In some embodiments, the MFIS score does not increase from the baseline MFIS score following administration of the CD 19 CAR-expressing cells. In some embodiments, the baseline MFIS score of the patient is the MFIS score measured prior to the first administration of the CD 19 CAR-expressing cells. In some embodiments, the baseline MFIS score of the patient is the first MFIS score measured after administration of the CD 19 CAR- expressing cells.

[0166] In some embodiments, a subject is identified as achieving a stable disease state as measured by no worsening of the MFIS score from baseline following administration of the CD19 CAR-expressing cells (e.g., rapcabtagene autoleucel) to the subject according to the methods described herein. In some embodiments, the MFIS score does not increase during the first 3 months following administration. In some embodiments, the MFIS score does not increase during the first 6 months following administration. In some embodiments, the MFIS score does not increase during the first 9 months following administration. In some embodiments, the MFIS score does not increase during the first 12 months following administration. In some embodiments, the MFIS score does not increase during the first 18 months following administration. In some embodiments, the MFIS score does not increase during the first 24 months following administration.

[0167] In some embodiments, the MFIS score of the patient does not increase from baseline following administration of the CD 19 CAR-expressing cells (e.g., rapcabtagene autoleucel) for at least 3. In some embodiments, the MFIS score does not increase for at least 6 months. In some embodiments, the MFIS score does not increase for at least 9 months. In some embodiments, the MFIS score does not increase for at least 12 months. In some embodiments, the MFIS score does not increase for at least 15 months. In some embodiments, the MFIS score does not increase for at least 18 months. In some embodiments, the MFIS score does not increase for at least 24 months. In some embodiments, the MFIS score of the patient remains stable indefinitely. PAT059856-PCT-SEC01

[0168] The 36-Item Short Form Survey (SF-36) is an outcome measure instrument that is often used, well-researched, self-reported measure of health. The SF-36 yields scores for eight scales: Physical Functioning (PF), Role Limitations due to Physical Health (RP), Bodily Pain (BP), General Health (GH), Vitality (VT), Social Functioning (SF), Role Limitations due to Emotional Health (RE), and Mental Health (MH). These scale scores can be combined to produce two component summary scores: the Physical Component Summary (PCS) and the Mental Component Summary (MCS), whose calculation produces a T-score ranging from 0 to 100, with a mean score of 50 and SD of 10, representing the reference score for the US general population. Higher scores indicate better health.

[0169] In some embodiments, a subject is identified as showing improvement as measured by an increase of the SF-36 score from baseline following administration of the CD19 CAR-expressing cells (e.g., rapcabtagene autoleucel) to the subject according to the methods described herein. In some embodiments, the baseline SF-36 score of the patient is the SF-36 score measured prior to the first administration of the CD 19 CAR-expressing cells. In some embodiments, the baseline SF-36 score of the patient is the first SF-36 score measured after administration of the CD 19 CAR-expressing cells.

[0170] In some embodiments, the SF-36 score is increased by 5 or more points. In some embodiments, the SF-36 score is increased by 10 or more points. In some embodiments, the SF- 36 score is increased by 15 or more points. In some embodiments, the SF-36 score is increased by 20 or more points. In some embodiments, the SF-36 score is increased by 25 or more points. In some embodiments, the SF-36 score is increased by 30 or more points. In some embodiments, the SF-36 score is increased by 35 or more points. In some embodiments, the SF-36 score is increased by 40 or more points. In some embodiments, the SF-36 score is increased by 45 or more points. In some embodiments, the SF-36 score is increased by 50 or more points.

[0171] In some embodiments, a subject is identified as showing improvement as measured by an increase of the SF-36 score from baseline following administration of the CD19 CAR-expressing cells (e.g., rapcabtagene autoleucel) to the subject according to the methods described herein. In some embodiments, the increase of the SF-36 score is achieved within 3 months of administration. In some embodiments, increase of the SF-36 score is achieved within 6 months of administration. In some embodiments, the increase of the SF-36 score is achieved within 9 months of administration. In some embodiments, the increase of the SF-36score is achieved PAT059856-PCT-SEC01 within 12 months of administration. In some embodiments, the increase of the SF-36 score is achieved within 18 months of administration. In some embodiments, the increase of the SF- 36score is achieved within 24 months of administration.

[0172] In some embodiments, the increase of the SF-36 score from baseline achieved by a subject following administration of the CD 19 CAR-expressing cells (e.g., rapcabtag ene autoleucel) is sustained for 3 months or more. In some embodiments, increase of the SF-36 score is sustained for 6 months or more. In some embodiments, the increase of the SF-36 score is sustained for 9 months or more. In some embodiments, the increase of the SF-36 score is sustained for 12 months or more. In some embodiments, the increase of the SF-36 score is sustained for 18 months or more. In some embodiments, the increase of the SF-36 score is sustained for 24 months or more. In some embodiments, the increase of the SF-36 score is sustained indefinitely.

[0173] In some embodiments, a subject is identified as achieving a stable disease state as measured by no worsening of the SF-36 score from baseline following administration of the CD19 CAR-expressing cells (e.g., rapcabtagene autoleucel) to the subject according to the methods described herein. In some embodiments, the SF-36 score does not decrease from the baseline SF-36 score following administration of the CD19 CAR-expressing cells. In some embodiments, the baseline SF-36 score of the patient is the SF-36 score measured prior to the first administration of the CD19 CAR-expressing cells. In some embodiments, the baseline SF- 36 score of the patient is the first SF-36 score measured after administration of the CD19 CAR- expressing cells.

[0174] In some embodiments, a subject is identified as achieving a stable disease state as measured by no worsening of the SF-36 score from baseline following administration of the CD19 CAR-expressing cells (e.g., rapcabtagene autoleucel) to the subject according to the methods described herein. In some embodiments, the SF-36 score does not decrease during the first 3 months following administration. In some embodiments, the SF-36 score does not decrease during the first 6 months following administration. In some embodiments, the SF-36 score does not decrease during the first 9 months following administration. In some embodiments, the SF-36 score does not decrease during the first 12 months following administration. In some embodiments, the SF-36 score does not decrease during the first 18 PAT059856-PCT-SEC01 months following administration. In some embodiments, the SF-36 score does not decrease during the first 24 months following administration.

[0175] In some embodiments, the SF-36 score of the patient does not decrease from baseline following administration of the CD 19 CAR-expressing cells (e.g., rapcabtag ene autoleucel) for at least 3. In some embodiments, the SF-36 score does not decrease for at least 6 months. In some embodiments, the SF-36 score does not decrease for at least 9 months. In some embodiments, the SF-36 score does not decrease for at least 12 months. In some embodiments, the SF-36 score does not decrease for at least 15 months. In some embodiments, the SF-36 score does not decrease for at least 18 months. In some embodiments, the SF-36 score does not decrease for at least 24 months. In some embodiments, the SF-36 score of the patient remains stable indefinitely.

[0176] The Nine-Hole Peg Test (NHPT) is considered as a gold standard measure of manual dexterity and is frequently used in MS research and clinical practice. It measures the time it takes a person to insert and remove nine pegs from a peg board. A decrease in completion time is associated with increased finger dexterity and therefore an overall improvement in the person’s health.

[0177] In some embodiments, a subject is identified as showing improvement as measured by a decrease of the NHPT time from baseline following administration of the CD 19 CAR-expressing cells (e.g., rapcabtagene autoleucel) to the subject according to the methods described herein. In some embodiments, the baseline NHPT time of the patient is the NHPT time measured prior to the first administration of the CD 19 CAR-expressing cells. In some embodiments, the baseline NHPT time of the patient is the first NHPT score measured after administration of the CD 19 CAR-expressing cells.

[0178] In some embodiments, the NHPT time is decreased by 2.5% or more. In some embodiments, the NHPT time is decreased by 5% or more. In some embodiments, the NHPT time is decreased by 7.5% or more. In some embodiments, the NHPT time is decreased by 10% or more. In some embodiments, the NHPT time is decreased by 12.5% or more. In some embodiments, the NHPT time is decreased by 15% or more. In some embodiments, the NHPT time is decreased by 20% or more. In some embodiments, the NHPT time is decreased by 25% or more. In some embodiments, the NHPT time is decreased by 30% or more. In some embodiments, the NHPT time is decreased by 35% or more. In some embodiments, the NHPT PAT059856-PCT-SEC01 time is decreased by 40% or more. In some embodiments, the NHPT time is decreased by 45% or more. In some embodiments, the NHPT time is decreased by 50% or more. In some embodiments, the NHPT time is decreased by 55% or more. In some embodiments, the NHPT time is decreased by 60% or more. In some embodiments, the NHPT time is decreased by 65% or more. In some embodiments, the NHPT time is decreased by 70% or more. In some embodiments, the NHPT time is decreased by 75% or more. In some embodiments, the NHPT time is decreased by 80% or more. In some embodiments, the NHPT time is decreased by 85% or more. In some embodiments, the NHPT time is decreased by 90% or more. In some embodiments, the NHPT time is decreased by 95% or more.

[0179] In some embodiments, a subject is identified as showing improvement as measured by a decrease in the NHPT time from baseline following administration of the CD 19 CAR-expressing cells (e.g., rapcabtagene autoleucel) to the subject according to the methods described herein. In some embodiments, the decrease in the NHPT time is achieved within 3 months of administration. In some embodiments, decrease in the NHPT time is achieved within 6 months of administration. In some embodiments, the decrease in the NHPT time is achieved within 9 months of administration. In some embodiments, the decrease in the NHPT time is achieved within 12 months of administration. In some embodiments, the decrease in the NHPT time is achieved within 18 months of administration. In some embodiments, the decrease in the NHPT time is achieved within 24 months of administration.

[0180] In some embodiments, the decrease in the NHPT time from baseline achieved by a subject following administration of the CD 19 CAR-expressing cells (e.g., rapcabtagene autoleucel) is sustained for 3 months or more. In some embodiments, the decrease in the NHPT time is sustained for 6 months or more. In some embodiments, the decrease in the NHPT time is sustained for 9 months or more. In some embodiments, the decrease in the NHPT time is sustained for 12 months or more. In some embodiments, the decrease in the NHPT time is sustained for 18 months or more. In some embodiments, the decrease in the NHPT time is sustained for 24 months or more. In some embodiments, the decrease in the NHPT time is sustained indefinitely.

[0181] In some embodiments, a subject is identified as achieving a stable disease state as measured by no worsening of the NHPT time from baseline following administration of the CD19 CAR-expressing cells (e.g., rapcabtagene autoleucel) to the subject according to the PAT059856-PCT-SEC01 methods described herein. In some embodiments, the NHPT time does not increase from the baseline NHPT time following administration of the CD 19 CAR-expressing cells. In some embodiments, the baseline NHPT time of the patient is the NHPT time measured prior to the first administration of the CD 19 CAR-expressing cells. In some embodiments, the baseline NHPT time of the patient is the first NHPT time measured after administration of the CD 19 CAR- expressing cells.

[0182] In some embodiments, a subject is identified as achieving a stable disease state as measured by no worsening of the NHPT time from baseline following administration of the CD19 CAR-expressing cells (e.g., rapcabtagene autoleucel) to the subject according to the methods described herein. In some embodiments, the NHPT time does not increase during the first 3 months following administration. In some embodiments, the NHPT time does not increase during the first 6 months following administration. In some embodiments, the NHPT time does not increase during the first 9 months following administration. In some embodiments, the NHPT time does not increase during the first 12 months following administration. In some embodiments, the NHPT time does not increase during the first 18 months following administration. In some embodiments, the NHPT time does not increase during the first 24 months following administration.

