Treating autoimmune disorders using chimeric antigen receptor therapy

Abstract

The present invention provides methods for generating immune effector cells (e.g., T cells, NK cells) that express chimeric antigen receptors (CARs) and compositions produced by such methods and their therapeutic uses for treating autoimmune diseases or disorders.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 375,776, filed September 15, 2022, and U.S. Provisional Patent Application No. 63 / 507,141, filed June 9, 2023, the contents of which are incorporated herein by reference in their entireties.

[0002] The present invention relates generally to methods for producing immune effector cells (e.g., T cells or NK cells) engineered to express chimeric antigen receptors (CARs), compositions comprising same, and therapeutic uses thereof for treating autoimmune diseases or disorders. [Background technology]

[0003] Current treatments for severe autoimmune diseases such as systemic lupus erythematosus (SLE) include traditional immunomodulatory and anti-inflammatory agents, such as antimalarials, glucocorticoids, and immunosuppressants (e.g., methotrexate, azathioprine, mycophenolate, and cyclophosphamide), and biologics (such as belimumab, and more recently anifrolumab and rituximab, which are commonly used in severe stages of the disease). Patients with severe, refractory SLE (srSLE), with or without renal involvement, have very limited treatment options after failure of immunosuppressive and biologic therapies. Autologous stem cell transplantation (ASCT) can be performed, but it remains experimental and is associated with severe toxicities, including death. Summary of the Invention [Problem to be solved by the invention]

[0004] Thus, there is an unmet need for new treatments for severe autoimmune diseases, including srSLE. [Means for solving the problem]

[0005] The present disclosure relates to methods of generating immune effector cells (e.g., T cells or NK cells) engineered to express a CAR, and compositions produced using such methods. The present disclosure relates to the treatment of diseases of interest, such as autoimmune diseases or disorders, including lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with severe pulmonary involvement (e.g., as seen in autoHSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-associated myositis, e.g., antisynthetase syndrome with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory sheath syndrome, and the like. Also disclosed are methods of using such compositions to treat rheumatoid arthritis, severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-associated disease (MOGAD), multiple sclerosis (MS)), severe refractory rheumatoid arthritis, antibody-mediated neuroimmune diseases (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG-associated disease), NMDAR+ encephalitis or antibody-related paraneoplastic neurological diseases), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis.

[0006] In one aspect, the present disclosure provides a method for treating an autoimmune disease or disorder, such as lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with severe pulmonary involvement (e.g., as seen in autoHSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-associated myositis, e.g., antisynthetase syndrome with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjogren's disease, severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), optic nerve spinal cord injury, 1. A method of treating a subject having a disease such as myelitis (NMO), MOG-associated disease (MOGAD), multiple sclerosis (MS), severe refractory rheumatoid arthritis, an antibody-mediated neuroimmune disease (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG associated disease), NMDAR+ encephalitis or antibody-associated paraneoplastic neurological disease), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis, comprising administering to the subject a population of cells (e.g., T cells) that express, or comprise a nucleic acid configured to express, a CD19 chimeric antigen receptor (CAR), wherein the population of cells (i) contacting (e.g., binding) a population of cells (e.g., T cells, e.g., T cells isolated from frozen or fresh leukapheresis products) with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells; (ii) contacting a population of cells (e.g., T cells) with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding a CAR, thereby providing a population of cells (e.g., T cells) comprising the nucleic acid molecule, wherein the CAR comprises a CD19 antigen-binding domain (a "CD19 CAR"); and (iii) recovering the population of cells (e.g., T cells) for storage (e.g., reformulating the population of cells in cryopreservation medium) or administration. and (a) step (ii) is carried out together with step (i) or within 20 hours after the start of step (i), such as within 12, 13, 14, 15, 16, 17 or 18 hours after the start of step (i), such as within 18 hours after the start of step (i); and step (iii) is carried out within 30 (e.g. 26) hours after the start of step (i), such as within 22, 23, 24, 25, 26, 27, 28, 29 or 30 hours after the start of step (i), for example within 24 hours after the start of step (i); (b) step (ii) is carried out together with step (i) or within 20 hours after the start of step (i), such as within 12, 13, 14, 15, 16, 17 or 18 hours after the start of step (i), such as within 18 hours after the start of step (i); and Step (iii) is carried out within 30 hours after the start of step (ii), such as within 22, 23, 24, 25, 26, 27, 28, 29 or 30 hours after the start of step (ii); or (c) the population of cells from step (iii) is not expanded or is expanded by no more than 5, 10, 15, 20, 25, 30, 35 or 40%, e.g., no more than 10%, compared to the population of cells at the start of step (i), e.g., as assessed by viable cell count; Optionally, the nucleic acid molecule of step (ii) is on a viral vector, and optionally the nucleic acid molecule of step (ii) is an RNA molecule on the viral vector, and optionally step (ii) comprises transducing a population of cells (e.g., T cells) with the viral vector comprising the nucleic acid molecule encoding the CAR.

[0007] In some embodiments, the agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3 (e.g., an anti-CD3 antibody), and the agent that stimulates a costimulatory molecule is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof; optionally, the agent that stimulates the CD3 / TCR complex or the agent that stimulates a costimulatory molecule is an antibody (e.g., a single domain antibody (e.g., a heavy chain variable domain antibody), a peptibody, a Fab fragment, or an scFv), a small molecule, or a ligand (e.g., a naturally occurring ligand, recombinant or chimeric ligand), optionally the agent that stimulates the CD3 / TCR complex or the agent that stimulates the costimulatory molecule does not comprise beads, optionally the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody, and the agent that stimulates the costimulatory molecule comprises an anti-CD28 antibody, optionally the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody covalently bound to a colloidal polymer nanomatrix, and the agent that stimulates the costimulatory molecule comprises an anti-CD28 antibody covalently bound to a colloidal polymer nanomatrix, optionally the agent that stimulates the CD3 / TCR complex and the agent that stimulates the costimulatory molecule comprise T Cell TransAct™.

[0008] In some embodiments, step (i) increases the percentage of CAR-expressing cells in the population of cells from step (iii), e.g., the population of cells from step (iii) exhibits a higher percentage of CAR-expressing cells (e.g., at least 10, 20, 30, 40, 50, or 60% higher) compared to cells produced by an otherwise similar method without step (i).

[0009] In some embodiments, (a) whether the percentage of naive cells, e.g., naive T cells, e.g., 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, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ cells, in the population of cells at the start of step (i); (b) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ T cells, in the population of cells from step (iii) is increased, e.g., by at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3-fold, compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ cells, in the population of cells at the start of step (i); (c) the percentage of CAR-expressing naive T cells, e.g., CAR-expressing CD45RA+ CD45RO- CCR7+ T cells, in the population of cells increases during step (ii), e.g., increases by at least 30, 35, 40, 45, 50, 55, or 60% in the 18-24 hours following initiation of step (ii); or (d) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ T cells, in the population of cells from step (iii) is not reduced, or is reduced by no more than 5 or 10%, compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ cells, in the population of cells at the start of step (i).

[0010] In some embodiments, (a) does the population of cells from step (iii) exhibit a higher percentage (e.g., at least 10, 20, 30, or 40% higher) of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ T cells, compared to cells produced by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (b) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ T cells, in the population of cells from step (iii) is higher (e.g., 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, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ T cells, in cells made by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (c) the percentage of CAR-expressing naive T cells, e.g., CAR-expressing CD45RA+ CD45RO- CCR7+ T cells, in the population of cells from step (iii) is higher (e.g., at least 4, 6, 8, 10, or 12-fold higher) than the percentage of CAR-expressing naive T cells, e.g., CAR-expressing CD45RA+ CD45RO- CCR7+ T cells, in cells made by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (d) does the population of cells from step (iii) exhibit a higher percentage (e.g., at least 10, 20, 30, or 40% higher) of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ T cells, compared to cells produced by a similar method but further comprising, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days; (e) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ T cells, in the population of cells from step (iii) is higher (e.g., 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, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ T cells, in cells made by a similar method but further including, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days; or (f) The percentage of CAR-expressing naive T cells, e.g., CAR-expressing CD45RA+ CD45RO- CCR7+ T cells, in the population of cells from step (iii) is higher (e.g., at least 4, 6, 8, 10, or 12-fold higher) than the percentage of CAR-expressing naive T cells, e.g., CAR-expressing CD45RA+ CD45RO- CCR7+ T cells, in cells made by a similar method but further including, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days.

[0011] In some embodiments, (a) the percentage of central memory cells, e.g., central memory T cells, e.g., 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, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells at the start of step (i); (b) the percentage of central memory cells, e.g., central memory T cells, e.g., 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% compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in the population of cells at the start of step (i); (c) the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+ CD45RO+ cells, decreases during step (ii), e.g., by at least 8, 10, 12, 14, 16, 18, or 20% in the 18-24 hours following initiation of step (ii); or (d) the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in the population of cells from step (iii) is not increased, or is increased by no more than 5 or 10%, compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in the population of cells at the start of step (i).

[0012] In some embodiments, (a) does the population of cells from step (iii) exhibit a lower percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells (e.g., at least 10, 20, 30, or 40% lower) than cells produced by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (b) the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in the population of cells from step (iii) is lower (e.g., at least 20, 30, 40, or 50% lower) than the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in cells produced by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (c) the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+ CD45RO+ T cells, in the population of cells from step (iii) is lower (e.g., at least 10, 20, 30, or 40% lower) than the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+ CD45RO+ T cells, in cells generated by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (d) the population of cells from step (iii) exhibits a lower percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells (e.g., at least 10, 20, 30, or 40% lower), compared to cells produced by a similar method but further comprising, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days; (e) the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in the population of cells from step (iii) is lower (e.g., at least 20, 30, 40, or 50% lower) than the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in cells made by a similar method but further including, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days; or (f) The percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+ CD45RO+ T cells, in the population of cells from step (iii) is lower (e.g., at least 10, 20, 30, or 40% lower) than the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+ CD45RO+ T cells, in cells made by a similar method but further including, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days.

[0013] In some embodiments, (a) whether the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) is increased compared to the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells at the start of step (i); (b) whether the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) is increased compared to the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells at the start of step (i); (c) the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) is greater than the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in cells made by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); or (d) is the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) higher than the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in cells made by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (e) the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) is greater than the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in cells made by a similar method but further comprising expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii); or (f) The percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) is higher than the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in cells made by a similar method but further including, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days.

[0014] In some embodiments, (a) the median GeneSetScore (Up TEM vs. Down TSCM) of the population of cells from step (iii) is approximately the same as or differs (e.g., increases by no more than) about 25, 50, 75, 100, or 125% from the median GeneSetScore (Up TEM vs. Down TSCM) of the population of cells at the start of step (i); (b) Median GeneSetScore (Up TEM vs. Down TSCM) of the population of cells from step (iii) or cells produced by a similar method, except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i); or Cells made by a similar method, but further comprising expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). lower (e.g., at least about 100, 150, 200, 250, or 300% lower) than the median GeneSetScore (Up TEM vs. Down TSCM); (c) the median GeneSetScore (Up Treg vs. Down Teff) of the population of cells from step (iii) is approximately the same as or differs (e.g., increases 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 at the start of step (i); (d) Median GeneSetScore of the population of cells from step (iii) (Up Treg vs. Down Teff) or cells produced by a similar method, except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i); or Cells made by a similar method, but further comprising expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). lower (e.g., at least about 50, 100, 125, 150, or 175% lower) than the median GeneSetScore (Up Treg vs. Down Teff) of (e) the median GeneSetScore (Down stemness) of the population of cells from step (iii) is approximately the same as or differs from (e.g., increases by) no more than about 25, 50, 100, 150, 200, or 250% of the median GeneSetScore (Down stemness) of the population of cells at the start of step (i); (f) The median GeneSetScore (Down stemness) of the population of cells from step (iii) is or cells produced by a similar method, except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i); or Cells made by a similar method, but further comprising expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). lower than the median GeneSetScore (Downstemness) (e.g., at least about 50, 100, or 125% lower); (g) the median GeneSetScore (Up hypoxia) of the population of cells from step (iii) is approximately the same as or differs (e.g., increases by no more than) about 125, 150, 175, or 200% from the median GeneSetScore (Up hypoxia) of the population of cells at the start of step (i); (h) The median GeneSetScore (Up hypoxia) of the population of cells from step (iii) is or cells produced by a similar method, except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i); or Cells made by a similar method, but further comprising expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). lower (e.g., at least about 40, 50, 60, 70, or 80% lower) than the median GeneSetScore (Up hypoxia); (j) the median GeneSetScore (Up autophagy) of the population of cells from step (iii) is about the same as or differs from (e.g., increases by) no more than about 180, 190, 200, or 210% of the median GeneSetScore (Up autophagy) of the population of cells at the start of step (i); or (k) The median GeneSetScore (Up autophagy) of the population of cells from step (iii) is or cells produced by a similar method, except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i); or Cells made by a similar method, but further comprising expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). Lower (e.g., at least 20, 30, or 40% lower) than the median GeneSetScore (Up autophagy).

[0015] In some embodiments, the population of cells from step (iii), after being incubated with cells expressing the antigen recognized by the CAR, secrete IL-2 at a higher (e.g., at least 2, 4, 6, 8, 10, 12 or 14 fold) level than cells made by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i), or than cells made by a similar method except that the method further includes expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8 or 9 days, after step (ii) and before step (iii).

[0016] In some embodiments, the population of cells from step (iii), after administration in vivo to a subject, persists longer or proliferates to a greater extent than cells made by a similar method except that step (iii) is performed more than 26 hours after the initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the initiation of step (i), or compared to cells made by a similar method except that step (iii) further includes expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8 or 9 days, after step (ii) and before step (iii).

[0017] In some embodiments, the population of cells from step (iii), after administration in vivo to a subject, exhibits a stronger activity (e.g., a lower dose, e.g., 0.15×10) than cells produced by a similar method except that step (iii) is performed more than 26 hours after the initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the initiation of step (i), or than cells produced by a similar method except that step (iii) further comprises expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). 6 , 0.2 × 10 6 , 0.25×10 6 or 0.3 x 10 6 (More potent activity at doses of 100 or fewer viable CAR-expressing cells).

[0018] In some embodiments, the population of cells from step (iii) is not expanded, or is expanded by no more than 5, 10, 15, 20, 25, 30, 35 or 40%, e.g., no more than 10%, compared to the population of cells at the start of step (i), e.g., as assessed by viable cell count, and optionally the number of viable cells in the population of cells from step (iii) is reduced from the number of viable cells in the population of cells at the start of step (i).

[0019] In some embodiments, the population of cells from step (iii) is not expanded or is expanded for less than 2 hours, for example less than 1 or 1.5 hours, compared to the population of cells at the start of step (i).

[0020] In some embodiments, steps (i) and / or (ii) are performed in cell culture medium (e.g., serum-free medium) containing IL-2, IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)), IL-7, IL-21, IL-6 (e.g., IL-6 / sIL-6Ra), an LSD1 inhibitor, a MALT1 inhibitor, or a combination thereof.

[0021] In some embodiments, steps (i) and / or (ii) are performed in a serum-free cell culture medium comprising a serum replacement, hi some embodiments, the serum replacement is CTS™ Immune Cell Serum Replacement (ICSR).

[0022] In some embodiments, the method further comprises, prior to step (i): (iv) (optionally) receiving a fresh leukapheresis product (or an alternative source of hematopoietic tissue, such as a fresh whole blood product, a fresh bone marrow product, or a fresh organ biopsy or resection (e.g., a fresh product from a thymus removal)) from an entity, e.g., a laboratory, hospital, or healthcare provider; and (v) isolating the population of cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells) contacted in step (i) from a fresh leukapheresis product (or an alternative source of hematopoietic tissue, such as a fresh whole blood product, a fresh bone marrow product, or a fresh organ biopsy or resection (e.g., a fresh product from a thymus removal)). and optionally further comprising: Step (iii) is carried out within 35 hours after the start of step (v), such as within 27, 28, 29, 30, 31, 32, 33, 34 or 35 hours after the start of step (v), such as within 30 hours after the start of step (v); or The population of cells from step (iii) is not expanded, or is expanded by no more than 5, 10, 15, 20, 25, 30, 35 or 40%, for example no more than 10%, compared to the population of cells at the end of step (v), e.g., as assessed by viable cell count.

[0023] In some embodiments, the method further comprises, prior to step (i), receiving cryopreserved T cells isolated from a leukapheresis product (or an alternative source of hematopoietic tissue, such as whole blood, bone marrow, or cryopreserved T cells isolated from an organ biopsy or resection (e.g., thymus removal)) from an entity, e.g., a laboratory, hospital, or healthcare provider.

[0024] In some embodiments, the method further comprises, prior to step (i): (iv) (optionally) receiving a cryopreserved leukapheresis product (or an alternative source of hematopoietic tissue, such as a cryopreserved whole blood product, a cryopreserved bone marrow product, or a cryopreserved organ biopsy or resection (e.g., a cryopreserved product from a thymus removal)) from an entity, e.g., a laboratory, hospital, or healthcare provider; and (v) isolating the population of cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells) contacted in step (i) from a cryopreserved leukapheresis product (or an alternative source of hematopoietic tissue, such as a cryopreserved whole blood product, a cryopreserved bone marrow product, or a cryopreserved organ biopsy or resection (e.g., a cryopreserved product from a thymus removal)). and optionally further comprising: Step (iii) is carried out within 35 hours after the start of step (v), such as within 27, 28, 29, 30, 31, 32, 33, 34 or 35 hours after the start of step (v), such as within 30 hours after the start of step (v); or The population of cells from step (iii) is not expanded, or is expanded by no more than 5, 10, 15, 20, 25, 30, 35 or 40%, for example no more than 10%, compared to the population of cells at the end of step (v), e.g., as assessed by viable cell count.

[0025] In some embodiments, the method further comprises step (vi): Culturing a portion of the population of cells from step (iii) for at least 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7 days, e.g., at least 2 days and no more than 7 days, and measuring the level of CAR expression in the portion (e.g., measuring the percentage of viable CAR-expressing cells in the portion). and optionally further comprising: Step (iii) involves harvesting and freezing a population of cells (e.g., T cells), and step (vi) involves thawing a portion of the population of cells from step (iii), culturing the portion for at least 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7 days, such as at least 2 days and not more than 7 days, and measuring the level of CAR expression in the portion (e.g., measuring the percentage of viable CAR-expressing cells in the portion).

[0026] In some embodiments, the population of cells at the start of step (i) or step (1) is enriched for IL6R-expressing cells (e.g., cells positive for IL6Rα and / or IL6Rβ). In some embodiments, the population of cells at the start of step (i) or step (1) comprises 50, 60, or 70% or more IL6R-expressing cells (e.g., cells positive for IL6Rα and / or IL6Rβ).

[0027] In some embodiments, steps (i) and (ii) or steps (1) and (2) are performed in a cell culture medium containing IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)). In some embodiments, the IL-15 increases the ability of the population of cells to proliferate, e.g., after 10, 15, 20, or 25 days. In some embodiments, the IL-15 increases the percentage of IL6Rβ-expressing cells in the population of cells.

[0028] In one aspect, the present disclosure provides a method for treating an autoimmune disease or disorder, such as lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with severe pulmonary involvement (e.g., as seen in autoHSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-associated myositis, e.g., antisynthetase syndrome associated with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), 1. A method of treating a subject having refractory Sjogren's disease, a severe refractory neuroimmune disease (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-associated disease (MOGAD), multiple sclerosis (MS)), severe refractory rheumatoid arthritis, an antibody-mediated neuroimmune disease (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG-associated disease), NMDAR+ encephalitis or antibody-associated paraneoplastic neurological disease), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis, comprising administering to the subject a population of cells engineered to express a CD19 CAR (a "population of CAR-expressing cells"), wherein the population comprises: (a) approximately the same percentage of naive cells, e.g., naive T cells, e.g., CD45RO− CCR7+ T cells, compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RO− CCR7+ cells, in the same population of cells before they are engineered to express a CAR; (b) a change within about 5% to about 10% of naive cells, e.g., naive T cells, e.g., CD45RO− CCR7+ T cells, as compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RO− CCR7+ cells, in the same population of cells before they were engineered to express a CAR; (c) an increased percentage of naive cells, e.g., naive T cells, e.g., CD45RO- CCR7+ T cells, as compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RO- CCR7+ cells, in the same population of cells before they were engineered to express a CAR, e.g., by at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3-fold; (d) approximately the same percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in the same population of cells before they were engineered to express a CAR; (e) a change within about 5% to about 10% of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in the same population of cells before they were engineered to express a CAR; (f) a reduced percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in the same population of cells before they were engineered to express a CAR; (g) approximately the same percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, compared to the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the same population of cells before they were engineered to express a CAR; (h) a change within about 5% to about 10% of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, compared to the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the same population of cells before they were engineered to express a CAR; or (i) an increased percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, compared to the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the same population of cells before they were engineered to express a CAR; The present invention provides a method comprising:

[0029] In one aspect, the present disclosure provides a method for treating an autoimmune disease or disorder, such as lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with severe pulmonary involvement (e.g., as seen in autoHSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-associated myositis, e.g., antisynthetase syndrome associated with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), 1. A method of treating a subject with relapsing Sjogren's disease, a severe refractory neuroimmune disease (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-associated disease (MOGAD), multiple sclerosis (MS)), severe refractory rheumatoid arthritis, an antibody-mediated neuroimmune disease (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG-associated disease), NMDAR+ encephalitis or antibody-associated paraneoplastic neurological disease), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis, comprising administering to the subject a population of cells engineered to express a CD19 CAR (a "population of CAR-expressing cells"); (a) whether the median GeneSetScore (Up TEM vs. Down TSCM) of a population of cells is approximately the same as or differs by (e.g., increases 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 before they were engineered to express a CAR; (b) the median GeneSetScore (Up Treg vs. Down Teff) of the population of cells is approximately the same as or differs (e.g., increases by less than) about 25, 50, 100, 150, or 200% from the median GeneSetScore (Up Treg vs. Down Teff) of the population of cells before they were engineered to express a CAR; (c) the median GeneSetScore (Down stemness) of the population of cells is approximately the same as or differs (e.g., increases by no more than) about 25, 50, 100, 150, 200, or 250% from the median GeneSetScore (Down stemness) of the population of cells before they were engineered to express a CAR; (d) the median GeneSetScore (Up hypoxia) of the population of cells is about the same as or differs (e.g., increases by no more than) about 125, 150, 175, or 200% from the median GeneSetScore (Up hypoxia) of the population of cells before they were engineered to express a CAR; or (e) The median GeneSetScore (Up autophagy) of the population of cells is about the same as or differs by (e.g., is increased by) no more than about 180, 190, 200, or 210% from the median GeneSetScore (Up autophagy) of the population of cells before they were engineered to express a CAR.

[0030] In one aspect, the present disclosure relates to an autoimmune disease or disorder, such as lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with severe pulmonary involvement (e.g., as seen in autoHSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-associated myositis, e.g., antisynthetase syndrome with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjogren's disease, severe refractory neuroimmune disease, Provided are methods for treating a subject with an infectious disease (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-associated disease (MOGAD), multiple sclerosis (MS)), severe refractory rheumatoid arthritis, an antibody-mediated neuroimmune disease (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG-associated disease), NMDAR+ encephalitis or antibody-related paraneoplastic neurological disease), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis, the methods comprising the step of administering lapcaptagene autoleucel to the subject.

[0031] In one aspect, the disclosure provides a method of treating a subject having a severe, intractable autoimmune disease, the method comprising administering to the subject rapcaptagene autoleucel.

[0032] In some embodiments, the severe, intractable autoimmune disease is selected from systemic lupus erythematosus, lupus nephritis, idiopathic inflammatory myopathy, systemic sclerosis, and ANCA-associated vasculitis.

[0033] In some embodiments, the lupus is systemic lupus erythematosus. In some embodiments, the SLE is severe refractory SLE (srSLE).

[0034] In some embodiments, the CD19 CAR comprises a CD19 binding domain, a transmembrane domain, and an intracellular signaling domain.

[0035] In some embodiments, (a) the transmembrane domain comprises a transmembrane domain of a protein selected from the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154; or (b) the transmembrane domain comprises the transmembrane domain of CD8; (c) the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; or (d) the nucleic acid molecule comprises a nucleic acid sequence encoding a transmembrane domain, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 17 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0036] In one aspect, the disclosure provides a method of treating a subject with severe refractory systemic lupus erythematosus (srSLE), comprising administering to the subject a population of cells comprising a CD19 chimeric antigen receptor (CD19 CAR) or comprising a nucleic acid encoding a CD19 CAR, CAR comprises a CD19 binding domain, a transmembrane domain, and an intracellular signaling domain, the transmembrane domain of which is linked to the transmembrane domain of the CD8 protein. Amounts sufficient to treat srSLE The present invention provides a method for treating srSLE, comprising the steps of:

[0037] In some embodiments, (a) the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; or (a) The nucleic acid molecule comprises a nucleic acid sequence encoding a transmembrane domain, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 17 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0038] In some embodiments, the population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is about 0.5 x 10 6 ~50×10 6 viable CAR-expressing cells, e.g., approximately 5 x 10 6 and optionally, the population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is administered at a dose of 5×10 6 The dose of viable CAR-expressing cells is administered.

[0039] In some embodiments, the population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is about 2.5 x 10 6 ~2.5×10 8 viable CAR-expressing cells, e.g., about 1.25 x 10 7 and optionally, the population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is administered at a dose of 1.25 x 10 viable CAR-expressing cells or 7 The dose of viable CAR-expressing cells is administered.