[0183] In some embodiments, the NHPT time of the patient does not increase from baseline following administration of the CD 19 CAR-expressing cells (e.g., rapcabtagene autoleucel) for at least 3. In some embodiments, the NHPT time does not increase for at least 6 months. In some embodiments, the NHPT time does not increase for at least 9 months. In some embodiments, the NHPT time does not increase for at least 12 months. In some embodiments, the NHPT time does not increase for at least 15 months. In some embodiments, the NHPT time does not increase for at least 18 months. In some embodiments, the NHPT time does not increase for at least 24 months. In some embodiments, the NHPT time of the patient remains stable indefinitely.

[0184] The timed 25 -foot walk (T25FW) is the most commonly used standalone measure of ambulation in patients with multiple sclerosis. The T25FW is typically conducted as a component of the MS functional composite (MSFC), though it has been used as a standalone measure in clinical studies. The patient is directed to one end of a clearly marked 25-foot course and is instructed to walk 25 feet as quickly as possible, but safely. The time is calculated from the initiation of the instruction and ends when the patient has reached the 25-foot mark. The task PAT059856-PCT-SEC01 is immediately administered again by having the patient walk back the same distance. Patients may use assistive devices when doing this task. The score for the T25-FW is the average of the two completed trials.

[0185] In some embodiments, a subject is identified as showing improvement as measured by a decrease of the T25FW time from baseline following administration of the CD 19 CAR- expressing cells (e.g., rapcabtag ene autoleucel) to the subject according to the methods described herein. In some embodiments, the baseline T25FW time of the patient is the T25FW time measured prior to the first administration of the CD 19 CAR-expressing cells. In some embodiments, the baseline T25FW time of the patient is the first T25FW time measured after administration of the CD 19 CAR-expressing cells.

[0186] In some embodiments, the T25FW time is decreased by 2.5% or more. In some embodiments, the T25FW time is decreased by 5% or more. In some embodiments, the T25FW time is decreased by 7.5% or more. In some embodiments, the T25FW time is decreased by 10% or more. In some embodiments, the T25FW time is decreased by 12.5% or more. In some embodiments, the T25FW time is decreased by 15% or more. In some embodiments, the T25FW time is decreased by 20% or more. In some embodiments, the T25FW time is decreased by 25% or more. In some embodiments, the T25FW time is decreased by 30% or more. In some embodiments, the T25FW time is decreased by 35% or more. In some embodiments, the T25FW time is decreased by 40% or more. In some embodiments, the T25FW time is decreased by 45% or more. In some embodiments, the T25FW time is decreased by 50% or more. In some embodiments, the T25FW time is decreased by 55% or more. In some embodiments, the T25FW time is decreased by 60% or more. In some embodiments, the T25FW time is decreased by 65% or more. In some embodiments, the T25FW time is decreased by 70% or more. In some embodiments, the T25FW time is decreased by 75% or more. In some embodiments, the T25FW time is decreased by 80% or more. In some embodiments, the T25FWtime is decreased by 85% or more. In some embodiments, the T25FW time is decreased by 90% or more. In some embodiments, the T25FW time is decreased by 95% or more.

[0187] In some embodiments, a subject is identified as showing improvement as measured by a decrease in the T25FW time from baseline following administration of the CD 19 CAR- expressing cells (e.g., rapcabtag ene autoleucel) to the subject according to the methods described herein. In some embodiments, the decrease in the T25FW time is achieved within 3 months of PAT059856-PCT-SEC01 administration. In some embodiments, decrease in the T25FW time is achieved within 6 months of administration. In some embodiments, the decrease in the T25FW time is achieved within 9 months of administration. In some embodiments, the decrease in the T25FW time is achieved within 12 months of administration. In some embodiments, the decrease in the T25FW time is achieved within 18 months of administration. In some embodiments, the decrease in the T25FW time is achieved within 24 months of administration.

[0188] In some embodiments, the decrease in the T25FW time from baseline achieved by a subject following administration of the CD 19 CAR-expressing cells (e.g., rapcabtag ene autoleucel) is sustained for 3 months or more. In some embodiments, the decrease in the T25FW time is sustained for 6 months or more. In some embodiments, the decrease in the T25FW time is sustained for 9 months or more. In some embodiments, the decrease in the T25FW time is sustained for 12 months or more. In some embodiments, the decrease in the T25FW time is sustained for 18 months or more. In some embodiments, the decrease in the T25FW time is sustained for 24 months or more. In some embodiments, the decrease in the T25FW time is sustained indefinitely.

[0189] In some embodiments, a subject is identified as achieving a stable disease state as measured by no worsening of the T25FW time from baseline following administration of the CD19 CAR-expressing cells (e.g., rapcabtagene autoleucel) to the subject according to the methods described herein. In some embodiments, the T25FW time does not increase from the baseline T25FW time following administration of the CD 19 CAR-expressing cells. In some embodiments, the baseline T25FW time of the patient is the T25FW time measured prior to the first administration of the CD 19 CAR-expressing cells. In some embodiments, the baseline T25FW time of the patient is the first T25FW time measured after administration of the CD 19 CAR-expressing cells.

[0190] In some embodiments, a subject is identified as achieving a stable disease state as measured by no worsening of the T25FW time from baseline following administration of the CD19 CAR-expressing cells (e.g., rapcabtagene autoleucel) to the subject according to the methods described herein. In some embodiments, the T25FW time does not increase during the first 3 months following administration. In some embodiments, the T25FW time does not increase during the first 6 months following administration. In some embodiments, the T25FW time does not increase during the first 9 months following administration. In some embodiments, PAT059856-PCT-SEC01 the T25FW time does not increase during the first 12 months following administration. In some embodiments, the T25FW time does not increase during the first 18 months following administration. In some embodiments, the T25FW time does not increase during the first 24 months following administration.

[0191] In some embodiments, the T25FW time of the patient does not increase from baseline following administration of the CD 19 CAR-expressing cells (e.g., rapcabtag ene autoleucel) for at least 3. In some embodiments, the T25FW time does not increase for at least 6 months. In some embodiments, the T25FW time does not increase for at least 9 months. In some embodiments, the T25FW time does not increase for at least 12 months. In some embodiments, the T25FW time does not increase for at least 15 months. In some embodiments, the T25FW time does not increase for at least 18 months. In some embodiments, the T25FW time does not increase for at least 24 months. In some embodiments, the T25FW time of the patient remains stable indefinitely.

[0192] Dosage regimen

[0193] In some embodiments, a dose of viable CD 19 CAR-expressing cells comprises about 0.5 x 106viable CAR-expressing cells to about 1.25 x 109viable CAR-expressing cells (for example, 0.5 x 106viable CAR-expressing cells to 1.25 x 109viable CAR-expressing cells). In some embodiments, a dose of viable CAR-expressing cells comprises about 1 x 106, about 2.5 x 106, about 5 x 106, about 1.25 x 107, about 2.5 x 107, about 5 x 107, about 5.75 x 107, or about 8 x 107viable CAR-expressing cells. In some embodiments, a dose of viable CAR-expressing cells comprises about 0.5 x 106to 90 x 106viable CAR-expressing cells. In some embodiments, a dose of viable CAR-expressing cells comprises about 5 x 106viable CAR-expressing cells. In some embodiments, a dose of viable CAR-expressing cells comprises about 2.5 x 106to 2.5 x 108viable CAR-expressing cells. In some embodiments, a dose of viable CAR-expressing cells comprises about 1.25 x 107viable CAR-expressing cells. In some embodiments, a dose of viable CAR-expressing cells comprises about 1.25 x 107to 1.25 x 109viable CAR-expressing cells. In some embodiments, a dose of viable CAR-expressing cells comprises about 1.25 x 108viable CAR-expressing cells. In some embodiments, a dose of viable CAR-expressing cells comprises about 2.5 x 106to 2.5 x 108viable CAR-expressing cells. In some embodiments, a dose of viable PAT059856-PCT-SEC01

[0194] CAR-expressing cells comprises about 1 x 107or 5 x 107viable CAR-expressing cells. In some embodiments, the viable CD 19 CAR-expressing cells are rapcabtagene autoleucel.

[0195] In some embodiments, a dose of CAR-expressing cells comprises about 1 x 106, 1.1 x

[0196] 106, 2 x 106, 3.6 x 106, 5 x 106, 1 x 107, 1.8 x 107, 2 x 107, 5 x 107, 1 x 108, 2 x 108, or 5 x 108cells / kg. In some embodiments, a dose of CAR-expressing cells comprises at least about 1 x 106, 1.1 x 106, 2 x 106, 3.6 x 106, 5 x 106, 1 x 107, 1.8 x 107, 2 x 107, 5 x 107, 1 x 108, 2 x 108, or 5 x 108cells / kg. In some embodiments, a dose of CD 19 CAR-expressing cells up to about 1 x 106, 1.1 x 106, 2 x 106, 3.6 x 106, 5 x 106, 1 x 107, 1.8 x 107, 2 x 107, 5 x 107, 1 x 108, 2 x 108, or 5 x 108cells / kg. In some embodiments, a dose of CD 19 CAR-expressing cells comprises about 1.1 X 106- 1.8 X 107cells / kg. In some embodiments, a dose of CD 19 CAR-expressing cells comprises about 1 x 107, 2 x 107, 5 x 107, 1 x 108, 2 x 108, 5 x 108, 1 x 109, 2 x 109, or 5 x 109cells. In some embodiments, a dose of CD 19 CAR-expressing cells comprises at least about 1 x

[0197] 107, 2 x 107, 5 x 107, 1 x 108, 2 x 108, 5 x 108, 1 x 109, 2 x 109, or 5 x 109cells. In some embodiments, a dose of CD 19 CAR expressing cells comprises up to about 1 x 107, 2 x l07, 5 x 107, 1 x 108, 2 x 108, 5 x 108, 1 x 109, 2 x 109, or 5 x 109cells. In some embodiments, the CD 19 CAR-expressing cells are rapcabtagene autoleucel.

[0198] The level of CAR-positive cells can be determined according to the methods disclosed in Example 8 of WO / 2021 / 173985. Briefly, for CAR T cells manufactured using a continuous Activated Rapid Manufacturing (ARM) process, e.g., ARM-CD19 CAR T cells, a sentinel vial of cryopreserved cells may be thawed and cultured for up to 5 days and the CAR expression analyzed by flow cytometry. The measurement of CAR expression on, e.g., day 2 or day 3 may be used to determine the dose of viable CAR-positive T cells.

[0199] In some embodiments, the disclosure provides a method of treating a patient, e.g., a patient having multiple sclerosis (e.g. progressive multiple sclerosis), comprising administering to said patient CAR-expressing cells produced as described herein, at a dose of viable CAR- expressing or CAR-positive cells (for example, viable CD 19 CAR-expressing cells, viable CD 19 CAR-positive cells, or any dual CARs thereof) from about 0.5 x 106viable CAR-expressing or CAR-positive cells to about 50 x 106viable CAR-expressing or CAR-positive cells (for example, from about 0.5 x 106viable CD 19 CAR-expressing or CAR-positive cells to about 90 x 106viable CD19 CAR-expressing or CAR-positive cells), e.g. at a dose of viable CAR-expressing or CAR-positive cells (for example, viable CD 19 CAR-expressing cells or viable CD 19 CAR- PAT059856-PCT-SEC01 positive cells) from about 2 x 106viable CAR-expressing or CAR-positive cells to about 40 x 106viable CAR-expressing or CAR-positive cells.