[0040] In some embodiments, the population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is about 1.25 x 10 7 ~1.25×10 9 viable CAR-expressing cells, e.g., about 1.25 x 10 8 and optionally, the population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is administered at a dose of 1.25 x 10 viable CAR-expressing cells or 8 The dose of viable CAR-expressing cells is administered.

[0041] In some embodiments, the population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is about 2.5 x 10 6 ~2.5×10 8 viable CAR-expressing cells, e.g., approximately 1 x 10 7or 5 x 10 7 The dose of viable CAR-expressing cells is administered.

[0042] In one aspect, the disclosure provides a method of treating a subject with severe refractory systemic lupus erythematosus (srSLE), comprising administering to the subject rapcavtagene autoleucel, Amounts sufficient to treat srSLE thereby treating srSLE.

[0043] In some embodiments, the rapcavtagene autoleucel is about 0.5×10 6 ~50×10 6 viable CAR-positive cells, e.g., approximately 5 x 10 6 viable CAR-positive cells are administered at a dose of 5 x 10, and optionally, rapcvtagene autoleucel 6 The dose of viable CAR-positive cells is administered.

[0044] In some embodiments, the rapcavtagene autoleucel is about 2.5×10 6 ~2.5×10 8 viable CAR-positive cells, e.g., approximately 1.25 x 10 7 viable CAR-positive cells are administered at a dose of 1.25 x 10, and optionally, rapcavtagene autoleucel 7 The dose of viable CAR-positive cells is administered.

[0045] In some embodiments, the rapcavtagene autoleucel is about 1.25×10 7 ~1.25×10 9 viable CAR-positive cells, e.g., approximately 1.25 x 10 8 viable CAR-positive cells are administered at a dose of 1.25 x 10, and optionally, rapcavtagene autoleucel 8 The dose of viable CAR-positive cells is administered.

[0046] In some embodiments, the rapcavtagene autoleucel is about 2.5×10 6 ~2.5×10 8viable CAR-positive cells, e.g., approximately 1 x 10 7 or 5 x 10 7 The dose of viable CAR-positive cells is administered.

[0047] In one aspect, the disclosure provides a method of treating a subject having lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), comprising administering to the subject a population of cells that express, or comprise a nucleic acid configured to express, a CD19 chimeric antigen receptor (CD19 CAR), wherein the cells are between 0.5 and 50 x 10 6 viable CAR+ T cells (e.g., 5–12.5 × 10 6 The method provides a method in which the patient is administered at a dose of 100 mg / kg of a therapeutically effective amount of CAR+ T cells (100 mg / kg of viable CAR+ T cells).

[0048] In one aspect, the disclosure provides a method of treating a subject having lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), comprising administering to the subject lapcaptagen autoleucel, wherein the lapcaptagen autoleucel is administered in a concentration of 0.5 to 50 x 10 6 viable CAR+ T cells (e.g., 5–12.5 × 10 6 The method provides a method in which the patient is administered at a dose of 100 mg / kg of a therapeutically effective amount of CAR+ T cells (100 mg / kg of viable CAR+ T cells).

[0049] In some embodiments, the lupus is systemic lupus erythematosus. In some embodiments, the SLE is severe refractory SLE (srSLE), and optionally, the subject has renal involvement.

[0050] In some embodiments, the CAR comprises a CD19 binding domain, a transmembrane domain, and an intracellular signaling domain.

[0051] In some embodiments, (a) the transmembrane domain comprises a transmembrane domain of a protein selected from the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154; or (b) the transmembrane domain comprises the transmembrane domain of CD8; (c) the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; or (d) the nucleic acid molecule comprises a nucleic acid sequence encoding a transmembrane domain, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 17 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0052] In some embodiments, the CD19 binding domain comprises heavy chain complementarity determining region 1 (HC CDR1), HC CDR2, HC CDR3, light chain complementarity determining region 1 (LC CDR1), LC CDR2, and LC CDR3; (a) HC CDR1 comprises the amino acid sequence of SEQ ID NO: 295; (b) HC CDR2 comprises the amino acid sequence of SEQ ID NO: 296; (c) HC CDR3 comprises the amino acid sequence of SEQ ID NO: 297; (d) LC CDR1 comprises the amino acid sequence of SEQ ID NO: 298; (e) LC CDR2 comprises the amino acid sequence of SEQ ID NO: 299; and (f) LC CDR3 comprises the amino acid sequence of SEQ ID NO: 300.

[0053] In some embodiments, the CD19 binding domain comprises a VH and a VL, wherein the VH and VL are connected by a linker, and optionally the linker comprises the amino acid sequence of SEQ ID NO: 63 or 104.

[0054] In some embodiments, the CD19 binding domain is linked to the transmembrane domain by a hinge region, and optionally (a) the hinge region comprises the amino acid sequence of SEQ ID NO: 2, 3, or 4, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; or (b) the nucleic acid molecule comprises a nucleic acid sequence encoding a hinge region, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 13, 14, or 15, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0055] In some embodiments, the intracellular signaling domain comprises a primary signaling domain, and optionally the primary signaling domain comprises a functional signaling domain derived from CD3ζ, TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (ICOS), FcεRI, DAP10, DAP12, or CD66d, and optionally (a) the primary signaling domain comprises a functional signaling domain derived from CD3ζ; (b) the primary signaling domain comprises the amino acid sequence of SEQ ID NO: 9 or 10, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereof; or (c) the nucleic acid molecule comprises a nucleic acid sequence encoding a primary signaling domain, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 20 or 21, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereof.

[0056] In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain, and optionally the costimulatory signaling domain is selected from the group consisting of an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), an activating NK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, C DS, ICAM-1, 4-1BB (CD137), B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp4 4, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, I TGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG 2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160( BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD28-OX40, CD28-4-1BB, or a ligand that specifically binds CD83; and optionally, (a) the costimulatory signaling domain comprises a functional signaling domain derived from 4-1BB; (b) the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; or (c) the nucleic acid molecule comprises a nucleic acid sequence encoding a costimulatory signaling domain, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 18, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereof.

[0057] In some embodiments, the intracellular signaling domain comprises a functional signaling domain derived from 4-1BB and a functional signaling domain derived from CD3ζ, and optionally, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:7 (or an amino acid sequence with at least about 85%, 90%, 95%, or 99% sequence identity thereof) and the amino acid sequence of SEQ ID NO:9 or 10 (or an amino acid sequence with at least about 85%, 90%, 95%, or 99% sequence identity thereof), and optionally, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:7 and the amino acid sequence of SEQ ID NO:9 or 10.

[0058] In some embodiments, the CAR further comprises a leader sequence comprising the amino acid sequence of SEQ ID NO:1.

[0059] In some embodiments, the CD19 CAR comprises the amino acid sequence of SEQ ID NO: 301 or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto.

[0060] In some embodiments, the nucleic acid molecule encoding the CD19 CAR comprises the nucleotide sequence of SEQ ID NO: 302, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto.

[0061] In some embodiments, the subject has been previously treated with or is concurrently being treated with one or more of an antimalarial drug (e.g., hydroxychloroquine or quinacrine), a glucocorticoid (e.g., prednisone), a calcineurin inhibitor, an immunomodulatory agent (e.g., methotrexate, azathioprine, mycophenolate mofetil, cyclophosphamide, or tacrolimus), a biologic agent (e.g., belimumab, rituximab, a disease-modifying antirheumatic drug (DMARD) (e.g., leflunomide).

[0062] In some embodiments, the subject has been identified as having failed treatment including two or more immunosuppressive therapies (e.g., a combination of mycophenolate or cyclophosphamide with a glucocorticoid) and one biologic agent.

[0063] In some embodiments, the subject has not previously received a therapy involving a CD19 CAR (e.g., rapcavtagene autoleucel), adoptive T cell therapy, or gene therapy product.

[0064] In some embodiments, prior to administration of the CD19 CAR (e.g., rapcavtagene autoleucel), the subject undergoes lymphodepletion therapy.

[0065] In some embodiments, the subject undergoes lymphodepletion therapy about two weeks prior to administration of the CD19 CAR (e.g., rapcavtagene autoleucel).

[0066] In some embodiments, the lymphodepletion therapy includes fludarabine (e.g., 25 mg / m 2 IV daily for three doses) and cyclophosphamide (e.g., 250 mg / m 2 IV three doses daily).

[0067] In some embodiments, the method further comprises administering a second therapeutic agent to the subject.

[0068] In some embodiments, the second therapeutic agent is administered before, simultaneously with, or after administration of the population of CAR-expressing cells or the rapcaptagene autoleucel.

[0069] In some embodiments, the subject is monitored for signs of cytokine release syndrome, eg, for at least 2, 2.5, 3, 3.5, or 4 days, eg, about 3 days.

[0070] In some embodiments, leukapheresis is performed (i) before administration of a corticosteroid, and / or (ii) after an absolute T cell count of 300 cells / mm 3 This is done when the

[0071] In one aspect, the present disclosure provides a method of generating a population of cells (e.g., T cells) expressing a chimeric antigen receptor (CAR), the method comprising: (i) contacting (e.g., binding) a population of cells (e.g., T cells, e.g., T cells isolated from frozen or fresh leukapheresis products) with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, wherein the population of cells is selected from the group consisting of an autoimmune disease or disorder, such as lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with severe pulmonary involvement (e.g., as seen in autoHSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, the subject has overlap myositis, cancer-associated myositis (e.g., antisynthetase syndrome with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjogren's disease, severe refractory neuroimmune disease (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-associated disease (MOGAD), multiple sclerosis (MS)), severe refractory rheumatoid arthritis, antibody-mediated neuroimmune disease (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG-associated disease), NMDAR+ encephalitis or antibody-associated paraneoplastic neurological disease), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis; (ii) contacting a population of cells (e.g., T cells) with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding a CAR, thereby providing a population of cells (e.g., T cells) comprising the nucleic acid molecule, wherein optionally, the CAR comprises a CD19 antigen-binding domain; and (iii) recovering the population of cells (e.g., T cells) for storage (e.g., reformulating the population of cells in cryopreservation medium) or administration. Including, (a) step (ii) is carried out together with step (i) or within 20 hours after the start of step (i), such as within 12, 13, 14, 15, 16, 17 or 18 hours after the start of step (i), such as within 18 hours after the start of step (i); and step (iii) is carried out within 30 (e.g. 26) hours after the start of step (i), such as within 22, 23, 24, 25, 26, 27, 28, 29 or 30 hours after the start of step (i), for example within 24 hours after the start of step (i); (b) step (ii) is carried out together with step (i) or within 20 hours after the start of step (i), such as within 12, 13, 14, 15, 16, 17 or 18 hours after the start of step (i), such as within 18 hours after the start of step (i); and Step (iii) is carried out within 30 hours after the start of step (ii), such as within 22, 23, 24, 25, 26, 27, 28, 29 or 30 hours after the start of step (ii); or (c) the population of cells from step (iii) is not expanded or is expanded by no more than 5, 10, 15, 20, 25, 30, 35 or 40%, e.g., no more than 10%, compared to the population of cells at the start of step (i), e.g., as assessed by viable cell count; Optionally, the nucleic acid molecule of step (ii) is on a viral vector, and optionally the nucleic acid molecule of step (ii) is an RNA molecule on the viral vector, and optionally step (ii) comprises transducing a population of cells (e.g., T cells) with the viral vector comprising the nucleic acid molecule encoding the CAR.

[0072] In some embodiments, the agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3 (e.g., an anti-CD3 antibody), and the agent that stimulates a costimulatory molecule is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof; optionally, the agent that stimulates the CD3 / TCR complex or the agent that stimulates a costimulatory molecule is an antibody (e.g., a single domain antibody (e.g., a heavy chain variable domain antibody), a peptibody, a Fab fragment, or an scFv), a small molecule, or a ligand (e.g., a naturally occurring ligand, recombinant or chimeric ligand), optionally the agent that stimulates the CD3 / TCR complex or the agent that stimulates the costimulatory molecule does not comprise beads, optionally the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody, and the agent that stimulates the costimulatory molecule comprises an anti-CD28 antibody, optionally the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody covalently bound to a colloidal polymer nanomatrix, and the agent that stimulates the costimulatory molecule comprises an anti-CD28 antibody covalently bound to a colloidal polymer nanomatrix, optionally the agent that stimulates the CD3 / TCR complex and the agent that stimulates the costimulatory molecule comprise T Cell TransAct™.

[0073] In some embodiments, step (i) enriches the population of cells from step (iii), e.g., the population of cells from step (iii) exhibits a higher percentage of CAR-expressing cells (e.g., at least 10, 20, 30, 40, 50, or 60% higher) compared to cells produced by a similar method but without step (i).

[0074] In some embodiments, (a) the percentage of naive cells, e.g., naive T cells, e.g., 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, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ cells, in the population of cells at the start of step (i); (b) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ T cells, in the population of cells from step (iii) is increased, e.g., by at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3-fold compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ cells, in the population of cells at the start of step (i); (c) the percentage of CAR-expressing naive T cells, e.g., CAR-expressing CD45RA+ CD45RO- CCR7+ T cells, in the population of cells increases during step (ii), e.g., increases by at least 30, 35, 40, 45, 50, 55, or 60% 18 to 24 hours after initiation of step (ii); or (d) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ T cells, in the population of cells from step (iii) is not reduced, or is reduced by no more than 5 or 10%, compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ cells, in the population of cells at the start of step (i).

[0075] In some embodiments, (a) the population of cells from step (iii) exhibits a higher percentage (e.g., at least 10, 20, 30, or 40% higher) of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ T cells, compared to cells produced by a similar method except that step (iii) is performed more than 26 hours after the initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the initiation of step (i); (b) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ T cells, in the population of cells from step (iii) is higher (e.g., at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3 times higher) than the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ T cells, in cells made by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (c) the percentage of CAR-expressing naive T cells, e.g., CAR-expressing CD45RA+ CD45RO-CCR7+ T cells, in the population of cells from step (iii) is higher (e.g., at least 4, 6, 8, 10, or 12 times higher) than the percentage of CAR-expressing naive T cells, e.g., CAR-expressing CD45RA+ CD45RO-CCR7+ T cells, in cells made by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (d) the population of cells from step (iii) exhibits a higher percentage (e.g., at least 10, 20, 30, or 40% higher) of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ T cells, compared to cells produced by a similar method but further comprising, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days; (e) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ T cells, in the population of cells from step (iii) is higher (e.g., at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3 times higher) than the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ T cells, in cells made by a similar method but further including, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days; or (f) The percentage of CAR-expressing naive T cells, e.g., CAR-expressing CD45RA+ CD45RO-CCR7+ T cells, in the population of cells from step (iii) is higher (e.g., at least 4, 6, 8, 10, or 12 fold) than the percentage of CAR-expressing naive T cells, e.g., CAR-expressing CD45RA+ CD45RO-CCR7+ T cells, in cells made by a similar method but further including, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days.

[0076] In some embodiments, (a) the percentage of central memory cells, e.g., central memory T cells, e.g., 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, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells at the start of step (i); (b) the percentage of central memory cells, e.g., central memory T cells, e.g., 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% compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in the population of cells at the start of step (i); (c) the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+ CD45RO+ cells, decreases during step (ii), e.g., decreases by at least 8, 10, 12, 14, 16, 18, or 20% 18 to 24 hours after initiation of step (ii); or (d) the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in the population of cells from step (iii) is not increased, or is increased by no more than 5 or 10%, compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in the population of cells at the start of step (i).

[0077] In some embodiments, (a) the population of cells from step (iii) exhibits a lower percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells (e.g., at least 10, 20, 30, or 40% lower), compared to cells produced by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (b) the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in the population of cells from step (iii) is lower (e.g., at least 20, 30, 40, or 50% lower) than the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in cells produced by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (c) the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+ CD45RO+ T cells, in the population of cells from step (iii) is lower (e.g., at least 10, 20, 30, or 40% lower) than the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+ CD45RO+ T cells, in cells generated by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (d) the population of cells from step (iii) exhibits a lower percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells (e.g., at least 10, 20, 30, or 40% lower), compared to cells produced by a similar method but further comprising, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days; (e) the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in the population of cells from step (iii) is lower (e.g., at least 20, 30, 40, or 50% lower) than the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in cells made by a similar method but further including, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days; or (f) The percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+ CD45RO+ T cells, in the population of cells from step (iii) is lower (e.g., at least 10, 20, 30, or 40% lower) than the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+ CD45RO+ T cells, in cells made by a similar method but further including, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days.

[0078] In some embodiments, (a) the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) is increased compared to the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells at the start of step (i); (b) whether the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) is increased compared to the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells at the start of step (i); (c) the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) is higher than the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in cells made by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); or (d) whether the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) is higher than the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in cells produced by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (e) the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) is higher than the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in cells made by a similar method but further including, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days; or (f) The percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) is higher than the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in cells made by a similar method but further including, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days.

[0079] In some embodiments, (a) the median GeneSetScore (Up TEM vs. Down TSCM) of the population of cells from step (iii) is about the same as or differs by (e.g., increases by) no more than about 25, 50, 75, 100, or 125% from the median GeneSetScore (Up TEM vs. Down TSCM) of the population of cells at the start of step (i); (b) Median GeneSetScore (Up TEM vs. Down TSCM) of the population of cells from step (iii) or cells produced by a similar method, except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i); or Cells made by a similar method, but further comprising expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). lower (e.g., at least about 100, 150, 200, 250, or 300% lower) than the median GeneSetScore (Up TEM vs. Down TSCM); (c) the median GeneSetScore (Up Treg vs. Down Teff) of the population of cells from step (iii) is approximately the same as or differs (e.g., increases 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 at the start of step (i); (d) Median GeneSetScore of the population of cells from step (iii) (Up Treg vs. Down Teff) or cells produced by a similar method, except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i); or Cells made by a similar method, but further comprising expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). lower (e.g., at least about 50, 100, 125, 150, or 175% lower) than the median GeneSetScore (Up Treg vs. Down Teff) of (e) the median GeneSetScore (Down stemness) of the population of cells from step (iii) is approximately the same as or differs from (e.g., increases by) no more than about 25, 50, 100, 150, 200, or 250% of the median GeneSetScore (Down stemness) of the population of cells at the start of step (i); (f) The median GeneSetScore (Down stemness) of the population of cells from step (iii) is or cells produced by a similar method, except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i); or Cells made by a similar method, but further comprising expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). lower than the median GeneSetScore (Downstemness) (e.g., at least about 50, 100, or 125% lower); (g) the median GeneSetScore (Up hypoxia) of the population of cells from step (iii) is approximately the same as or differs (e.g., increases by no more than) about 125, 150, 175, or 200% from the median GeneSetScore (Up hypoxia) of the population of cells at the start of step (i); (h) The median GeneSetScore (Up hypoxia) of the population of cells from step (iii) is or cells produced by a similar method, except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i); or Cells made by a similar method, but further comprising expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). lower (e.g., at least about 40, 50, 60, 70, or 80% lower) than the median GeneSetScore (Up hypoxia); (j) the median GeneSetScore (Up autophagy) of the population of cells from step (iii) is about the same as or differs from (e.g., increases by) no more than about 180, 190, 200, or 210% of the median GeneSetScore (Up autophagy) of the population of cells at the start of step (i); or (k) The median GeneSetScore (Up autophagy) of the population of cells from step (iii) is or cells produced by a similar method, except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i); or Cells made by a similar method, but further comprising expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). lower (e.g., at least about 20, 30, or 40% lower) than the median GeneSetScore (Up autophagy).

[0080] In some embodiments, the population of cells from step (iii), after being incubated with cells expressing the antigen recognized by the CAR, secrete IL-2 at a higher (e.g., at least 2, 4, 6, 8, 10, 12 or 14 fold) level than cells made by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i), or that further includes expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8 or 9 days, after step (ii) and before step (iii).

[0081] In some embodiments, the population of cells from step (iii), after administration in vivo to a subject, persists longer or proliferates to a greater extent than cells made by a similar method except that step (iii) is performed more than 26 hours after the initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the initiation of step (i), or compared to cells made by a similar method except that step (iii) further includes, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8 or 9 days.

[0082] In some embodiments, the population of cells from step (iii), after administration in vivo to a subject, exhibits a stronger activity (e.g., a lower dose, e.g., 0.15×10) than cells produced by a similar method except that step (iii) is performed more than 26 hours after the initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the initiation of step (i), or than cells produced by a similar method except that step (iii) further comprises expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). 6 , 0.2 × 10 6 , 0.25×10 6 or 0.3 x 10 6 (more potent activity at doses of 1000 surviving CAR-expressing cells).

[0083] In some embodiments, the population of cells from step (iii) is not expanded, or is expanded by no more than 5, 10, 15, 20, 25, 30, 35, or 40% compared to the population of cells at the start of step (i), e.g., as assessed by viable cell count, and optionally the number of viable cells in the population of cells from step (iii) is reduced from the number of viable cells in the population of cells at the start of step (i).

[0084] In some embodiments, the population of cells from step (iii) is not expanded or is expanded for less than 2 hours, for example less than 1 or 1.5 hours, compared to the population of cells at the start of step (i).

[0085] In some embodiments, steps (i) and / or (ii) are performed in cell culture medium (e.g., serum-free medium) containing IL-2, IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)), IL-7, IL-21, IL-6 (e.g., IL-6 / sIL-6Ra), an LSD1 inhibitor, a MALT1 inhibitor, or a combination thereof.

[0086] In some embodiments, steps (i) and / or (ii) are carried out in a serum-free cell culture medium that includes a serum replacement.

[0087] In some embodiments, the serum replacement is CTS™ Immune Cell Serum Replacement (ICSR).

[0088] In some embodiments, the method further comprises, prior to step (i): (iv) (optionally) receiving a fresh leukapheresis product (or an alternative source of hematopoietic tissue, such as a fresh whole blood product, a fresh bone marrow product, or a fresh organ biopsy or resection (e.g., a fresh product from a thymus removal)) from an entity, e.g., a laboratory, hospital, or healthcare provider; and (v) isolating the population of cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells) contacted in step (i) from a fresh leukapheresis product (or an alternative source of hematopoietic tissue, such as a fresh whole blood product, a fresh bone marrow product, or a fresh organ biopsy or resection (e.g., a fresh product from a thymus removal)). and optionally further comprising: Step (iii) is carried out within 35 hours after the start of step (v), such as within 27, 28, 29, 30, 31, 32, 33, 34 or 35 hours after the start of step (v), such as within 30 hours after the start of step (v); or The population of cells from step (iii) is not expanded or is expanded by no more than 5, 10, 15, 20, 25, 30, 35 or 40%, for example no more than 10%, compared to the population of cells at the end of step (v), as assessed by viable cell count.

[0089] In some embodiments, the method further comprises, prior to step (i), receiving cryopreserved T cells isolated from a leukapheresis product (or an alternative source of hematopoietic tissue, such as whole blood, bone marrow, or cryopreserved T cells isolated from an organ biopsy or resection (e.g., thymus removal)) from an entity, e.g., a laboratory, hospital, or healthcare provider.

[0090] In some embodiments, the method further comprises, prior to step (i): (iv) (optionally) receiving a cryopreserved leukapheresis product (or an alternative source of hematopoietic tissue, such as a cryopreserved whole blood product, a cryopreserved bone marrow product, or a cryopreserved organ biopsy or resection (e.g., a cryopreserved product from a thymus removal)) from an entity, e.g., a laboratory, hospital, or healthcare provider; and (v) isolating the population of cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells) contacted in step (i) from a cryopreserved leukapheresis product (or an alternative source of hematopoietic tissue, such as a cryopreserved whole blood product, a cryopreserved bone marrow product, or a cryopreserved organ biopsy or resection (e.g., a cryopreserved product from a thymus removal)). and optionally further comprising: Step (iii) is carried out within 35 hours after the start of step (v), such as within 27, 28, 29, 30, 31, 32, 33, 34 or 35 hours after the start of step (v), such as within 30 hours after the start of step (v); or The population of cells from step (iii) is not expanded or is expanded by no more than 5, 10, 15, 20, 25, 30, 35 or 40%, for example no more than 10%, compared to the population of cells at the end of step (v), as assessed by viable cell count.

[0091] In some embodiments, the method includes step (vi): Culturing a portion of the population of cells from step (iii) for at least 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7 days, e.g., at least 2 days and no more than 7 days, and measuring the level of CAR expression in the portion (e.g., measuring the percentage of viable CAR-expressing cells in the portion). and optionally further comprising: Step (iii) involves harvesting and freezing a population of cells (e.g., T cells), and step (vi) involves thawing a portion of the population of cells from step (iii), culturing the portion for at least 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7 days, such as at least 2 days and not more than 7 days, and measuring the level of CAR expression in the portion (e.g., measuring the percentage of viable CAR-expressing cells in the portion).

[0092] In some embodiments, the population of cells at the start of step (i) or step (1) is enriched for IL6R-expressing cells (e.g., cells positive for IL6Rα and / or IL6Rβ). In some embodiments, the population of cells at the start of step (i) or step (1) comprises at least 50, 60, or 70% IL6R-expressing cells (e.g., cells positive for IL6Rα and / or IL6Rβ).

[0093] In some embodiments, steps (i) and (ii) or steps (1) and (2) are performed in a cell culture medium containing IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)). In some embodiments, the IL-15 increases the ability of the population of cells to proliferate, e.g., after 10, 15, 20, or 25 days. In some embodiments, the IL-15 increases the percentage of IL6Rβ-expressing cells in the population of cells.

[0094] In some embodiments, the lupus is systemic lupus erythematosus. In some embodiments, the SLE is severe refractory SLE (srSLE).

[0095] In some embodiments, the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

[0096] In some embodiments, the antigen binding domain binds to a lupus-associated B cell antigen (e.g., CD19).

[0097] In some embodiments, the antigen binding domain comprises a CDR, VH, VL, scFv, or CAR sequence disclosed herein.