[0200] In some embodiments, the disclosure provides a method of treating a patient, e.g., a patient having a neuroimmune disease or disorder, e.g., multiple sclerosis or progressive multiple sclerosis (PMS), comprising administering to said patient CAR-expressing or CAR-positive cells produced as described herein, at a dose of viable CAR-expressing or CAR-positive cells from about 0.5 x 106viable CAR-expressing or CAR-positive cells to about 50 x 106viable CAR- expressing or CAR-positive cells (for example, from about 0.5 x 106viable CD 19 CAR- expressing or CAR-positive cells to about 50 x 106viable CD 19 CAR-expressing or CARpositive cells).

[0201] In some embodiments, the disclosure provides a method of treating a patient, e.g., a patient having a neuroimmune disease or disorder, e.g., multiple sclerosis or progressive multiple sclerosis (PMS), comprising administering to said patient CAR-expressing or CAR-positive cells produced as described herein at a dose of about 2.5 x 106viable CD 19 CAR-expressing, e.g., rapcabtagene autoleucel.

[0202] In some embodiments, the disclosure provides a method of treating a patient, e.g., a patient having a neuroimmune disease or disorder, e.g., multiple sclerosis or progressive multiple sclerosis (PMS), comprising administering to said patient CAR-expressing or CAR-positive cells produced as described herein at a dose of about 7.5 x 106viable CD 19 CAR-expressing, e.g., rapcabtagene autoleucel.

[0203] In some embodiments, the disclosure provides a method of treating a patient, e.g., a patient having a neuroimmune disease or disorder, e.g., multiple sclerosis or progressive multiple sclerosis (PMS), comprising administering to said patient CAR-expressing or CAR-positive cells produced as described herein at a dose of about 12.5 x 106viable CD 19 CAR-expressing, e.g., rapcabtagene autoleucel.

[0204] In some embodiments, the disclosure provides a method of treating a patient, e.g., a patient having a neuroimmune disease or disorder, e.g., multiple sclerosis or progressive multiple sclerosis (PMS), comprising administering to said patient CAR-expressing or CAR-positive cells produced as described herein, at a dose of from about 2.5 x 106viable CD 19 CAR-expressing or CAR-positive cells to about 40 x 106viable CD 19 CAR-expressing or CAR-positive cells. PAT059856-PCT-SEC01

[0205] In some embodiments, the disclosure provides a method of treating a patient, e.g., a patient having a neuroimmune disease or disorder, e.g., multiple sclerosis or progressive multiple sclerosis (PMS), comprising administering to said patient CAR-expressing or CAR-positive cells produced as described herein, at a dose of from about 5 x 106viable CD 19 CAR-expressing or CAR-positive cells to about 12.5 x 106viable CD 19 CAR-expressing or CAR-positive cells.

[0206] In some embodiments, the disclosure provides a method of treating a patient, e.g., a patient having a neuroimmune disease or disorder, e.g., multiple sclerosis or progressive multiple sclerosis (PMS), comprising administering to said patient CAR-expressing or CAR-positive cells produced as described herein, at a dose of from about 25 x 106viable CD 19 CAR-expressing or CAR-positive cells to about 40 x 106viable CD 19 CAR-expressing or CAR-positive cells.

[0207] Evaluating CAR Safety

[0208] In some embodiments of any of the therapeutic methods disclosed herein, the method further involves evaluating the safety of the CAR-expressing cell therapy in a subject. In some embodiments, safety of the CAR-expressing cell therapy is evaluated by measuring or recording one or more of a subject’s vital signs, adverse events experienced by the subject, various laboratory parementers, and / or an electrocardiogram of the subject.

[0209] In some embodiments, the subject does not experience an adverse event of grade 4 or higher. In some embodiments, the subject does not experience cytokine release syndrome (CRS). In some embodiments, the subject does not experience CRS of grade 3 or higher. In some embodiments, the subject does not experience immune effector cell-associated neurotoxicity syndrome (ICANS).

[0210] Methods of Manufacturing

[0211] Provided herein are methods of manufacturing immune effector cells (for example, T cells or NK cells) engineered to express a CAR, for example, a CAR described herein, compositions comprising such cells, and methods of using such cells for treating a disease, such as multiple sclerosis (e.g. progressive multiple sclerosis), in a subject. In some embodiments, the methods disclosed herein may manufacture immune effector cells engineered to express a CAR in less than 24 hours. Without wishing to be bound by theory, the methods provided herein PAT059856-PCT-SEC01 preserve the undifferentiated phenotype of T cells, such as naive T cells, during the manufacturing process. These CAR-expressing cells with an undifferentiated phenotype may persist longer and / or expand better in vivo after infusion. In some embodiments, CART cells produced by the manufacturing methods provided herein comprise a higher percentage of stem cell memory T cells, compared to CART cells produced by the traditional manufacturing process, e.g., as measured using scRNA-seq. (FIG. 3). In some embodiments, CART cells produced by the manufacturing methods provided herein comprise a higher percentage of effector T cells, compared to CART cells produced by the traditional manufacturing process, e.g., as measured using scRNA-seq. (FIG. 3). In some embodiments, CART cells produced by the manufacturing methods provided herein better preserve the sternness of T cells, compared to CART cells produced by the traditional manufacturing process. (FIG. 3). In some embodiments, CART cells produced by the manufacturing methods provided herein show a lower level of hypoxia, compared to CART cells produced by the traditional manufacturing process, e.g., as measured using scRNA-seq. (FIG. 3). In some embodiments, CART cells produced by the manufacturing methods provided herein show a lower level of autophagy, compared to CART cells produced by the traditional manufacturing process. (FIG. 3).

[0212] In some embodiments, the CART cells manufactured by the methods disclosed herein may be administered to a subject with minimal ex vivo expansion, for example, less than 1 day, less than 12 hours, less than 8 hours, less than 6 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, or no ex vivo expansion. Accordingly, the methods described herein provide a fast manufacturing process of making improved CAR-expressing cell products for use in treating a disease in a subject.

[0213] In some embodiments, the present disclosure provides methods of making a population of cells (for example, T cells) that express a chimeric antigen receptor (CAR) comprising: (i) contacting a population of cells (for example, T cells, for example, T cells isolated from a frozen or fresh leukapheresis product from a subject having multiple sclerosis (e.g. progressive multiple sclerosis)) with (A) an agent that stimulates a CD3 / TCR complex and / or (B) an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells; (ii) contacting the population of cells (for example, T cells) with a nucleic acid molecule (for example, a DNA or RNA molecule) encoding the CAR, thereby providing a population of cells (for example, T cells) comprising the nucleic acid molecule, and (iii) harvesting the population PAT059856-PCT-SEC01 of cells (for example, T cells) for storage (for example, reformulating the population of cells in cryopreservation media) or administration, wherein: (a) step (ii) is performed together with step

[0214] (i) or no later than 20 hours after the beginning of step (i), for example, no later than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 hours after the beginning of step (i), for example, no later than 18 hours after the beginning of step (i), and step (iii) is performed no later than 26 hours after the beginning of step (i), for example, no later than 22, 23, or 24 hours after the beginning of step (i), for example, no later than 24 hours after the beginning of step (i); (b) step

[0215] (ii) is performed together with step (i) or no later than 20 hours after the beginning of step (i), for example, no later than 12, 13, 14, 15, 16, 17, or 18 hours after the beginning of step (i), for example, no later than 18 hours after the beginning of step (i), and step (iii) is performed no later than 30, 36, or 48 hours after the beginning of step (ii), for example, no later than 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours after the beginning of step (ii); or (c) the population of cells from step (iii) are not expanded, or expanded by no more than 5, 10, 15, 20, 25, 30, 35, or 40%, for example, no more than 10%, for example, as assessed by the number of living cells, compared to the population of cells at the beginning of step (i).

[0216] In some embodiments, the nucleic acid molecule in step (ii) is a DNA molecule. In some embodiments, the nucleic acid molecule in step (ii) is an RNA molecule. In some embodiments, the nucleic acid molecule in step (ii) is on a viral vector, for example, a viral vector chosen from a lentivirus vector, an adenoviral vector, or a retrovirus vector. In some embodiments, the nucleic acid molecule in step (ii) is on a non-viral vector. In some embodiments, the nucleic acid molecule in step (ii) is on a plasmid. In some embodiments, the nucleic acid molecule in step (ii) is not on any vector. In some embodiments, step (ii) comprises transducing the population of cells (for example, T cells) a viral vector comprising a nucleic acid molecule encoding the CAR.

[0217] In some embodiments, the population of cells (for example, T cells) is collected from an apheresis sample (for example, a leukapheresis sample) from a subject having multiple sclerosis (e.g. progressive multiple sclerosis).

[0218] In some embodiments, the apheresis sample (for example, a leukapheresis sample) is collected from the subject and shipped as a fresh product or a frozen sample (for example, a cryopreserved sample) to a cell manufacturing facility. Then the T cells (for example, CD4+ T cells and / or CD8+ T cells) are selected from the apheresis sample, for example, using a cell PAT059856-PCT-SEC01 sorting machine (for example, a CliniMACS® Prodigy® device). The selected T cells (for example, CD4+ T cells and / or CD8+ T cells) are then seeded for CART manufacturing using the process described herein. In some embodiments, the selected T cells (for example, CD4+ T cells and / or CD8+ T cells) undergo one or more rounds of freeze-thaw before being seeded for CART manufacturing.

[0219] In some embodiments, cells (for example, T cells) are contacted with anti-CD3 and anti- CD28 antibodies for, for example, 12 hours, followed by transduction with a vector (for example, a lentiviral vector) encoding a CAR. 24 hours after culture initiation, the cells are washed and formulated for storage or administration. Without wishing to be bound by theory, brief CD3 and CD28 stimulation may promote efficient transduction of self-renewing T cells. Compared to traditional CART manufacturing approaches, the activation process provided herein does not involve prolonged ex vivo expansion. (FIG. 3). Similar to the cytokine process, the activation process provided herein also preserves undifferentiated T cells during CART manufacturing.

[0220] In some embodiments, the population of cells is contacted with (A) an agent that stimulates a CD3 / TCR complex and / or (B) an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells. In some embodiments, the agent that stimulates a CD3 / TCR complex is an agent that stimulates CD3. In some embodiments, the agent that stimulates a costimulatory molecule and / or growth factor receptor is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, 0X40, DR3, GITR, CD30, HM1 , CD2, CD226, or any combination thereof. In some embodiments, the agent that stimulates a costimulatory molecule and / or growth factor receptor is an agent that stimulates CD28.