[0098] In some embodiments, the antigen binding domain comprises a CD19 binding domain comprising heavy chain complementarity determining region 1 (HC CDR1), HC CDR2, HC CDR3, light chain complementarity determining region 1 (LC CDR1), LC CDR2, and LC CDR3; (a) HC CDR1 comprises the amino acid sequence of SEQ ID NO: 295; (b) HC CDR2 comprises the amino acid sequence of SEQ ID NO: 296; (c) HC CDR3 comprises the amino acid sequence of SEQ ID NO: 297; (d) LC CDR1 comprises the amino acid sequence of SEQ ID NO: 298; (e) LC CDR2 comprises the amino acid sequence of SEQ ID NO: 299; and (f) LC CDR3 comprises the amino acid sequence of SEQ ID NO: 300.

[0099] In some embodiments, the antigen-binding domain comprises a VH and a VL, wherein the VH and VL are connected by a linker, and optionally the linker comprises the amino acid sequence of SEQ ID NO: 63 or 104.

[0100] In some embodiments, (a) the transmembrane domain comprises a transmembrane domain of a protein selected from the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154; or (b) the transmembrane domain comprises the transmembrane domain of CD8; (c) the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; or (d) the nucleic acid molecule comprises a nucleic acid sequence encoding a transmembrane domain, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 17 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0101] In some embodiments, the antigen binding domain is linked to the transmembrane domain by a hinge region, and optionally (a) the hinge region comprises the amino acid sequence of SEQ ID NO: 2, 3, or 4, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; or (b) the nucleic acid molecule comprises a nucleic acid sequence encoding a hinge region, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 13, 14, or 15, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0102] In some embodiments, the intracellular signaling domain comprises a primary signaling domain, and optionally the primary signaling domain comprises a functional signaling domain derived from CD3ζ, TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (ICOS), FcεRI, DAP10, DAP12, or CD66d, and optionally (a) the primary signaling domain comprises a functional signaling domain derived from CD3ζ; (b) the primary signaling domain comprises the amino acid sequence of SEQ ID NO: 9 or 10, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereof; or (c) the nucleic acid molecule comprises a nucleic acid sequence encoding a primary signaling domain, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 20 or 21, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereof.

[0103] In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain, and optionally the costimulatory signaling domain is selected from the group consisting of an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), an activating NK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, C D40, CDS, ICAM-1, 4-1BB (CD137), B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF 1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA- 6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB 7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9( a ligand that specifically binds to CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD28-OX40, CD28-4-1BB, or CD83, and optionally (a) the costimulatory signaling domain comprises a functional signaling domain derived from 4-1BB; (b) the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; or (c) the nucleic acid molecule comprises a nucleic acid sequence encoding a costimulatory signaling domain, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 18, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereof.

[0104] In some embodiments, the intracellular signaling domain comprises a functional signaling domain derived from 4-1BB and a functional signaling domain derived from CD3ζ, and optionally, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:7 (or an amino acid sequence with at least about 85%, 90%, 95%, or 99% sequence identity thereof) and the amino acid sequence of SEQ ID NO:9 or 10 (or an amino acid sequence with at least about 85%, 90%, 95%, or 99% sequence identity thereof), and optionally, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:7 and the amino acid sequence of SEQ ID NO:9 or 10.

[0105] In some embodiments, the CAR further comprises a leader sequence comprising the amino acid sequence of SEQ ID NO:1.

[0106] In some embodiments, the CAR comprises a CD19 CAR comprising the amino acid sequence of SEQ ID NO: 301, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto.

[0107] In some embodiments, the nucleic acid molecule encoding the CD19 CAR comprises the nucleotide sequence of SEQ ID NO: 302, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto.

[0108] In some embodiments, the subject has been previously treated with one or more of an antimalarial drug (e.g., hydroxychloroquine or quinacrine), a glucocorticoid (e.g., prednisone), a calcineurin inhibitor, an immunomodulatory agent (e.g., methotrexate, azathioprine, mycophenolate mofetil, cyclophosphamide, or tacrolimus), a biologic agent (e.g., belimumab, rituximab, a disease-modifying antirheumatic drug (DMARD) (e.g., leflunomide).

[0109] In some embodiments, the subject has been identified as having failed treatment including two or more immunosuppressive therapies (e.g., a combination of mycophenolate or cyclophosphamide with a glucocorticoid) and one biologic agent.

[0110] In some embodiments, the subject has not previously received a therapy comprising a CD19 CAR, adoptive T cell therapy, or gene therapy product.

[0111] In some embodiments, leukapheresis is performed (i) before administration of a corticosteroid, and / or (ii) after an absolute T cell count of 300 cells / mm 3 This is done when the

[0112] In one aspect, the present disclosure provides a population of CAR-expressing cells (e.g., autologous or allogeneic CAR-expressing T cells or NK cells) produced by the methods described herein.

[0113] In some embodiments, the population comprises autoreactive B cells (e.g., autoreactive B cells that do not express a CAR).

[0114] In one aspect, the present disclosure provides a pharmaceutical composition comprising a population of CAR-expressing cells described herein and a pharmaceutically acceptable carrier.

[0115] In one aspect, the disclosure provides a population of CAR-expressing cells, or a pharmaceutical composition comprising the same, for use in a method of modulating the immune response of a subject with lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), the method comprising administering to the subject an effective amount of the population of CAR-expressing cells or an effective amount of the pharmaceutical composition.

[0116] In one aspect, the disclosure provides a method of treating a subject having an autoimmune disease, e.g., lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), comprising administering to the subject: a population of cells that express, or contain a nucleic acid configured to express, a CD19 chimeric antigen receptor (CD19 CAR); and A second therapy selected from antimalarials or stable immunosuppressants wherein the second therapy and the CD19 CAR cells are present in the subject at the same time, e.g., the second therapy is administered at a time when the CD19 CAR cells are present in the subject.

[0117] In one aspect, the disclosure provides a method of treating a subject having an autoimmune disease, e.g., lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), comprising administering to the subject: Lapcaptagene Autoleucel, and A second therapy selected from antimalarials or stable immunosuppressants wherein the second therapy and lapcaptagene autoleucel are present in the subject at the same time, for example, the second therapy is administered at the same time that lapcaptagene autoleucel is present in the subject.

[0118] In some aspects, the present disclosure provides a method for treating an autoimmune disease or disorder, such as lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with severe pulmonary involvement (e.g., as seen in autoHSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-associated myositis, e.g., antisynthetase syndrome associated with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjögren's disease, or The present invention provides rapcavtagene autoleucel produced from autologous cells of subjects with rheumatoid arthritis, severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-associated disease (MOGAD), multiple sclerosis (MS)), severe refractory rheumatoid arthritis, antibody-mediated neuroimmune diseases (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG-associated disease), NMDAR+ encephalitis or antibody-related paraneoplastic neurological diseases), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis.

[0119] In one aspect, the present disclosure provides a pharmaceutical composition comprising lapcaptagene autoleucel and a pharmaceutically acceptable carrier.

[0120] In one aspect, the present disclosure provides a method for treating an autoimmune disease or disorder, such as lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with severe pulmonary involvement (e.g., as seen in autoHSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-associated myositis, e.g., antisynthetase syndrome associated with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjogren's disease, severe refractory neuroimmune disease (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO)), , MOG-associated disease (MOGAD), multiple sclerosis (MS)), severe refractory rheumatoid arthritis, antibody-mediated neuroimmune disease (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG-associated disease), NMDAR+ encephalitis or antibody-related paraneoplastic neurological disease), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis, the method comprising the step of administering to the subject an effective amount of a population of lapcabutagen autoleucel or an effective amount of the pharmaceutical composition.

[0121] In one aspect, the present disclosure provides lapcaptagene autoleucel or a pharmaceutical composition comprising same for use in a method of modulating the immune response of a subject with lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), the method comprising administering to the subject an effective amount of lapcaptagene autoleucel or an effective amount of the pharmaceutical composition.

[0122] Autoimmune diseases or disorders, such as lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with severe pulmonary involvement (e.g., as seen in autoHSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-associated myositis, e.g., antisynthetase syndrome with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjogren's disease, severe refractory rapcavtagene autoleucel for use in the treatment of a subject with a chronic neuroimmune disease (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-associated disease (MOGAD), multiple sclerosis (MS)), severe refractory rheumatoid arthritis, an antibody-mediated neuroimmune disease (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG-associated disease), NMDAR+ encephalitis or antibody-associated paraneoplastic neurological disease), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris or amyotrophic lateral sclerosis.

[0123] 1. Lapcabtagene autoleucel for use in treating a subject with severe refractory systemic lupus erythematosus (srSLE), the lapcabtagene autoleucel being formulated for administration in an amount sufficient to treat srSLE.

[0124] 0.5 to 50 x 10 lapcaptagen autoleucel for use in treating a subject with lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), 6 viable CAR+ T cells (e.g., 5–12.5 × 10 6 rapcavtagene autoleucel formulated to result in administration of a dose of 1000 mg / kg of rapcavtagene autoleucel (1000 mg / kg of viable CAR+ T cells).

[0125] 1. A compound comprising lapcaptagen autoleucel and a second therapy for use in treating a subject having an autoimmune disease, such as lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), comprising: the second therapy is selected from an antimalarial or a stable immunosuppressant; and The second therapy and the lapcaptagene autoleucel are present in the subject at the same time, for example, the second therapy is administered at a time when the lapcaptagene autoleucel is present in the subject, and the lapcaptagene autoleucel and the second therapy.

[0126] 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 (e.g., sequence database reference numbers) mentioned herein are incorporated by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences mentioned herein (e.g., in any table herein) are incorporated by reference. When a gene or protein references multiple sequence accession numbers, all sequence variants are encompassed.

[0127] Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting. Headings, subheadings, or numbered or lettered elements, e.g., (a), (b), (i), etc., are provided merely for ease of reading. The use of headings or numbered or lettered elements herein does not require the steps or elements to be performed in alphabetical order or that the steps or elements are necessarily distinct from one another. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0128] [Figure 1]FIG. 1 is a schematic showing the clinical trial design for an open-label, multicenter, Phase 1 / 2 study to determine the safety, efficacy, and cellular kinetics of ARM-CD19 CAR T cells in participants with severe, refractory autoimmune disorders. DETAILED DESCRIPTION OF THE INVENTION

[0129] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0130] The terms "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical referent of the article. By way of example, "an element" means one element or more than one element.

[0131] The term "about," when referring to a measurable value, such as an amount, a temporal duration, or the like, means that variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value are encompassed, as such variations are appropriate for the practice of the methods of the present disclosure.

[0132] The compositions and methods of the invention encompass polypeptides and nucleic acids having a designated sequence or sequences substantially identical or similar thereto, e.g., sequences at least 85%, 90%, or 95% or more identical to the designated sequence. The term "substantially identical," in the context of amino acid sequences, is used herein to refer to an amino acid sequence containing a common structural domain that has at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a first amino acid sequence, e.g., a reference sequence, e.g., a sequence described herein, that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of, aligned amino acid residues in a second amino acid sequence, such that the first and second amino acid sequences may have a common structural domain and / or a common functional activity.

[0133] The term "substantially identical," in the context of nucleotide sequences, is used herein to refer to a nucleotide sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a first nucleic acid sequence, e.g., a reference sequence, e.g., a sequence described herein, that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence, such that the first and second nucleotide sequences encode polypeptides having a common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity.

[0134] The term "variant" refers to a polypeptide having substantially the same amino acid sequence as, or encoded by substantially the same nucleotide sequence as, a reference amino acid sequence. In some embodiments, the variant is a functional variant.

[0135] The term "functional variant" refers to a polypeptide that has substantially the same amino acid sequence as a reference amino acid sequence, or is encoded by substantially the same nucleotide sequence, and that may have one or more activities of the reference amino acid sequence.

[0136] The term cytokine (e.g., IL-2, IL-7, IL-15, IL-21, or IL-6) encompasses full-length naturally occurring cytokines, fragments, or variants, e.g., functional variants (including fragments and functional variants thereof having at least 10%, 30%, 50%, or 80% of the activity, e.g., immunomodulatory activity, of a naturally occurring cytokine). In some embodiments, a cytokine has an amino acid sequence that is substantially identical (e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a naturally occurring cytokine or encoded by a nucleotide sequence that is substantially identical (e.g., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a naturally occurring nucleotide sequence encoding the cytokine. In some embodiments, as understood from the context, the cytokine further comprises a receptor domain, eg, a cytokine receptor domain (eg, IL-15 / IL-15R).

[0137] The term "chimeric antigen receptor" or alternatively "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 a CAR polypeptide construct comprise within the same polypeptide chain, e.g., a chimeric fusion protein. In some embodiments, the domains in a CAR polypeptide construct are not contiguous to one another, e.g., provided within different polypeptide chains, e.g., within an RCAR as described herein.