[0221] In some embodiments, the agent that stimulates a CD3 / TCR complex is chosen from an antibody (for example, a single-domain antibody (for example, a heavy chain variable domain antibody), a peptibody, a Fab fragment, or a scFv), a small molecule, or a ligand (for example, a naturally existing, recombinant, or chimeric ligand). In some embodiments, the agent that stimulates a costimulatory molecule and / or growth factor receptor is chosen from an antibody (for example, a single-domain antibody (for example, a heavy chain variable domain antibody), a peptibody, a Fab fragment, or a scFv), a small molecule, or a ligand (for example, a naturally existing, recombinant, or chimeric ligand). In some embodiments, the agent that stimulates a PAT059856-PCT-SEC01

[0222] CD3 / TCR complex does not comprise a bead. In some embodiments, the agent that stimulates a costimulatory molecule and / or growth factor receptor does not comprise a bead. In some embodiments, the agent that stimulates a CD3 / TCR complex comprises an anti-CD3 antibody. In some embodiments, the agent that stimulates a costimulatory molecule and / or growth factor receptor comprises an anti-CD28 antibody. In some embodiments, the agent that stimulates a CD3 / TCR complex comprises an anti-CD3 antibody covalently attached to a colloidal polymeric nanomatrix. In some embodiments, the agent that stimulates a costimulatory molecule and / or growth factor receptor comprises an anti-CD28 antibody covalently attached to a colloidal polymeric nanomatrix.

[0223] In some embodiments, the agent that stimulates a costimulatory molecule and / or growth factor receptor is an agent that stimulates CD28, ICOS, CD27, CD25, 4-1BB, IL6RA, IL6RB, or CD2. In some embodiments, the agent that stimulates a costimulatory molecule and / or growth factor receptor comprises one or more of a CD28, ICOS, CD27, CD25, 4-1BB, IL6RB, and / or CD2 antigen binding domain, such as but not limited to an anti- CD28, anti-ICOS, anti-CD27, anti-CD25, anti-4-lBB, anti-IL6RA, anti-IL6RB, or anti-CD2 antibody or an antibody fragment comprising one or more CDRs, heavy chain, and / or light chain thereof - such as but not limited to an anti- CD28, anti-ICOS, anti-CD27, anti-CD25, anti-4-lBB, anti-IL6RA, anti-IL6RB, or anti-CD2 antibody provided in Table 27 of WO / 2021 / 173985, hereby incorporated by reference in its entirety. In some embodiments, the agent that stimulates a CD3 / TCR complex and the agent that stimulates a costimulatory molecule and / or growth factor receptor comprise T Cell TransAct™. In some embodiments, the agent that stimulates a CD3 / TCR complex and the agent that stimulates a costimulatory molecule and / or growth factor receptor are comprised in a multispecific binding molecule. In some embodiments, the multispecific binding molecule comprises a CD3 antigen binding domain and a CD28 or CD2 antigen-binding domain

[0224] In some embodiments, the population of cells is contacted with a nucleic acid molecule encoding a CAR. In some embodiments, the population of cells is transduced with a DNA molecule encoding a CAR. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR occurs simultaneously with contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule PAT059856-PCT-SEC01 encoding the CAR occurs no later than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR occurs no later than 20 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR occurs no later than 19 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR occurs no later than 18 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR occurs no later than 17 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR occurs no later than 16 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR occurs no later than 15 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR occurs no later than 14 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that PAT059856-PCT-SEC01 stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR occurs no later than 14 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR occurs no later than 13 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR occurs no later than 12 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR occurs no later than 11 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR occurs no later than 10 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR occurs no later than 9 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR occurs no later than 8 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR occurs no later than 7 hours after the beginning of contacting the PAT059856-PCT-SEC01 population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR occurs no later than 6 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR occurs no later than 5 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR occurs no later than 4 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR occurs no later than 3 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR occurs no later than 2 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR occurs no later than 1 hour after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR occurs no later than 30 minutes after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. PAT059856-PCT-SEC01

[0225] In some embodiments, the population of cells is harvested for storage or administration. In some embodiments, the population of cells is harvested for storage or administration no later than 72, 60, 48, 36, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, the population of cells is harvested for storage or administration no later than 26 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, the population of cells is harvested for storage or administration no later than 25 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, the population of cells is harvested for storage or administration no later than 24 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, the population of cells is harvested for storage or administration no later than 23 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, the population of cells is harvested for storage or administration no later than 22 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above.

[0226] In some embodiments, the population of cells is not expanded ex vivo. In some embodiments, the population of cells is expanded by no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or 60%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, PAT059856-PCT-SEC01 the population of cells is expanded by no more than 5%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 10%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 15%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 20%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 25%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 30%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 35%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 40%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that PAT059856-PCT-SEC01 stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells described above.

[0227] In some embodiments, the activation process is conducted in serum free cell media. In some embodiments, the activation process is conducted in cell media comprising one or more cytokines chosen from: IL-2, IL-15 (for example, hetIL-15 (IL15 / sIL-15Ra)), or IL-6 (for example, IL-6 / sIL-6Ra). In some embodiments, the activation process is conducted in cell media comprising a LSD1 inhibitor. In some embodiments, the activation process is conducted in cell media comprising a MALT1 inhibitor. In some embodiments, the serum free cell media comprises a serum replacement. In some embodiments, the serum replacement is CTS™ Immune Cell Serum Replacement (ICSR). In some embodiments, the level of ICSR can be, for example, up to 5%, for example, about 1%, 2%, 3%, 4%, or 5%.

[0228] In some embodiments, the present disclosure provides methods of making a population of cells (for example, T cells) that express a chimeric antigen receptor (CAR) comprising: (a) providing an apheresis sample (for example, a fresh or cryopreserved leukapheresis sample) collected from a subject with multiple sclerosis (e.g. progressive multiple sclerosis); (b) selecting T cells from the apheresis sample (for example, using negative selection, positive selection, or selection without beads); (c) seeding isolated T cells at, for example, 1 x 106to 1 x 107cells / mL; (d) contacting T cells with an agent that stimulates T cells, for example, an agent that stimulates a CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells (for example, contacting T cells with anti-CD3 and / or anti-CD28 antibody, for example, contacting T cells with TransAct); (e) contacting T cells with a nucleic acid molecule (for example, a DNA or RNA molecule) encoding the CAR (for example, contacting T cells with a virus comprising a nucleic acid molecule encoding the CAR) for, for example, 6-48 hours, for example, 20-28 hours; and (f) washing and harvesting T cells for storage (for example, reformulating T cells in cryopreservation media) or administration. In some embodiments, step (f) is performed no later than 30, 36, or 48 hours after the beginning of step (d) or (e), for example, no later than 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours after the beginning of step (d) or (e).

[0229] In some embodiments of the aforementioned methods, the methods are performed in a closed system. In some embodiments, T cell separation, activation, transduction, incubation, and washing are all performed in a closed system. In some embodiments of the aforementioned PAT059856-PCT-SEC01 methods, the methods are performed in separate devices. In some embodiments, T cell separation, activation and transduction, incubation, and washing are performed in separate devices.

[0230] In some embodiments of the aforementioned methods, the methods further comprise adding an adjuvant or a transduction enhancement reagent in the cell culture medium to enhance transduction efficiency. In some embodiments, the adjuvant or transduction enhancement reagent comprises a cationic polymer. In some embodiments, the adjuvant or transduction enhancement reagent is chosen from: LentiBOOST™ (Sirion Biotech), vectofusin-1, F108 (Poloxamer 338 or Pluronic® F-38), protamine sulfate, hexadimethrine bromide (Polybrene), PEA, Pluronic F68, Pluronic Fl 27, Synperonic or LentiTrans™. In some embodiments, the transduction enhancement reagent is LentiBOOST™ (Sirion Biotech). In some embodiments, the transduction enhancement reagent is Fl 08 (Poloxamer 338 or Pluronic® F-38)

[0231] In some embodiments of the aforementioned methods, the transducing the population of cells (for example, T cells) with a viral vector comprises subjecting the population of cells and viral vector to a centrifugal force under conditions such that transduction efficiency is enhanced. In an embodiment, the cells are transduced by spinoculation.

[0232] In some embodiments of the aforementioned methods, cells (e.g., T cells) are activated and transduced in a cell culture flask comprising a gas-permeable membrane at the base that supports large media volumes without substantially compromising gas exchange. In some embodiments, cell growth is achieved by providing access, e.g., substantially uninterrupted access, to nutrients through convection.

[0233] Pharmaceutical Compositions

[0234] The methods described herein can further include formulating a CD 19 CAR-expressing cell in a pharmaceutical composition. Pharmaceutical compositions may comprise a CD 19 CAR-expressing cell, for example, a plurality of CD 19 CAR-expressing cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating PAT059856-PCT-SEC01 agents such as EDTA or glutathione; adjuvants (for example, aluminum hydroxide); and preservatives. Compositions can be formulated, for example, for intravenous administration.

[0235] In some embodiments, the pharmaceutical composition is substantially free of, for example, there are no detectable levels of a contaminant, for example, selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3 / anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus. In some embodiments, the bacterium is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes group A.

[0236] When “an immunologically effective amount” or “therapeutic amount” is indicated, the precise amount of the compositions to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the immune effector cells (for example, T cells, NK cells) described herein may be administered at a dosage of about 0.5 x 106to 50 x 106viable CAR-expressing cells, in some instances about 2.5 x 106, 7.5 x 106, or 12.5 x 106viable CAR-expressing cells, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).

[0237] The administration of the subject compositions may be carried out in any convenient manner. The compositions described herein may be administered to a patient trans arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally, for example, by intradermal or subcutaneous injection. The compositions of immune effector cells (for example, T cells, NK cells) may be injected directly into a lymph node or site of disease. PAT059856-PCT-SEC01

[0238] EXAMPLES

[0239] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0240] Example 1: Description of the Activated Rapid Manufacturing (ARM) process

[0241] In some embodiments, CART cells are manufactured using a continuous Activated Rapid Manufacturing (ARM) process, over approximately 2 days, which will potentially allow for a greater number of less differentiated T cells (T naive and TSCM (stem central memory T) cells) to be returned to a patient for in vivo cellular expansion. The short manufacturing time period allows the early differentiated T cells profile to proliferate in the body for their desired terminal differentiated state rather that in an ex vivo culture vessel.

[0242] In some embodiments, CART cells are manufactured using cryopreserved leukapheresis source material, for example, non-mobilized autologous peripheral blood leukapheresis (LKPK) material. Cryopreserved source material undergoes processing steps for T cell enrichment on the first day of production (Day 0) by means of anti-CD4 / anti-CD8 immunomagnetic system. Positive fraction is then seeded in G-rex culture vessel, activated with an anti-CD3 / CD28 system (TransACT™) and on the same day transduced with a lentiviral vector (LV) encoding a CAR. On the following day, after 20-28 hours of transduction, the T cells are harvested, washed four times, formulated in freezing medium, and then frozen by a Controlled Rate Freezer (CRF). From the start of the process on Day 0 to the initiation of harvest on the following day, cells are cultured for 20 - 28 hours with a target of 24 hours after Day 0 seeding.

[0243] Media for Day 0 were prepared according to Table 1.

[0244] Table 1: Media type and point of use during CART manufacturing PAT059856-PCT-SEC01

[0245] The cryopreserved leukapheresis material is thawed. The thawed cells are diluted with the Rapid Buffer (Table 21) and washed on the CliniMACS® Prodigy® device. The T cells are selected by CliniMACS® CD4 and CD8 microbeads. Once the program is finished for T cell selection (approximately 3h 40 min to 4h 40 min), the reapplication bag containing the cells suspended in Rapid Media (Table 21) are transferred in a transfer pack. A sample is taken for viability and cell count. The cell count and viability data from the positive fraction bag is used to determine the cell concentration when seeding the culture vessel for activation and vector transduction.

[0246] Following positive selection of T cells via the CliniMACS® microbeads (CD4 and CD8), the cells are seeded in the culture vessel, G-Rex. Once the cells are seeded, the activation reagent (TransACT™) is then added to the culture vessel. The cells are then transduced with a lentiviral vector encoding a CAR at a target MOI of 1.0 (0.8-1.2). Following the vector addition, the culture vessel is transported to an incubator where it is incubated for a target of 24 hours (operating range 20-28 hours) at a nominal temperature of 37 °C (operating range 36-38 °C) with nominal 5% CO2 (operating range 4.5-5.5%). Following the incubation, the cells are washed with Harvest Wash Solution (Table 1) four times to remove any non-integrated vector and residual viral particles, as well as any other process related impurities. Then, the cells are eluted and a sample for cell count and viability is taken for testing and the results are used to determine the volume required to re-suspend the cells for final formulation with CryoStor® CS10. The cells are then centrifugated to remove the Harvest Wash Solution and proceed with cryopreservation.

[0247] In some embodiments, the CAR expressed in CART cells binds to CD 19. In some embodiments, the CAR made by the ARM process is rapcabtagene autoleucel. In some embodiments, IL-2 used in the Rapid Media (RM) (Table 1) can be replaced with IL-15, hetlL- 15 (IL-15 / sIL-15Ra), IL-6, or IL-6 / sIL-6Ra. PAT059856-PCT-SEC01

[0248] Example 2: Phase 1 / 2 study, open-label, multi-center, to assess safety, efficacy and cellular kinetics of rapcabtagene autoleucel cells in participants with non-active progressive multiple sclerosis.

[0249] The purpose of this clinical study is to assess safety, disease progression, and cellular kinetics of rapcabtagene autoleucel treatment, a CD19-directed, genetically engineered, chimeric antigen receptor T-cell immunotherapy product (CAR-T), in patients with non-active Progressive Multiple Sclerosis (PMS).

[0250] Rapcabtagene autoleucel is an autologous CD19-directed CAR-T cell therapy that is comprised of CD4+ / CD8+ T cells that have undergone ex vivo T cell activation and gene modification. Rapcabtagene autoleucel utilizes the FMC63 scFv domain for CD 19 recognition and the same lentiviral vector as tisagenlecleucel (Kymriah, CTL019) and is manufactured via the activated rapid manufacturing (ARM) process. The ARM process reduces the turnaround time compared to traditional manufacturing processes (FIG. 3) and preserves T cell sternness, the ability to self-renew and mature, resulting in a product with greater proliferative potential and fewer exhausted T cells compared to traditionally manufactured CAR-T cells. With ARM, CAR- T cell expansion occurs primarily within a patient’s body (in vivo), eliminating the need for an extended culture time outside of the body (ex vivo). These unique characteristics may lead to better and more durable responses, improved long-term outcomes and a reduced risk of severe adverse events compared to CAR-T cell products manufactured via traditional manufacturing methods. Non-clinical studies show that rapcabtagene autoleucel is a product with potentially superior efficacy, a similar safety profile, and delayed expansion compared to other CD 19- directed CAR-T cell therapy relying on a traditional manufacturing process (e.g. tisagenlecleucel). The expected improved safety profile of rapcabtagene autoleucel compared to other CAR-T therapies, together with the capacity of CD 19 CAR-T cells to traffic across central and peripheral tissues, and to efficiently destroy B cell subsets including CD20-negative plasmablasts and short-lived plasma cells offers the opportunity to eliminate CNS-resident B cell subsets in MS patients resistant to approved highly efficacious therapy (HET), including B celldepleting antibody therapies.

[0251] Overall Study Design PAT059856-PCT-SEC01

[0252] The study design utilizes an ascending single dose design initiated with sentinel dosing at each new dose level. The cohort design is shown in FIG. 2. Approximately 28 patients will receive treatment with rapcabtagene autoleucel. The study design / parti cipant journey is illustrated in FIG. 1. The total study duration for a participant will be up to 27 months. After this, a long-term follow-up will be initiated until 15 years after rapcabtagene autoleucel administration.

[0253] After consenting, participants will first be evaluated for clinical eligibility (Day -70 to Day —42). If clinically eligible, leukapheresis will be scheduled as soon as clinical eligibility is confirmed (including availability of Central Lab results as applicable) and the washout of immunosuppressive therapy is completed. Once the leukapheresis product has been confirmed to be suitable for rapcabtagene autoleucel manufacturing, the manufacturing process will commence. Before the planned apheresis and CAR-T cell administration, Disease Modifying Therapies (DMTs) will be stopped. The washout periods for the DMTs are relatively short to minimize the risk of disease worsening. Corticosteroids (administered for non-MS reasons) should be tapered to a maximum dose of 10 mg per day (prednisone or equivalent) 1 week prior to leukapheresis and rapcabtagene autoleucel administration. After leukapheresis and before rapcabtagene autoleucel administration, increased dosing of corticosteroids is allowed as clinically indicated but will need to be tapered to 10 mg per day (prednisone or equivalent) 1 week prior to rapcabtagene autoleucel administration. After the final product has been confirmed to be available, participants will receive lymphodepleting therapy (Day -9 to Day -2). Following pre-treatment check on Day 1 and premedication, rapcabtagene autoleucel will be given as a single i.v. infusion on Day 1.

[0254] The study design is comprised of 3 cohorts of 3 planned increasing doses where a sentinel dosing for each participant and each cohort will be applied, followed by an expansion cohort (FIG. 2). The study will be initiated with Cohort 1 at a dose of 2.5 x 106CAR-positive viable T cells given to 3 participants in a sentinel fashion for each participant. The first participant will be observed and monitored (vital signs, ECG, laboratory parameters, clinical examination, imaging (MRI) and AEs) for a minimum of 42 days post CAR-T administration for detection of any safety events. This clinical information will be evaluated by the treating Investigator and reviewed by the sponsor and, if no relevant safety issues occur (with consideration of study defined dose limiting toxicities [DLTs] and study stopping criteria), the PAT059856-PCT-SEC01 second participant in the Cohort 1 can be dosed. The same process will apply prior to dosing of the third participant. Once the third participant in Cohort 1 has reached 42 days post dose, a full review of safety data to date will be completed by the sponsor and independent DMC.

[0255] • If there are no safety concerns with the 3 sentinel Cohort 1 participants, this cohort will be closed and Cohort 2 (Provisional Dose of 7.5 x 106CAR-positive viable T cells) will be initiated with the same sentinel dosing procedure as in Cohort 1.

[0256] • If DLTs are observed and DMC evaluation recommends continuation in Cohort 1, an additional 3 patients will be recruited. When the 6thpatient in the Cohort reaches 42 days post treatment, a similar review will take place to either proceed to Cohort 2 or to initiate the expansion cohort at the 2.5 X 106dose level. If similar safety concerns persist, a de- escalation cohort at a lower dose may be triggered.

[0257] • If there are major safety concerns (including defined study stopping criteria), then a de- escalation cohort at a lower dose may be triggered, or recruitment into the study may be terminated prematurely.

[0258] If there is complete B cell depletion at the dose of 2.5 x 106viable CAR-positive T cells an exploratory lower dose cohort may be added.

[0259] If Cohort 2 is initiated (7.5 x 106cells), a similar process as in Cohort 1 will take place with the first 3 participants dosed in a sentinel fashion. The same decision mechanism as described above will be followed to evaluate the escalation decision. Similarly, depending on the safety concerns and the recommendation of the DMC either the cohort will be expanded at the same dose level with additional 3 subjects, or the lower dose at 2.5x106viable CAR-positive T cells will be studied. If the safety and tolerability is deemed to be acceptable the next dose level will be studied in Cohort 3. Again, the same process will be followed if Cohort 3 (Provisional Dose of 12.5 x 106CAR-positive viable T cells) is initiated as well.

[0260] • If there are no safety concerns with the 3 sentinel Cohort 3 participants, then Cohort 4 (Expansion Cohort - Dose of 12.5 x 106CAR-positive viable T cells) will be initiated and enroll up to 19 participants.

[0261] • If DLTs occur and DMC evaluation recommends continuation in Cohort 3 an additional 3 participants will be recruited. When the 6thpatient in Cohort 3 reaches 42 days post treatment, another comprehensive data review will take place to inform the decision to either PAT059856-PCT-SEC01 complete recruitment with Cohort 3 dose or decrease the dose and initiate the expansion cohort at the 7.5 xlO6dose level.

[0262] • If the safety concerns lead to decision of not continuing in Cohort 3, the recruitment for the expansion cohort will continue in Cohort 2. The review of the DMC will be taken into account for the decision on the expansion dose based on the data from the sentinel and if needed additional subjects to the cohorts.

[0263] Data reviewed will include available safety, and MRI data for escalation decisions. Additionally, the PK / PD data will be evaluated to confirm CAR-T cell expansion and B cell aplasia if available. An interim analysis (IA) will be performed after the last participant in the selected cohort for expansion reaches at least 42 days post treatment.

[0264] As there will be continuous safety review by the sponsor and the independent DMC, if at any time the current enrolling cohort shows safety concerns, additional dose cohorts may be investigated.

[0265] Study Population

[0266] The study population is of ambulatory (EDSS <6.5) non-active (na) SPMS and PPMS patients, aged 18-60 with evidence of progression. Non-active disease and progression are defined if all of the below criteria are met:

[0267] • No relapse in the past year

[0268] • EDSS progression of >0.5 points in the past year

[0269] • No Gd-enhancing lesion on MRI at screening

[0270] Study Treatment

[0271] Eligible participants undergo the following sequence of events prior to rapcabtagene autoleucel administration: (1) leukapheresis, (2) pre-lymphodepletion evaluation, (3) lymphodepletion, (4) premedication, and (5) pre- rapcabtagene autoleucel administration check.

[0272] Lymphodepleting therapy starts within one week before rapcabtagene autoleucel injection, which means that rapcabtagene autoleucel is injected 2 to 6 days after lymphodepleting therapy is completed. Lymphodepleting therapy may be repeated in the case rapcabtagene autoleucel has been delayed by more than 2 weeks. The lymphodepleting therapy regime is as follows: (1) fludarabine administered 25 mg / m2intravenously [i.v.] daily for 3 doses (for PAT059856-PCT-SEC01 participants with renal impairment, the dose may be reduced as per local approved labels of fludarabine), and (2) cyclophosphamide administered 250 mg / m2i.v. daily for 3 doses starting with the first dose of fludarabine.

[0273] All participants are pre-medicated with acetaminophen (paracetamol, 500-1000 mg, orally) and diphenhydramine (25-50 mg, i.v. or orally) or another Hl antihistamine approximately 30 to 60 minutes prior to injection. These medications can be repeated every 6 hours as needed. Non-steroidal anti-inflammatory medication may be prescribed if the participant continues to have fever not relieved with acetaminophen (paracetamol). Steroids should not be used for premedication.