[0138] In some embodiments, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., a primary signaling domain of CD3ζ). In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains from at least one costimulatory molecule, as defined below. In some embodiments, the costimulatory molecule is selected from 41BB (i.e., CD137), CD27, ICOS, and / or CD28. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain from a costimulatory molecule and a functional signaling domain from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains from one or more costimulatory molecules and a functional signaling domain from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises an optional leader sequence at the amino-terminus (N-terminus) of the CAR fusion protein. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, which is optionally cleaved from the antigen recognition domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane.

[0139] A CAR comprising an antigen-binding domain (e.g., an scFv, a single domain antibody, or a TCR (e.g., a TCRα-binding domain or a TCRβ-binding domain)) that targets a particular antigen X (X can be an antigen as described herein), such as those described herein, is also referred to as an XCAR. For example, a CAR comprising an antigen-binding domain that targets CD19 is referred to as a CD19 CAR. CARs can be expressed in any cell, such as an immune effector cell (e.g., a T cell or an NK cell) described herein.

[0140] The term "signaling domain" refers to a functional portion of a protein that acts by transmitting information within the cell to regulate cellular activity through a specified signaling pathway by generating second messengers or functioning as an effector by responding to such messengers.

[0141] The term "antibody," as used herein, refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain or intact immunoglobulins, and can be derived from natural or recombinant sources. An antibody can be a tetramer of immunoglobulin molecules.

[0142] The term "antibody fragment" refers to at least a portion of an intact antibody or a recombinant variant thereof, and refers to an antigen-binding domain, e.g., the antigenic determining variable region of an intact antibody, sufficient to confer recognition and specific binding of the antibody fragment to a target, e.g., an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies (either VL or VH) such as sdAbs, camelid VHH domains, and multispecific antibodies formed from antibody fragments such as two or more, e.g., two Fab fragments, linked by a disulfide bridge at the hinge region, or a bivalent fragment comprising two or more linked antibodies, e.g., two isolated CDRs or other epitope fragments. Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antibody fragments can also be grafted onto polypeptide-based scaffolds, such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies). The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are closely linked by a short flexible polypeptide linker, capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, an scFv can have the VL and VH variable regions in either order, e.g., relative to the N- and C-terminal ends of the polypeptide, and can comprise a VL-linker-VH or a VH-linker-VL.

[0143] In some embodiments, the scFv is NH2-V L-Linker-V H -COOH or NH2-V H -Linker-V L It may contain the structure -COOH.

[0144] As used herein, the term "complementarity-determining region" or "CDR" refers to the sequence of amino acids within an antibody variable region that confers antigen specificity and binding affinity. For example, there are typically three CDRs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3), and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). The exact amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme), or a combination thereof. In the combined Kabat and Chothia numbering scheme, in some embodiments, the CDRs correspond to amino acid residues that are part of a Kabat CDR, part of a Chothia CDR, or both.

[0145] The portion of the CAR composition of the invention comprising an antibody or antibody fragment thereof can exist in various forms, for example, the antigen-binding domain is expressed as part of a polypeptide chain, including, for example, a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), or a humanized antibody (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In some embodiments, the antigen-binding domain of a CAR of the invention comprises an antibody fragment. In some embodiments, a CAR comprises an antibody fragment, including an scFv.

[0146] As used herein, the term "binding domain" or "antibody molecule" (also referred to herein as "anti-target binding domain") refers to a protein, e.g., an immunoglobulin chain or fragment thereof, that comprises at least one immunoglobulin variable domain sequence. The term "binding domain" or "antibody molecule" encompasses antibodies and antibody fragments. In some embodiments, an antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In certain embodiments, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.

[0147] The terms "bispecific antibody" and "bispecific antibodies" refer to a molecule that combines the antigen-binding sites of two antibodies in a single molecule. Thus, a bispecific antibody can bind to two different antibodies simultaneously or sequentially. Methods for producing bispecific antibodies are known in the art. Various formats for combining two antibodies are also known in the art. Forms of bispecific antibodies of the present invention include, but are not limited to, diabodies, single-chain antibodies, Fab dimerization (Fab-Fab), Fab-scFv, and tandem antibodies, as are well known to those skilled in the art.

[0148] The term "antibody heavy chain" refers to the larger of the two polypeptide chains present in antibody molecules in their naturally occurring conformation, and which usually determines the class to which the antibody belongs.

[0149] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0150] The term "recombinant antibody" refers to an antibody made using recombinant DNA techniques, such as, for example, antibodies expressed in bacteriophage or yeast expression systems. The term should also be construed to mean an antibody made by synthesis of a DNA molecule encoding an antibody and expressing the antibody protein, or an amino acid sequence specifying that antibody, where the DNA or amino acid sequence was obtained using well-known recombinant DNA or amino acid sequence techniques available in the art.

[0151] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may include either or both antibody production or activation of specific immunologically competent cells. Those skilled in the art will understand that any macromolecule can be an antigen, including virtually any protein or peptide. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will therefore understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response encodes an "antigen" as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of two or more genes, and that these nucleotide sequences are arranged in various combinations to encode a polypeptide that elicits a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be synthetically produced, obtained from a biological sample, or be a macromolecule other than a polypeptide. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells or bodily fluids, along with other biological components.

[0152] The term "autologous" refers to any material originating from the same individual that is later reintroduced into that individual.

[0153] The term "allogeneic" refers to any material derived from a different animal of the same species as the individual into which 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 genetically different to interact antigenically.

[0154] The term "xenogeneic" refers to a graft derived from an animal of a different species.

[0155] As used herein, the term "apheresis" refers to an art-recognized extracorporeal procedure in which a donor's or patient's blood is withdrawn from the donor or patient, passed through a device that separates certain selected components, and then the remainder is returned to the donor or patient, e.g., by reinfusion. Thus, reference to an "apheresis sample" refers to a sample obtained using apheresis.

[0156] As used herein, "lupus" refers to all types and manifestations of lupus. Lupus manifestations include, without limitation, systemic lupus erythematosus (severe refractory SLE (srSLE); lupus nephritis; cutaneous manifestations (e.g., manifestations seen in cutaneous lupus erythematosus, including, for example, skin lesions or rashes); CNS lupus; cardiovascular, pulmonary, hepatic, hematologic, gastrointestinal, and musculoskeletal manifestations; neonatal lupus erythematosus; pediatric systemic lupus erythematosus; drug-induced lupus erythematosus; antiphospholipid syndrome; and complement deficiency syndromes that result in lupus manifestations.

[0157] "Derived from," as the term is used herein, refers to the relationship between a first molecule and a second molecule. This generally refers to the structural similarity between the first and second molecules and does not imply or include a limitation on the manner or source by which the first molecule is derived from the second molecule. For example, in the case of an intracellular signaling domain derived from the CD3ζ molecule, the intracellular signaling domain retains sufficient CD3ζ structure such that it has the desired function, i.e., the ability to generate a signal under appropriate conditions. This does not imply or include a limitation to a particular method of making the intracellular signaling domain, e.g., it does not mean that one must start with the CD3ζ sequence and delete unnecessary sequence or make mutations to arrive at the intracellular signaling domain in order to provide the intracellular signaling domain.

[0158] The term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing that amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into an antibody or antibody fragment of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a CAR of the invention can be replaced with other amino acid residues from the same side chain family, and the altered CAR can be tested using the functional assays described herein.

[0159] In the context of stimulation by stimulatory and / or costimulatory molecules, the term "stimulation" refers to a response, e.g., a primary or secondary response, induced by the binding of a stimulatory molecule (e.g., TCR / CD3 complex) and / or costimulatory molecule (e.g., C28 or 4-1BB) to its cognate ligand, thereby mediating a signaling event, such as, but not limited to, signaling through the TCR / CD3 complex. Stimulation can mediate, for example, changes in the expression of certain molecules and / or reorganization of cytoskeletal structures.

[0160] The term "stimulatory molecule" refers to a molecule that is expressed by a T cell and provides a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex for at least some aspects of the T cell signaling pathway. In some embodiments, an ITAM-containing domain within a CAR recapitulates primary TCR signaling independent of the endogenous TCR complex. In some embodiments, the primary signal is, for example, a primary signal initiated by binding of the TCR / CD3 complex to a peptide-bearing MHC molecule, which results in mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, etc. The primary cytoplasmic signaling sequence (also referred to as a "primary signaling domain") that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of ITAM-containing primary cytoplasmic signaling sequences that are particularly useful in the present invention include, but are not limited to, those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, and CD66d, DAP10, and DAP12. In a specific CAR of the present invention, the intracellular signaling domain of any one or more CARS of the present invention comprises an intracellular signaling sequence, e.g., a primary signaling sequence of CD3-ζ. The term "antigen-presenting cell" or "APC" refers to immune system cells such as accessory cells (e.g., B cells, dendritic cells, etc.) that present foreign antigens complexed with major histocompatibility complexes (MHC) on their surface. T cells can recognize such complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

[0161] "Intracellular signaling domain," as the term is used herein, refers to the intracellular portion of a molecule. In embodiments, the intracellular signaling domain transduces an effector function signal, instructing the cell to perform a specific function. While the entire intracellular signaling domain can be used, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion may be used in place of the complete chain, so long as it transmits the effector function signal. The term intracellular signaling domain, therefore, is intended to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0162] The intracellular signaling domain generates a signal that promotes immune effector function of the CAR-containing cell, e.g., a CART cell, such as helper activity, including cytolytic activity and cytokine secretion.

[0163] In some embodiments, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules involved in primary or antigen-dependent stimulation. In some embodiments, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules involved in costimulatory signals or antigen-independent stimulation. For example, in the case of CART, the primary intracellular signaling domain can comprise a cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain can comprise a cytoplasmic sequence from a co-receptor or costimulatory molecule.

[0164] A primary intracellular signaling domain can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of primary cytoplasmic signaling sequences containing ITAMs include, but are not limited to, those derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, CD66d, DAP10, and DAP12.

[0165] The term "zeta" or alternatively "zeta chain," "CD3-zeta," or "TCR-zeta" refers to CD247. Swiss-Prot Accession No. P20963 provides an exemplary human CD3zeta amino acid sequence. "zeta stimulatory domain" or alternatively "CD3-zeta stimulatory domain" or "TCR-zeta stimulatory domain" refers to the stimulatory domain of CD3zeta or a variant thereof (e.g., a molecule having a mutation, e.g., a point mutation, a fragment, an insertion, or a deletion). In some embodiments, the cytoplasmic domain of zeta comprises residues 52-164 of GenBank Accession No. BAG36664.1 or a variant thereof (e.g., a molecule having a mutation, e.g., a point mutation, a fragment, an insertion, or a deletion). In some embodiments, the "zeta stimulatory domain" or "CD3-zeta stimulatory domain" is the sequence provided as SEQ ID NO: 9 or 10 or a variant thereof (e.g., a molecule having a mutation, e.g., a point mutation, a fragment, an insertion, or a deletion).

[0166] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for an efficient immune response. Costimulatory molecules include MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD5, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CD S, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD1 9, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, IT GAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, T NFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160( BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD28-OX40, CD28-4-1BB, and ligands that specifically bind to CD83.

[0167] A costimulatory intracellular signaling domain refers to the intracellular portion of a costimulatory molecule.

[0168] The intracellular signaling domain can comprise the entire intracellular portion of the molecule from which it is derived or the entire naturally occurring intracellular signaling domain, or a functional fragment thereof.

[0169] The term "4-1BB" refers to CD137 or tumor necrosis factor receptor superfamily member 9. Swiss-Prot accession number P20963 provides an exemplary human 4-1BB amino acid sequence. A "4-1BB costimulatory domain" refers to the costimulatory domain of 4-1BB or a variant thereof (e.g., a molecule having a mutation, e.g., a point mutation, a fragment, an insertion, or a deletion). In some embodiments, the "4-1BB costimulatory domain" is the sequence provided as SEQ ID NO: 7 or a variant thereof (e.g., a molecule having a mutation, e.g., a point mutation, a fragment, an insertion, or a deletion).

[0170] "Immune effector cells," as that term is used herein, refer to cells that are involved in an immune response, e.g., promoting an immune effector response. Examples of immune effector cells include T cells, e.g., α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.

[0171] "Immune effector function or immune effector response," as the term is used herein, refers to a function or response, e.g., 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 to a property of a T cell or NK cell that promotes the killing or growth or proliferation inhibition of a target cell. In the case of T cells, primary stimulation and costimulation are examples of immune effector functions or responses.

[0172] The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including secretion of cytokines.

[0173] The term "encoding" refers to the inherent property of a specific nucleotide sequence within a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have either a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence. Thus, a gene, cDNA, or RNA encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand used as a transcription template for a gene or cDNA can be said to encode the protein or other product of that gene's cDNA.

[0174] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, to the extent that the nucleotide sequence encoding the protein may, in any version, contain one or more introns.

[0175] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material or composition described herein that is effective in achieving a particular biological result.

[0176] The term "endogenous" refers to any substance from or produced within an organism, cell, tissue, or system.

[0177] The term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0178] The term "expression" refers to the transcription and / or translation of a particular nucleotide sequence. In some embodiments, expression includes the translation of mRNA introduced into a cell.