[0274] Rapcabtag ene autoleucel treatment will consist of a single intravenous (i.v.) administration of CAR-positive viable T cells. Participants will be assigned to either one of three sentinel cohorts in a generally ascending dose order as per design of the study or they will be assigned to the expansion cohort. Provisional target doses (release specification range) selected for this adaptive single ascending dose study include:

[0275] • Cohort 1 : 2.5 x 106CAR-positive viable T cells (range: 1-2.5 x 106CAR-positive viable T cells)

[0276] • Optional de-escalation cohort: lower dose to be based on DMC and Sponsor decision

[0277] • Cohort 2: 7.5 x 106CAR-positive viable T cells (range: 5-7.5 x 106CAR-positive viable T cells)

[0278] • Cohort 3: 12.5 x 106CAR-positive viable T cells (range: 5-12.5 x 106CAR-positive viable T cells)

[0279] • Cohort 4 (expansion): up to 12.5 x 106CAR-positive viable T cells (range: 1-12.5 x 106CAR-positive viable T cells)

[0280] Safety, Pharmacokinetic (PK) and Efficacy Assessments

[0281] The primary aim of the study is to assess the safety of single ascending doses of rapcabtagene autoleucel in PMS patients. For the assessment of safety, all safety data including laboratory measurements, vital signs, adverse events (AEs), AEs of special interest (AESIs), serious adverse events (SAEs), laboratory ECGs, neurological status, and brain MRI (either new abnormal MRI findings atypical for MS [e.g., signs of cerebral edema or new microbleeds], or PAT059856-PCT-SEC01

[0282] MRI changes reflecting increased MS disease activity [e.g., new or enlarging T2 lesions]) are considered as primary safety endpoints.

[0283] The primary safety question of interest is to assess the frequency and severity of AESIs in the 42 days following single ascending doses in patients with PMS irrespective of intercurrent events. The AESIs are defined as:

[0284] • Cytokine Release Syndrome (CRS)

[0285] • Immune effector Cell- Associated Neurotoxic Syndrome (ICANS), including clinical and imaging assessments

[0286] The primary estimand is described by the following attributes:

[0287] • Population: PMS patients who received CAR T-cell administration.

[0288] • Primary Variable / Endpoint: The occurrence and severity of selected AESIs (as defined above) within 42 days after rapcabtagene autoleucel administration.

[0289] • Treatment of interest: pre-treatment / treatment procedures ending with rapcabtagene autoleucel administration.

[0290] • Remaining intercurrent events: lost to follow-up (FU), significant disease exacerbation (worsening of >1 EDSS points), patient withdrawal, and initiation of new therapy for worsening of disease will be addressed with treatment policy strategy.

[0291] • Summary measure: frequency and severity of AESIs in pre-specified interval of 42 days following rapcabtagene autoleucel administration.

[0292] The secondary clinical question of interest is related to the efficacy objective for the study, i.e., to assess whether rapcabtagene autoleucel, a CAR-T product, following lymphodepletion, impacts MS disease activity. To assess the effect of rapcabtagene autoleucel on MS disease activity, the following assessments will be analyzed at 2 years post administration (and possibly earlier timepoints): clinical measures for relapses and disability (including EDSS, SF-36, T25FW, 9HPT, SDMT, MFIS) and MRI changes in disease activity (including new or enlarging T2 lesions and Gd-enhancing lesions).

[0293] Objectives and related endpoints are summarized in Table 2. PAT059856-PCT-SEC01

[0294] Table 2 Objectives and related endpoints

[0295] Objective(s) Endpoint(s)

[0296] Primary objective(s) Endpoint(s) for primary objective(s)

[0297] • To assess the safety of single • Occurrence, severity and frequency of Adverse ascending doses of rapcabtagene Events (AEs) and change from baseline over 2 autoleucel in PMS patients. years in safety parameters including, but not limited to vital signs, laboratory, ECG, neurological status, and safety measures from brain MRI.

[0298] Secondary objective(s) Endpoint(s) for secondary objective(s)

[0299] • To assess the effect of • Change from baseline for clinical measures of rapcabtagene autoleucel on MS disability (includes EDSS, SF-36, T25FW, 9HPT, disease progression. SDMT, MFIS) over 2 years.

[0300] • To characterize the in vivo • rapcabtagene autoleucel transgene expression cellular kinetics levels by qPCR over time in blood; cellular

[0301] (pharmacokinetics, PK) of kinetics parameters (Cmax, AUC, Tmax, Clast, rapcabtagene autoleucel in blood. Tlast).

[0302] • To characterize the incidence and • Pre-existing and treatment-induced prevalence of pre-existing and immunogenicity (humoral, anti- rapcabtagene treatment induced autoleucel antibody; and cellular, presence of immunogenicity (cellular and CAR19 specific CD4 and CD8 T cells measuring humoral) of rapcabtagene interferon gamma production). autoleucel.

[0303] Several reported outcomes are included in this study for all participants:

[0304] • Symbol Digit Modalities Test (SDMT) The SDMT is a timed cognition test administered by a trained administrator. The test assesses sustained attention, processing speed, visual scanning, and motor speed to determine cognitive impairment. Participants are given a coding key which contains abstract symbols that correspond to specific numbers. Participants are timed how quickly and accurately they are able to substitute the symbols for the numbers, and is scored by the number of correctly coded items. PAT059856-PCT-SEC01

[0305] • Modified Fatigue Impact Scale (MFIS)

[0306] The MFIS is a questionnaire to assess fatigue-related symptoms in patients with MS.

[0307] • Short Form Health Survey (SF-36)

[0308] The Short Form Health Survey (SF-36 v2) is a widely used and extensively studied instrument to measure health-related quality of life among healthy participants and participants with acute and chronic conditions. It consists of eight subscales that can be scored individually: Physical Functioning, Role-Physical, Bodily Pain, General Health, Vitality, Social Functioning, Role-Emotional, and Mental Health. Two overall summary scores, the Physical Component Summary (PCS) and the Mental Component Summary (MCS) also can be computed. The SF-36 has proven useful in monitoring general and specific populations, comparing the relative burden of different diseases, differentiating the health benefits produced by different treatments, and in screening individual participants.

[0309] • 9 Hole Peg Test (9HPT)

[0310] The 9HPT is a finger dexterity test that is administered by a trained administrator. The participant is directed to put 9 pegs, one by one, onto and then off the holder board as quickly as possible starting with using only the dominant hand, and then repeated with the non-dominant hand. Longer completion times are associated with decreased finger dexterity.

[0311] • Timed 25 Foot Walk (T25FW)

[0312] The T25FW is a mobility test based on a timed walk of 25 feet that is administered by a trained administrator. The participant is directed to walk the clearly marked 25 foot distance as quickly as possible. Longer completion time corresponds with decreased mobility.

[0313] • Expanded Disability Status Scale (EPSS)

[0314] EPSS is used to measure the change in disability level in participants using a scale from 0 to 10. The higher the score, the greater the degree of disability. The EPSS is the standard scale for clinical trials in patients with Multiple Sclerosis (MS).

[0315] • Magnetic Resonance Imaging (MRI)

[0316] All participants will undergo MRI scanning of the brain according to the assessment schedule. MRI scans will be transmitted (including Screening) by the sites to the central MRI reading center, designated by Novartis, for quality checks and central read. Scans should be sent promptly after acquisition, generally within three (3) days of capture. If a scan is incomplete or incorrectly performed, the study site will be asked to repeat it as soon as possible. Further details PAT059856-PCT-SEC01 on the image acquisition can be found in the MRI manual of the central MRI reading center. Details regarding the central read can be found in the central MRI reading center independent review charter.

[0317] Gadolinium MRI contrast enhancement will be administered in this study for each participant using the same approach for consistency. Sequences include but are not limited to T1 hypointense images and T2-weighted images will be performed by the sites following the instructions in the central MRI reading center MRI manual. The central MRI reading center will perform lesion count and volume analysis to support the study endpoints and assess for safety findings.

[0318] MRI endpoints related to MS disease progression will include but not be limited to whole brain volume and changes, substructure volumes and changes, and volumes of T2 lesions and T1 -hypointense lesions. Safety MRI endpoints will include but not be limited to extent of T2 hyperintensity edema, new cerebral microbleeds, new lacunes, lateral ventricular volume changes, new or enlarging T2 lesions, and T1 Gd-enhancing lesion count. Additional MRI analyses and endpoints may be included to further explore the effects of rapcabtagene autoleucel in progressive MS.

[0319] Appropriateness of Efficacy Assessments

[0320] The neurostatus.net EDSS (Kurtzke 1983) has been the gold standard for evaluation of disability worsening (1 point worsening when EDSS scores < 5.5; 0.5 points if baseline score is > 5.5) in patients with MS which will be supported with valid and specific additional tools. The evaluation of the secondary outcome concerning the disease progression of rapcabtagene autoleucel administration is planned at 2 years which is considered as an adequate duration allowing observation of any potential progression in the study population of patient with PMS.

[0321] Of the additional disease progression assessments the SF-36 is a validated tool to measure quality of life in MS patients (Nortvedt et al 2000); the T25FW is the most commonly used measure for ambulation of MS patients (Goldman et al 2013); the 9HPT is a brief, quantitative test of upper extremity function (Mathiowetz et al 1985); the SDMT is a sensitive measure of impaired cognition (Smith 1982), and the MFIS, is a valid, reliable measure of fatigue-related symptoms and impacts in patients with PMS (Hudgens et al 2019). PAT059856-PCT-SEC01

[0322] Pharmacokinetics and Immunogenicity

[0323] For pharmacokinetic (PK) analysis, serial blood samples will be collected at specific time points to measure rapcabtagene autoleucel cellular kinetics in peripheral blood by flow cytometry and by qPCR, and to measure humoral and cellular immunogenicity, including neutralizing antibodies. Analytical methods for PK and immunogenicity assessments are listed in Table 3.

[0324] Table 3. Analytical methods associated with the PK and immunogenicity assessments PAT059856-PCT-SEC01

[0325] The absolute number of CD 19+ B cells in peripheral blood will be used as the PD marker to characterize B cell depletion resulting from rapcabtagene autoleucel treatment. The CD 19+ B cell counts will be measured by flow cytometry. The flow cytometry analysis will be performed using a validated panel that also includes the analysis of T cells and NK cells (TBNK panel).

[0326] EQUIVALENTS

[0327] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to certain embodiments, it is apparent that further embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

PAT059856-PCT-SEC01What is claimed is:

1. A method of treating a subject diagnosed with progressive multiple sclerosis comprising administering rapcabtagene autoleucel to the subject.

2. The method of claim 1, wherein the EDSS score of the subject is reduced by at least 0.5 points from a baseline EDSS score greater than or equal to 5.5.

3. The method of claim 1, wherein the EDSS score of the subject is reduced by at least 1.0 points from a baseline EDSS score less than 5.5.

4. The method of claim 1 or 2, wherein the reduction in the EDSS score is sustained for at least 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months.

5. The method of any of claims 1-4, wherein the total EDSS score of the patient is reduced to 6 or below, 5 or below, 4 or below, 3 or below, 2 or below, 1 or below, or 0.

6. The method of any of claims 1-5, wherein the subject has a reduction in the size and number of brain lesions as detected by magnetic resonance imaging (MRI).

7. The method of any of claims 1-6, wherein the subject has no new or enlarged T2 lesions, gadolinium-enhanced lesions, paramagnetic rim lesions, or slowly expanding lesions as detected by MRI.

8. The method of any of claims 1-7, wherein the subject has no new or additional brain atrophy as detected by MRI.

9. The method of claim 6, wherein the reduction in the size and number of brain lesions as detected by MRI is sustained for at least 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months.

10. The method of any of claims 7-8, wherein the subject has no new or enlarged T2 lesions, gadolinium-enhanced lesions, paramagnetic rim lesions, or slowly expanding lesions as detected by MRI for at least 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months.

11. The method of any of claims 1-10, wherein the subject achieves a reduction in SDMT score of at least 4 points, 5 points, 6 points, 7 points, 8 points, 9 points, or 10 points.PAT059856-PCT-SEC0112. The method of claim 11, wherein reduction in SDMT score is sustained for at least 3 months, 6 months, 12 months, 18 months, or 24 months.

13. The method of any of claims 1-12, wherein the subject achieves a reduction in SDMT score of at least 10%, 15%, 20%, 25%, or 30%.

14. The method of claim 13, wherein the reduction in SDMT score is sustained for at least 3 months, 6 months, 12 months, 18 months, or 24 months.

15. The method of any of claims 1-14, wherein the subject has a reduction MFIS score of at least 6 points, 10 points, 15 points, 20 points, or 25 points.

16. The method of claim 15, wherein the subject has a reduction in MFIS score of at least 6.3 points.

17. The method of any of claims 1-16, wherein reduction in MFIS score is sustained for at least 3 months, 6 months, 12 months, 18 months, or 24 months.

18. The method of claim 1-17, wherein the subject has an increase in a SF-36 score of at least 5 points, 10 points, 15 points, 20 points, 25 points, 30 points, 35 points, 40 points, 45 points, or 50 points.

19. The method of any of claims 1-18, wherein the increase in SF-36 score is sustained for at least 3 months, 6 months, 12 months, 18 months, or 24 months.

20. The method of claim 19, wherein the response is sustained for at least 3 months, 6 months, 12 months, or 18 months.

21. The method of any of claims 1-20, wherein the subject has a decrease in a NHPT test of at least 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

22. The method of claim 21, wherein the decrease in NHPT test is sustained for at least 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months.

23. The method of any of claims 1-22, wherein the subject has a decrease in T25FW time of at least 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

24. The method of claim 23, wherein the decrease in T25FW test is sustained for at least 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months.PAT059856-PCT-SEC0125. The method of any of claims 1-24, wherein the rapcabtagene autoleucel is administered at a dose of about 0.5 x 106- 1.25 x 109viable CAR-expressing cells.

26. The method of claim 25, wherein the rapcabtagene autoleucel is administered at a dose of 2.5 x 106, 7.5 x 106, or 12.5 x 106viable CAR-expressing cells.

27. The method of claim 25, wherein the rapcabtagene autoleucel is administered at a dose of 12.5 x 106viable CAR-expressing cells.

28. The method of claim 25, wherein the rapcabtagene autoleucel is administered at a dose of 2.5 x 106viable CAR-expressing cells29. The method of claim 25, wherein the rapcabtagene autoleucel is administered at a dose of 7.5 x 106viable CAR-expressing cells.

30. The method of any of claims 1-29, wherein the rapcabtagene autoleucel is administered in combination with a second therapy.

31. A method of treating a subject diagnosed with progressive multiple sclerosis comprising administering to the subject a population of cells engineered to express a CD19 CAR, said population comprising:(i) about the same percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells, as compared to the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ cells, in the same population of cells prior to being engineered to express the CAR;(ii) a change within about 5% to about 10% of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells, for example, as compared to the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ cells, in the same population of cells prior to being engineered to express the CAR;(iii) an increased percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells, for example, increased by at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3 -fold, as compared to the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ cells, in the same population of cells prior to being engineered to express the CAR;(iv) about the same percentage of central memory cells, for example, central memory T cells, for example, CD45RA- CCR7+ CD45RO+ T cells, as compared to the percentage of central memory cells, for example, central memory T cells, for example, CD45RA- CCR7+ CD45RO+ T cells, in the same population of cells prior to being engineered to express the CAR;(v) a change within about 5% to about 10% of central memory cells, for example, central memory T cells, for example, CD45RA- CCR7+ CD45RO+ T cells, as compared to the percentage of central memory cells, for example, central memory TPAT059856-PCT-SEC01 cells, for example, CD45RA- CCR7+ CD45RO+ T cells, in the same population of cells prior to being engineered to express the CAR;(vi) a decreased percentage of central memory cells, for example, central memory T cells, for example, CD45RA- CCR7+ CD45RO+ T cells, for example, decreased by at least 20, 25, 30, 35, 40, 45, or 50%, as compared to the percentage of central memory cells, for example, central memory T cells, for example, CD45RA- CCR7+ CD45RO+ T cells, in the same population of cells prior to being engineered to express the CAR;(vii) about the same percentage of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, as compared to the percentage of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, in the same population of cells prior to being engineered to express the CAR;(viii) a change within about 5% to about 10% of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, as compared to the percentage of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, in the same population of cells prior to being engineered to express the CAR; or(ix) an increased percentage of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, as compared to the percentage of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, in the same population of cells prior to being engineered to express the CAR.

32. The method of claim 31, wherein the EDSS score of the subject is reduced by at least 0.5 points from a baseline EDSS score greater than or equal to 5.5.

33. The method of claim 31, wherein the EDSS score of the subject is reduced by at least 1.0 points from a baseline EDSS score less than 5.5.

34. The method of claim 31 or 32, wherein the reduction in the EDSS score is sustained for at least 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months.

35. The method of any of claims 31-34, wherein the total EDSS score of the patient is reduced to 6 or below, 5 or below, 4 or below, 3 or below, 2 or below, 1 or below, or 0.

36. The method of any of claims 31-35, wherein the subject has a reduction in the size and number of brain lesions as detected by magnetic resonance imaging (MRI).PAT059856-PCT-SEC0137. The method of any of claims 31-36, wherein the subject has no new or enlarged T2 lesions, gadolinium-enhanced lesions, paramagnetic rim lesions, or slowly expanding lesions as detected by MRI.

38. The method of any of claims 31-37, wherein the subject has no new or brain atrophy as detected by MRI.

39. The method of claim 36, wherein the reduction in the size and number of brain lesions as detected by MRI is sustained for at least 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months.

40. The method of any of claims 37-38, wherein the subject has no new or enlarged T2 lesions, gadolinium-enhanced lesions, paramagnetic rim lesions, or slowly expanding lesions as detected by MRI for at least 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months.

41. The method of any of claims 31-40, wherein the subject achieves a reduction in SDMT score of at least 4 points, 5 points, 6 points, 7 points, 8 points, 9 points, or 10 points.

42. The method of claim 41, wherein reduction in SDMT score is sustained for at least 3 months, 6 months, 12 months, 18 months, or 24 months.

43. The method of any of claims 31-42, wherein the subject achieves a reduction in SDMT score of at least 10%, 15%, 20%, 25%, or 30%.

44. The method of claim 43, wherein the reduction in SDMT score is sustained for at least 3 months, 6 months, 12 months, 18 months, or 24 months.

45. The method of any of claims 31-44, wherein the subject has a reduction MFIS score of at least 6 points, 10 points, 15 points, 20 points, or 25 points.

46. The method of claim 45, wherein the subject has a reduction in MFIS score of at least 6.3 points.

47. The method of any of claims 31 -46, wherein reduction in MFIS score is sustained for at least 3 months, 6 months, 12 months, 18 months, or 24 months.

48. The method of claim 31-47, wherein the subject has an increase in a SF-36 score of at least 5 points, 10 points, 15 points, 20 points, 25 points, 30 points, 35 points, 40 points, 45 points, or 50 points.PAT059856-PCT-SEC0149. The method of any of claims 31-48, wherein the increase in SF-36 score is sustained for at least 3 months, 6 months, 12 months, 18 months, or 24 months.

50. The method of claim 49, wherein the response is sustained for at least 3 months, 6 months, 12 months, or 18 months.

51. The method of any of claims 31-50, wherein the subject has a decrease in a NHPT test of at least 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

52. The method of claim 51, wherein the decrease in NHPT test is sustained for at least 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months.

53. The method of any of claims 31-52, wherein the subject has a decrease in T25FW time of at least 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

54. The method of claim 53, wherein the decrease in T25FW test is sustained for at least 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months.

55. The method of any of claims 31-54, wherein the population of cells engineered to express a CD 19 CAR is administered at a dose of about 0.5 x 106- 1.25 X 109viable CAR-expressing cells.

56. The method of claim 55, wherein the population of cells engineered to express a CD 19 CAR is administered at a dose of 2.5 x 106, 7.5 x 106, or 12.5 x 106viable CAR-expressing cells.

57. The method of claim 55, wherein the population of cells engineered to express a CD 19 CARis administered at a dose of 12.5 x 106viable CAR-expressing cells.

58. The method of claim 55, wherein the population of cells engineered to express a CD19 CARis administered at a dose of 2.5 x 106viable CAR-expressing cells59. The method of claim 55, wherein the population of cells engineered to express a CD19 CARis administered at a dose of 7.5 x 106viable CAR-expressing cells.

60. A method of reducing a EDSS score in a subject with progressive multiple sclerosis the method comprising administering rapcabtagene autoleucel to the subject, wherein the EDSS score is reduced by at least 0.5 points as compared to baseline.PAT059856-PCT-SEC0161. The method of claim 60, wherein the EDSS score is reduced by at least 1.0 points as compared to baseline.

62. The method of claim 60 or 61, wherein the reduction in EDSS score is sustained for at least 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months.

63. The method of any of claims 60-62, wherein the rapcabtag ene autoleucel is administered at a dose of 2.5-, 7.5-, or 12.5 x 10A6 viable CAR-expressing cells.

64. A method of reducing the size and number of brain lesions of a subject as detected by magnetic resonance imaging (MRI) comprising administering rapcabtagene autoleucel to the subject.

65. The method of claim 64, wherein the reduction in size and number of brain lesions is sustained for at least 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months.

66. The method of claim 64 or 65, wherein the rapcabtagene autoleucel is administered at a dose of 2.5-, 7.5-, or 12.5 x 10A6 viable CAR-expressing cells.

67. A method of causing stabilizing the disease progression of a subject with progressive multiple sclerosis, the method comprising administering rapcabtagene autoleucel to the subject, wherein the disease progression is determined to be stabilized by (1) no relapses, (2) no new or enlarged T2 or gadolinium-enhanced T1 lesions as determined by magnetic resonance imaging, and (3) no disability progression as measured by an increase in EDSS score.

68. The method of claim 67, wherein the disease progression is stable for at least 3 months, 6 months, 12 months, 18 months, or 24 months following administration.

69. The method of claim 67 or 68, wherein the rapcabtagene autoleucel is administered at a dose of 2.5-, 7.5-, or 12.5 x 10A6 viable CAR-expressing cells.

70. A method of reducing a SMDT score in a subject with progressive multiple sclerosis, the method comprising administering rapcabtagene autoleucel to the subject, wherein the SMDT score is reduced by 4 or more points.

71. The reducing a SMDT score in a subject with progressive multiple sclerosis, the method comprising administering rapcabtagene autoleucel to the subject, wherein the SMDT score is reduced by 10% or more.

72. The method of claim 70 or 71, wherein the reduction is sustained for at least 3 months, 6 months, 9 months, 12 months, 18 months or 24 months.PAT059856-PCT-SEC0173. The method of any of claims 70-72, wherein the rapcabtagene autoleucel is administered at a dose of 2.5-, 7.5-, or 12.5 x 10A6 viable CAR-expressing cells.

74. A method of reducing the MFIS score of a subject with progressive multiple sclerosis, the method comprising administering rapcabtagene autoleucel to the subject at a dose of 2.5-, 7.5-, or 12.5 x 10A6 viable CAR-expressing cells.

75. A method of increasing the SF-36 score of a subject with progressive multiple sclerosis, the method comprising administering rapcabtagene autoleucel to the subject at a dose of 2.5-, 7.5-, or 12.5 x 10A6 viable CAR-expressing cells.