[0179] The term "transfer vector" refers to a composition containing an isolated nucleic acid that can be used to deliver the isolated nucleic acid into a cell. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "transfer vector" includes self-replicating plasmids or viruses. This term should be interpreted to further include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

[0180] The term "expression vector" refers to a vector containing a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0181] The term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells, and they can deliver large amounts of genetic information into the DNA of host cells, making them one of the most efficient gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.

[0182] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, including, inter alia, self-inactivating lentiviral vectors as provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that may be used clinically include, but are not limited to, the LENTIVECTOR® gene delivery technology from Oxford BioMedica and the LENTIMAX™ vector system from Lentigen. Non-clinical types of lentiviral vectors are also available and will be known to those skilled in the art.

[0183] The term "homologous" or "identity" refers to the subunit sequence identity between two polymer molecules, e.g., between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomer subunit, e.g., if a position in each of the two DNA molecules is occupied by adenine, they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions. For example, if half of the positions in two sequences are homologous (e.g., 5 positions in a polymer 10 subunits long), the two sequences are 50% homologous. If 90% of the positions (e.g., 9 out of 10) are matched or homologous, the two sequences are 90% homologous.

[0184] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies and antibody fragments thereof are those in which residues from a complementarity-determining region (CDR) of a human immunoglobulin (recipient antibody or antibody fragment) are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies / antibody fragments can comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further refine and optimize antibody or antibody fragment performance. Generally, a humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or most of the FR regions being those of a human immunoglobulin sequence. The humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2:593-596, 1992.

[0185] "Fully human" refers to an immunoglobulin, such as an antibody or antibody fragment, whose entire molecule is of human origin or consists of an amino acid sequence identical to a human form of an antibody or immunoglobulin.

[0186] The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that has been partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0187] In the context of the present invention, the following abbreviations are used for commonly occurring nucleobases: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0188] The term "operably linked" or "transcriptional control" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, for example, in the same reading frame, as necessary to join two protein-coding regions.

[0189] The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, intratumoral, or infusion techniques.

[0190] The terms "nucleic acid," "nucleic acid molecule," "polypeptide," or "polynucleotide molecule" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the terms encompass nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. In some embodiments, a "nucleic acid," "nucleic acid molecule," "polypeptide," or "polynucleotide molecule" includes nucleotide / nucleoside derivatives or analogs. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions, e.g., conservative substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions, e.g., conservative substitutions, can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0191] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, with no limit on the maximum number of amino acids that can comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and to the numerous types of longer chains generally referred to in the art as proteins. "Polypeptide" specifically includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. A polypeptide includes natural peptides, recombinant peptides, or combinations thereof.

[0192] The term "promoter" refers to a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery necessary to initiate the specific transcription of a polynucleotide sequence.

[0193] The term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to that promoter / regulatory sequence. In some instances, this sequence may be a core promoter sequence, and in other instances, this sequence may also include an enhancer sequence and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that confers tissue-specific expression of the gene product.

[0194] The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the production of the gene product in a cell under most or all physiological conditions of the cell.

[0195] The term "inducible" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes production of the gene product in a cell substantially only when an inducer corresponding to the promoter is present in the cell.

[0196] The term "tissue-specific" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes the production of a gene product in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0197] As used herein, "B cell antigen" refers to an antigen associated with a B cell. Non-limiting examples of B cell-associated molecules include proteins expressed on the surface of B cells, such as CD19, BCMA, CD22, CD20, CD10, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

[0198] As used herein, the term "CD19" refers to the cluster of differentiation 19 protein. For human and mouse amino acid and nucleic acid sequences, public databases such as GenBank, UniProt, and Swiss-Prot can be referenced. For example, the amino acid sequence of human CD19 can be referenced under UniProt / Swiss-Prot accession number P15391, and the nucleic acid sequence encoding human CD19 can be referenced under accession number NM_001178098. It is also an early marker of B cell precursors. See, e.g., Nicholson et al. Mol. Immun. 34(16-17):1157-1165 (1997). In one embodiment, the antigen-binding portion of the CART recognizes and binds to an antigen within the extracellular domain of the CD19 protein. In one embodiment, the CD19 protein is expressed on autoreactive B cells. As used herein, "CD19" includes proteins that include mutations of full-length wild-type CD19, such as point mutations, fragments, insertions, deletions, and splice variants.

[0199] The term "flexible polypeptide linker" or "linker," when used in the context of an scFv, refers to a peptide linker composed of amino acids such as glycine and / or serine residues used alone or in combination to link the variable heavy and variable light chain regions together. In some embodiments, the flexible polypeptide linker is a Gly / Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser)n, where n is a positive integer equal to or greater than 1, e.g., n=1, n=2, n=3, n=4, n=5, and n=6, n=7, n=8, n=9, and n=10 (SEQ ID NO:41). In some embodiments, flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4 (SEQ ID NO:27) or (Gly4Ser)3 (SEQ ID NO:28). In some embodiments, the linker comprises multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser) (SEQ ID NO:25). Also included within the scope of the present invention are the linkers described in WO 2012 / 138475, which is incorporated herein by reference.

[0200] As used herein, a 5' cap (RNA cap, RNA 7-methylguanosine cap or RNA m 7 A 5' cap (also called a G-cap) is a modified guanine nucleotide added to the "pre" or 5' end of eukaryotic messenger RNA immediately after transcription initiation. The 5' cap consists of a terminal group attached to the first transcribed nucleotide. Its presence is important for ribosome recognition and protection from RNases. Capping is coupled to transcription and occurs cotranscriptionally, with each cap affecting the other. Shortly after transcription initiation, a cap-synthesizing complex associated with RNA polymerase binds to the 5' end of the mRNA being synthesized. This enzyme complex catalyzes the chemical reactions required for mRNA capping. Synthesis proceeds as a multistep biochemical reaction. The capping moiety can be modified to adjust mRNA functions such as its stability or translation efficiency.

[0201] As used herein, "in vitro transcribed RNA" refers to RNA synthesized in vitro. In some embodiments, the RNA is mRNA. Generally, in vitro transcribed RNA is generated from an in vitro transcription vector. The in vitro transcription vector contains a template used to generate the in vitro transcribed RNA.

[0202] As used herein, "poly(A)" is a series of adenosines attached to mRNA by polyadenylation. In some embodiments of constructs for transient expression, the poly(A) is between 50 and 5000. In some embodiments, the poly(A) is greater than 64. In some embodiments, the poly(A) is greater than 100. In some embodiments, the poly(A) is greater than 300. In some embodiments, the poly(A) is greater than 400. The poly(A) sequence can be modified chemically or enzymatically to adjust mRNA function, such as localization, stability, or translation efficiency.

[0203] As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylyl moiety or its modified variants to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at their 3' ends. The 3' poly(A) tail is a long sequence (often several hundred) of adenine nucleotides added to pre-mRNA by the action of the enzyme polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added to transcripts containing a specific sequence, the polyadenylation signal. The poly(A) tail and its associated proteins help protect the mRNA from exonuclease degradation. Polyadenylation is also important for transcription termination, mRNA nuclear export, and translation. Polyadenylation occurs in the nucleus immediately after DNA-to-RNA transcription, but can also occur later in the cytoplasm. After transcription is terminated, the mRNA strand is cleaved by the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA is cleaved, an adenosine residue is added to the free 3' end of the cleavage site.

[0204] As used herein, "transient" refers to expression of a non-integrated transgene for a period of hours, days, or weeks, which is shorter than the period of expression of the gene when integrated into the genome or contained in a stable plasmid replicon in a host cell.

[0205] As used herein, the terms "treat," "treatment," and "treating" refer to a reduction or amelioration of the progression, severity, and / or duration of an autoimmune disorder or an amelioration of one or more symptoms (preferably one or more discernible symptoms) of an autoimmune disorder resulting from the administration of one or more therapies (e.g., one or more therapeutic agents, such as a CAR of the invention). In specific embodiments, the terms "treat," "treatment," and "treating" refer to an improvement in at least one measurable physical parameter of an autoimmune disorder, such as the level of autoantibodies, which is not necessarily discernible by the patient. In other embodiments, the terms "treat," "treatment," and "treating" refer to either or both a physical inhibition of the progression of an autoimmune disorder, e.g., by stabilization of discernible symptoms, a physiological inhibition, e.g., by stabilization of physical parameters.

[0206] The term "signal transduction pathway" refers to the biochemical relationships between various signaling molecules that play a role in transmitting a signal from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecules and molecular complexes that have the ability to receive a signal and transmit the signal across the membrane of a cell.

[0207] The term "subject" is intended to include living organisms (eg, mammals, such as humans) in which an immune response can be generated.

[0208] The term "substantially purified" cells refers to cells that are essentially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types with which they are normally associated in their naturally occurring state. In some instances, a substantially purified cell population refers to a homogenous cell population. In other instances, the term simply refers to cells that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In some embodiments, the cells are not cultured in vitro.

[0209] The term "therapeutic" as used herein means treatment. A therapeutic effect is achieved by the reduction, suppression, amelioration, or eradication of the disease state.

[0210] As used herein, the term "prophylaxis" refers to the prevention or prophylactic treatment of a disease or condition.

[0211] The terms "transfected" or "transformed" or "transduced" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0212] The term "specifically binds" refers to an antibody or ligand that recognizes and binds to a cognate binding partner protein present in a sample (e.g., a stimulatory and / or costimulatory molecule present on a T cell) but does not substantially recognize or bind to other molecules in the sample.

[0213] "Membrane anchor" or "membrane tethering domain," as the term is used herein, refers to a polypeptide or moiety, e.g., a myristoyl group, sufficient to attach an extracellular or intracellular domain to the plasma membrane.

[0214] "Refractory," as used herein, refers to an autoimmune disease or disorder that does not respond to treatment, e.g., SLE. In embodiments, a refractory autoimmune disease or disorder may be resistant to treatment before or at the start of treatment. In other embodiments, a refractory autoimmune disease or disorder may become resistant during treatment. A refractory autoimmune disease or disorder may also be referred to as a resistant autoimmune disease or disorder.

[0215] As used herein, "severe refractory autoimmune disease" refers to an autoimmune disease manifestation that has failed (e.g., remains characterized by high disease activity) after at least one standard immunosuppressive therapy or at least one biologic agent. One example of a severe refractory autoimmune disease is severe refractory systemic lupus erythematosus.

[0216] As used herein, "severe refractory systemic lupus erythematosus" or "srSLE" refers to an SLE episode that has failed (e.g., remains characterized by high disease activity) after at least one standard immunosuppressive therapy (e.g., mycophenolate, cyclophosphamide), a glucocorticoid drug, or at least one biologic agent. In some embodiments, srSLE includes an SLE episode that has failed two or more standard immunosuppressive therapies in combination with a glucocorticoid drug. In some embodiments, srSLE includes an SLE episode that has failed at least one biologic agent.

[0217] "Recurrent" or "recurring," as used herein, refers to the return or reappearance of a disease (e.g., an autoimmune disease or disorder) or signs and symptoms of a disease, such as an autoimmune disease or disorder, after a period of improvement or response, e.g., after previous treatment with a therapy, e.g., a standard of care therapy. The initial period of response may involve cellular autoantibody levels falling below a certain threshold. Reappearance may involve autoantibody levels rising above a certain threshold.

[0218] Ranges: Throughout this disclosure, various embodiments of the invention may 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 construed to include all the possible subranges specifically disclosed as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be construed to include specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity includes those 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 width of the range.

[0219] As used herein, "administered in combination" means that two (or more) different treatments are delivered to a subject over the course of the subject's illness, e.g., after the subject is diagnosed with an illness and before the illness is cured or eliminated, or before treatment is discontinued for other reasons. In some embodiments, the delivery of one treatment is still ongoing when the delivery of the second treatment begins, thereby resulting in an overlap in administration. This may be referred to herein as "simultaneous" or "co-delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments, in either case, the treatments are more effective when administered in combination. For example, the second treatment is more effective, e.g., a comparable effect is seen with less of the second treatment, or the second treatment alleviates symptoms to a greater extent than, or a similar condition is seen as, the second treatment is administered without the first treatment. In some embodiments, the delivery is such that the relief of symptoms or other parameters related to the disorder is greater than that observed when one is delivered without the other. The effects of the two treatments can be partially additive, fully additive, or greater than additive. The delivery can be such that the effect of the first treatment delivered remains detectable when the second treatment is delivered.

[0220] The terms "depletion" or "depleting," as used interchangeably herein, refer to a decrease or reduction in the level or amount of cells, proteins, or macromolecules in a sample after a process, e.g., a selection step, e.g., negative selection, has been performed. Depletion can be a complete or partial depletion of cells, proteins, or macromolecules. In some embodiments, depletion is a decrease or reduction in the level or amount of cells, proteins, or macromolecules by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to the level or amount of cells, proteins, or macromolecules in the sample before the process is performed.