76. A method of reducing the NHPT time of a subject with progressive multiple sclerosis, the method comprising administering rapcabtagene autoleucel to the subject at a dose of 2.5-, 7.5-, or 12.5 x 10A6 viable CAR-expressing cells.

77. A method of reducing the T25FW time of a subject with progressive multiple sclerosis, the method comprising administering rapcabtagene autoleucel to the subject at a dose of 2.5-, 7.5-, or 12.5 x 10A6 viable CAR-expressing cells78. Rapcabtagene autoleucel for use in a method of treating a subject having progressive multiple sclerosis, said method comprising administering to the subject rapcabtagene autoleucel at a dose of 2.5-, 7.5-, or 12.5 x 10A6 cells.

79. A pharmaceutical composition comprising the rapcabtagene autoleucel of claim 78 and a pharmaceutically acceptable carrier.

80. A method of treating a subject with progressive multiple sclerosis comprising administering to the subject rapcabtagene autoleucel, wherein the rapcabtagene autoleucel is made by a method comprising:(i) contacting a population of T-cells derived from the subject with SLE with an agent that stimulates a CD3 / TCR complex;(ii) contacting the population of T-cells with a nucleic acid molecule encoding a CD 19 CAR, thereby providing a population T cells comprising the CD 19 CAR(iii) harvesting the population of T-cells for storage or administration, wherein(a) step (ii) is performed together with step (i) or no later than 20 hours after the beginning of step (i) and step (iii) is performed no later than 30 hours after the beginning of step (i);(b) step (ii) is performed together with step (i) or no later than 20 hours after the beginning of step (i) and step (iii) is performed no later than 30 hours after the beginning of step (ii); or,PAT059856-PCT-SEC01(c) the population of cells from step (iii) are not expanded as assessed by the number of living cells compared to the population of cells at the beginning of step (i).

81. The method of claim 80, wherein:(a) the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells, in the population of cells from step (iii) is the same as or differs by no more than 5 or 10% from the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ cells, in the population of cells at the beginning of step (i);(b) the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells, in the population of cells from step (iii) is increased by, for example, at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3-fold, as compared to the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ cells, in the population of cells at the beginning of step (i);(c) the percentage of CAR-expressing naive T cells, for example, CAR-expressing CD45RA+ CD45RO- CCR7+ T cells in the population of cells increases during the duration of step (ii), for example, increases by, for example, at least 30, 35, 40, 45, 50, 55, or 60%, between 18-24 hours after the beginning of step (ii); or(d) the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells, in the population of cells from step (iii) does not decrease, or decreases by no more than 5 or 10%, as compared to the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ cells, in the population of cells at the beginning of step (i).

82. The method of any one of claims 80 or 81, wherein:(a) the population of cells from step (iii) shows a higher percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells (for example, at least 10, 20, 30, or 40% higher), compared with cells made by an otherwise similar method in which step (iii) is performed more than 26 hours after the beginning ofPAT059856-PCT-SEC01 step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the beginning of step (i);(b) the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells, in the population of cells from step (iii) is higher (for example, at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3-fold higher) than the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells, in cells made by an otherwise similar method in which step (iii) is performed more than 26 hours after the beginning of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the beginning of step (i);(c) the percentage of CAR-expressing naive T cells, for example, CAR-expressing CD45RA+ CD45RO- CCR7+ T cells, in the population of cells from step (iii) is higher (for example, at least 4, 6, 8, 10, or 12-fold higher) than the percentage of CAR- expressing naive T cells, for example, CAR-expressing CD45RA+ CD45RO- CCR7+ T cells, in cells made by an otherwise similar method in which step (iii) is performed more than 26 hours after the beginning of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the beginning of step (i);(d) the population of cells from step (iii) shows a higher percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells (for example, at least 10, 20, 30, or 40% higher), compared with cells made by an otherwise similar method which further comprises, after step (ii) and prior to step (iii), expanding the population of cells (for example, T cells) in vitro for more than 3 days, for example, for 5, 6, 7, 8 or 9 days;(e) the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells, in the population of cells from step (iii) is higher (for example, at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3-fold higher) than the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells, in cells made by an otherwise similar method which further comprises, after step (ii) and prior to step (iii), expanding the population of cells (for example, T cells) in vitro for more than 3 days, for example, for 5, 6, 7, 8 or 9 days; or(f) the percentage of CAR-expressing naive T cells, for example, CAR-expressing CD45RA+ CD45RO- CCR7+ T cells, in the population of cells from step (iii) is higherPAT059856-PCT-SEC01(for example, at least 4, 6, 8, 10, or 12-fold higher) than the percentage of CAR- expressing naive T cells, for example, CAR-expressing CD45RA+ CD45RO- CCR7+ T cells, in cells made by an otherwise similar method which further comprises, after step (ii) and prior to step (iii), expanding the population of cells (for example, T cells) in vitro for more than 3 days, for example, for 5, 6, 7, 8 or 9 days.

83. The method of any one of claims 80-82, wherein:(a) the percentage of central memory cells, for example, central memory T cells, for example, CD95+ central memory T cells, in the population of cells from step (iii) is the same as or differs by no more than 5 or 10% from the percentage of central memory cells, for example, central memory T cells, for example, CD95+ central memory T cells, in the population of cells at the beginning of step (i);(b) the percentage of central memory cells, for example, central memory T cells, for example, CCR7+CD45RO+ T cells, in the population of cells from step (iii) is reduced by at least 20, 25, 30, 35, 40, 45, or 50%, as compared to the percentage of central memory cells, for example, central memory T cells, for example, CCR7+CD45RO+ T cells, in the population of cells at the beginning of step (i);(c) the percentage of CAR-expressing central memory T cells, for example, CAR- expressing CCR7+CD45RO+ cells, decreases during the duration of step (ii), for example, decreases by, for example, at least 8, 10, 12, 14, 16, 18, or 20%, between 18-24 hours after the beginning of step (ii); or(d) the percentage of central memory cells, for example, central memory T cells, for example, CCR7+CD45RO+ T cells, in the population of cells from step (iii) does not increase, or increases by no more than 5 or 10%, as compared to the percentage of central memory cells, for example, central memory T cells, for example, CCR7+CD45RO+ T cells, in the population of cells at the beginning of step (i).

84. The method of any one of claims 80-83, wherein:(a) the population of cells from step (iii) shows a lower percentage of central memory cells, for example, central memory T cells, for example, CD95+ central memoryPAT059856-PCT-SEC01T cells (for example, at least 10, 20, 30, or 40% lower), compared with cells made by an otherwise similar method in which step (iii) is performed more than 26 hours after the beginning of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the beginning of step (i);(b) the percentage of central memory cells, for example, central memory T cells, for example, CCR7+CD45RO+ T cells in the population of cells from step (iii) is lower (for example, at least 20, 30, 40, or 50% lower) than the percentage of central memory cells, for example, central memory T cells, for example, CCR7+CD45RO+ T cells, in cells made by an otherwise similar method in which step (iii) is performed more than 26 hours after the beginning of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the beginning of step (i);(c) the percentage of CAR-expressing central memory T cells, for example, CAR- expressing CCR7+CD45RO+ T cells in the population of cells from step (iii) is lower (for example, at least 10, 20, 30, or 40% lower) than the percentage of CAR-expressing central memory T cells, for example, CAR-expressing CCR7+CD45RO+ T cells, in cells made by an otherwise similar method in which step (iii) is performed more than 26 hours after the beginning of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the beginning of step (i);(d) the population of cells from step (iii) shows a lower percentage of central memory cells, for example, central memory T cells, for example, CD95+ central memory T cells (for example, at least 10, 20, 30, or 40% lower), compared with cells made by an otherwise similar method which further comprises, after step (ii) and prior to step (iii), expanding the population of cells (for example, T cells) in vitro for more than 3 days, for example, for 5, 6, 7, 8 or 9 days;(e) the percentage of central memory cells, for example, central memory T cells, for example, CCR7+CD45RO+ T cells in the population of cells from step (iii) is lower (for example, at least 20, 30, 40, or 50% lower) than the percentage of central memory cells, for example, central memory T cells, for example, CCR7+CD45RO+ T cells, in cells made by an otherwise similar method which further comprises, after step (ii) and prior to step (iii), expanding the population of cells (for example, T cells) in vitro for more than 3 days, for example, for 5, 6, 7, 8 or 9 days; orPAT059856-PCT-SEC01(f) the percentage of CAR-expressing central memory T cells, for example, CAR- expressing CCR7+CD45RO+ T cells in the population of cells from step (iii) is lower (for example, at least 10, 20, 30, or 40% lower) than the percentage of CAR-expressing central memory T cells, for example, CAR-expressing CCR7+CD45RO+ T cells, in cells made by an otherwise similar method which further comprises, after step (ii) and prior to step (iii), expanding the population of cells (for example, T cells) in vitro for more than 3 days, for example, for 5, 6, 7, 8 or 9 days.

85. The method of any one of claims 80-84, wherein:(a) the percentage of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, in the population of cells from step (iii) is increased, as compared to the percentage of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, in the population of cells at the beginning of step (i);(b) the percentage of CAR-expressing stem memory T cells, for example, CAR-expressing CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, in the population of cells from step (iii) is increased, as compared to the percentage of CAR- expressing stem memory T cells, for example, CAR-expressing CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, in the population of cells at the beginning of step (i);(c) the percentage of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, in the population of cells from step (iii) is higher than the percentage of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, in cells made by an otherwise similar method in which step (iii) is performed more than 26 hours after the beginning of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the beginning of step (i); or(d) the percentage of CAR-expressing stem memory T cells, for example, CAR-expressing CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, in the population of cells from step (iii) is higher than the percentage of CAR-expressing stem memory T cells, for example, CAR-expressing CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, in cells made by an otherwise similar method in which stepPAT059856-PCT-SEC01(iii) is performed more than 26 hours after the beginning of step (i), for example, more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the beginning of step (i);(e) the percentage of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, in the population of cells from step (iii) is higher than the percentage of stem memory T cells, for example, CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, in cells made by an otherwise similar method which further comprises, after step (ii) and prior to step (iii), expanding the population of cells (for example, T cells) in vitro for more than 3 days, for example, for 5, 6, 7, 8 or 9 days; or(f) the percentage of CAR-expressing stem memory T cells, for example, CAR-expressing CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, in the population of cells from step (iii) is higher than the percentage of CAR-expressing stem memory T cells, for example, CAR-expressing CD45RA+CD95+IL-2 receptor P+CCR7+CD62L+ T cells, in cells made by an otherwise similar method which further comprises, after step (ii) and prior to step (iii), expanding the population of cells (for example, T cells) in vitro for more than 3 days, for example, for 5, 6, 7, 8 or 9 days.

86. A method of depleting the B cells of a subject with progressive multiple sclerosis, the method comprising administering rapcabtagene autoleucel to the subject at a dose of 2.5-, 7.5-, or 12.5 x 10A6 viable CAR-expressing cells.

87. The method of any of claims 1-86, wherein the subject has experienced breakthrough disease progression while on a therapy.

88. The method of claim 87, wherein the highly efficacious therapy is beta interferon, dimethylfumarate, fingolimod, ponesimod, ozanlmod, siponimod, teriflunomide, miltoxantrone, cyclophosphamide, rituximab, ocrelizumab, natalizumab, ofatumumab, ublituximab, or alemtuzumab.

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