[0221] As used herein, "naive T cells" refer to antigen-naive T cells. In some embodiments, antigen-naive T cells have encountered their cognate antigen in the thymus but not in the periphery. In some embodiments, naive T cells are precursors of memory T 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 isoforms. In some embodiments, naive T cells express CD62L, IL-7 receptor alpha, IL-6 receptor, and CD132, but do not express CD25, CD44, CD69, or CD45RO. In some embodiments, naive T cells express CD45RA, CCR7, and CD62L, but do not express CD95 or IL-2 receptor β. In some embodiments, the surface expression levels of the markers are assessed using flow cytometry.

[0222] The term "central memory T cells" refers to a subset of T cells that are CD45RO positive and express CCR7 in humans. 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 beta, CCR7, and CD62L. In some embodiments, the surface expression levels of markers are assessed using flow cytometry.

[0223] The terms "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" refer to a subset of memory T cells with stem cell-like capabilities, e.g., the ability to self-renew and / or multipotency to reconstitute memory and / or effector T cell subsets. In some embodiments, stem memory T cells express CD45RA, CD95, IL-2 receptor beta, CCR7, and CD62L. In some embodiments, the 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, the entire contents of which are incorporated herein by reference.

[0224] For purposes of clarity, unless otherwise noted, classifying a cell or population of cells as "not expressing" or "absent from" or "negative for" a particular marker does not necessarily mean the absence of the marker. One skilled in the art can readily compare cells to positive and / or negative controls and / or set a predetermined threshold and use conventional detection methods, such as flow cytometry, to classify a cell or population of cells as not expressing or negative for a marker when the cell has an expression level below the predetermined threshold or the population of cells has an overexpression level below the predetermined threshold, as described in the Examples herein.

[0225] As used herein, the term "GeneSetScore (Up TEM vs. Down TSCM)" of a cell refers to a score that represents the degree to which the cell exhibits an effector memory T cell (TEM) phenotype relative to a stem cell memory T cell (TSCM) phenotype. A higher GeneSetScore (Up TEM vs. Down TSCM) indicates an increase in the TEM phenotype, whereas a lower GeneSetScore (Up TEM vs. Down TSCM) indicates an increase in the TSCM phenotype. In some embodiments, the GeneSetScore (Up TEM vs. Down TSCM) TSCM) is a gene that is upregulated in TEM cells and / or downregulated in TSCM, e.g., MXRA7, CLIC1, NAT13, TBC1D2B, GLCCI1, DUSP10, APOBEC3D, CACNB3, ANXA2P2, TPRG1, EOMES, MATK, ARHGAP10, ADAM8, MAN1A1, SLFN12L, SH2D2A, EIF2C4, CD58, MYO1F, RA In some embodiments, the GeneSetScore (Up TEM vs. Down TSCM) is determined for each cell using RNA-seq, e.g., single-cell RNA-seq (scRNA-seq), as exemplified in Example 10 with respect to Figure 39A for WO 2020 / 047452, which is incorporated by reference in its entirety. In some embodiments, the GeneSetScore (Up TEM vs. Down TSCM) is calculated by taking the average log-normalized gene expression value of all the genes in the gene set.

[0226] As used herein, the term "GeneSetScore (Up Treg vs. Down Teff)" of a cell refers to a score that represents the extent to which the cell exhibits a regulatory T cell (Treg) versus an effector T cell (Teff) phenotype. A higher GeneSetScore (Up Treg vs. Down Teff) indicates an increase in the Treg phenotype, whereas a lower GeneSetScore (Up Treg vs. Down Teff) indicates an increase in the Teff phenotype. In some embodiments, the GeneSetScore (Up Treg vs. Down Teff) can be expressed as one or more genes that are upregulated in Treg cells and / or downregulated in Teff cells, such as C12orf75, SELPLG, SWAP70, RGS1, PRR11, SPATS2L, SPATS2L, TSHR, C14orf145, CASP8, SYT11, ACTN4, ANXA5, GLRX, HLA-DMB, PMCH, RAB11FIP1, IL32, FAM160B1, SHMT2, FRMD4B, CCR3, TNFRSF13B, NTNG2, CLDND1, BARD1, FCER1G, TYMS, ATP1B1, GJB6, FGL2, TK1, SLC2A8, CDKN2 A, SKAP2, GPR55, CDCA7, S100A4, GDPD5, PMAIP1, ACOT9, CEP55, SGMS1, ADPRH, AKAP2, HDAC9, IKZF4, CARD17, VAV 3, OBFC2A, ITGB1, CIITA, SETD7, HLA-DMA, CCR10, KIAA0101, SLC14A1, PTTG3P, DUSP10, FAM164A, PYHIN1, MYO1 F, 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,The determination is made by measuring the expression of one or more genes selected from the group consisting of LGALS1, ANXA2, PI16, 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, TRIB1, FCRL3 and FOXP3. In some embodiments, GeneSetScore (Up Treg vs. Down Teff) is determined using RNA-seq, e.g., single-cell RNA-seq (scRNA-seq), e.g., as exemplified in Example 10 with respect to Figure 39B for WO 2020 / 047452, which is incorporated by reference in its entirety. In some embodiments, GeneSetScore (Up Treg vs. Down Teff) is calculated by taking the average log-normalized gene expression value of all genes in the gene set.

[0227] As used herein, the term "GeneSetScore (Down stemness)" of a cell refers to a score that represents the extent to which the cell exhibits a stem cell phenotype. A lower GeneSetScore (Down stemness) indicates an increased stem cell phenotype. In some embodiments, GeneSetScore (Down stemness) is measured by measuring the expression of one or more genes that are downregulated in hematopoietic stem cells and upregulated in differentiated stem cells, e.g., one or more genes selected from the group consisting of ACE, BATF, CDK6, CHD2, ERCC2, HOXB4, MEOX1, SFRP1, SP7, SRF, TAL1, and XRCC5. In some embodiments, GeneSetScore (Down stemness) is determined using RNA-seq, e.g., single-cell RNA-seq (scRNA-seq), as exemplified in Example 10 with respect to Figure 39C of WO 2020 / 047452, which is incorporated by reference in its entirety. In some embodiments, the GeneSetScore(Down stemness) is calculated by taking the average log-normalized gene expression value of all the genes in the gene set.

[0228] As used herein, the term "GeneSetScore(UP hypoxia)" of a cell refers to a score that represents the degree to which the cell exhibits a hypoxic phenotype. A higher GeneSetScore(UP hypoxia) indicates an increased hypoxic phenotype. In some embodiments, the GeneSetScore(UP hypoxia) is determined by the expression of one or more genes that are upregulated in cells experiencing hypoxia, such as ABCB1, ACAT1, ADM, ADORA2B, AK2, AK3, ALDH1A1, ALDH1A3, ALDOA, ALDOC, ANGPT2, ANGPTL4, ANXA1, ANXA2, ANXA5, ARHGAP5, ARSE, ART1, BACE2, BATF3, BCL2L1, BCL2L2, BHLHE40, BHLHE41, BIK, BIRC2, BNIP3, BNI P3L, BPI, BTG1, C11orf2, C7orf68, CA12, CA9, CALD1, CCNG2, CCT6A, CD99, CDK1, CDKN1A, CDKN1B, CITED2, CLK1, CNOT7, COL4A5, CO L5A1, COL5A2, COL5A3, CP, CTSD, CXCR4, D4S234E, DDIT3, DDIT4, 1-Dec, DKC1, DR1, EDN1, EDN2, EFNA1, EGF, EGR1, EIF4A3, ELF3, EL L2, ENG, ENO1, ENO3, ENPEP, EPO, ERRFI1, ETS1, F3, FABP5, FGF3, FKBP4, FLT1, FN1, FOS, FTL, GAPDH, GBE1, GLRX, GPI, GPRC5A, HAP 1, HBP1, HDAC1, HDAC9, HERC3, HERPUD1, HGF, HIF1A, HK1, HK2, HLA-DQB1, HMOX1, HMOX2, HSPA5, HSPD1, HSPH1, HYOU1, ICAM1, ID2, I FI27, IGF2, IGFBP1, IGFBP2, IGFBP3, IGFBP5, IL6, IL8, INSIG1, IRF6, ITGA5, JUN, KDR, KRT14, KRT18, KRT19, LDHA, LDHB, LEP, LGA LS1, 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, P LOD2, PNN, PNP, POLM, PPARA, PPAT, PROK1, PSMA3, PSMD9, PTGS1, PTGS2, QSOX1, RBPJ, RELA, RIOK3, 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, GeneSetScore(UP hypoxia) is determined using RNA-seq, e.g., single-cell RNA-seq (scRNA-seq), e.g., as exemplified in Example 10 with respect to Figure 39D in International Publication No. WO 2020 / 047452, which is incorporated by reference in its entirety. In some embodiments, GeneSetScore(UP hypoxia) is calculated by taking the average log-normalized gene expression value of all genes in the gene set.

[0229] As used herein, the term "GeneSetScore(UP autophagy)" of a cell refers to a score that represents the degree to which the cell exhibits an autophagic phenotype. A higher GeneSetScore(UP autophagy) indicates an increased autophagic phenotype. In some embodiments, the GeneSetScore(UP autophagy) is a score that represents the degree to which the cell exhibits an ... TG5, ATG7, ATG9A, ATG9B, ATP13A2, ATP1B1, ATPAF1-AS1, ATPIF1, BECN1, BECN1P1, BLOC1S1, BMP2KL, BNIP1, BNIP3, BOC, C11orf 2, C11orf41, C12orf44, C12orf5, C14orf133, C1orf210, C5, C6orf106, C7orf59, C7orf68, C8orf59, C9orf72, CA7, CALCB, CALC OCO2, CAPS, CCDC36, CD163L1, CD93, CDC37, CDKN2A, CHAF1B, CHMP2A, CHMP2B, CHMP3, CHMP4A, CHMP4B, CHMP4C, CHMP6, CHST3, CI SD2, CLDN7, CLEC16A, CLN3, CLVS1, COX8A, CPA3, CRNKL1, CSPG5, CTSA, CTSB, CTSD, CXCR7, DAP, DKKL1, DNAAF2, DPF3, DRAM1, DRA M2, DYNLL1, DYNLL2, DZANK1, EI24, EIF2S1, EPG5, EPM2A, FABP1, FAM125A, FAM131B, FAM134B, FAM13B, FAM176A, FAM176B, FAM48 A, FANCC, FANCF, FANCL, FBXO7, FCGR3B, FGF14, FGF7, FGFBP1, FIS1, FNBP1L, FOXO1, FUNDC1, FUNDC2, FXR2, GABARAP, 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、KIAA022 6、KIAA1324、KRCC1、KRT15、KRT73、LAMP1、LAMP2、LAMTOR1、LAMTOR2、LAMTOR3、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、MT MR14、MTMR3、MTOR、MTSS1、MYH11、MYLK、MYOM1、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、P RKAA1、PRKAA2、PRKAB1、PRKAB2、PRKAG1、PRKAG2、PRKAG3、PRKD2、PRKG1、PSEN1、PTPN22、RAB12、RAB1A、RAB1B、RAB23、RAB24、RAB33B、RAB39、RAB7A、RB1 CC1、RBM18、REEP2、REP15、RFWD3、RGS19、RHEB、RIMS3、RNF185、RNF41、RPS27A、RPTOR、RRAGA、RRAGB、RRAGC、RRAGD、S100A8、S100A9、SCN1A、SERPIN10、SESN2, SFRP4, SH3GLB1, SIRT2, SLC1A3, SLC1A4, SLC22A3, SLC25A19, SLC35B3, SLC35C1, SLC37A4, SLC6A1, SLCO1A2, SMURF1 , SNAP29, SNAPIN, SNF8, SNRPB, SNRPB2, SNRPD1, SNRPF, SNTG1, SNX14, SPATA18, SQSTM1, SRPX, STAM, STAM2, STAT2, STBD1, ST K11, STK32A, STOM, STX12, STX17, SUPT3H, TBC1D17, TBC1D25, TBC1D5, TCIRG1, TEAD4, TECPR1, TECPR2, TFEB, TM9SF1, TMBIM 6, TMEM203, TMEM208, TMEM39A, TMEM39B, TMEM59, TMEM74, TMEM93, TNIK, TOLLIP, TOMM20, TOMM22, TOMM40, TOMM5, TOMM6, TOM M7, TOMM70A, TP53INP1, TP53INP2, TRAPPC8, TREM1, TRIM17, TRIM5, TSG101, TXLNA, UBA52, UBB, UBC, UBQLN1, UBQLN2, UBQLN 4, ULK1, ULK2, ULK3, USP10, USP13, USP30, UVRAG, VAMP7, VAMP8, VDAC1, VMP1, VPS11, VPS16, VPS18, VPS25, VPS28, VPS33A, VP In some embodiments, the GeneSetScore(UP autophagy) is determined using RNA-seq, e.g., single-cell RNA-seq (scRNA-seq), as exemplified in Example 10 with respect to Figure 39E of International Publication No. WO 2020 / 047452, which is incorporated herein by reference in its entirety.GeneSetScore(UP autophagy) is calculated by taking the average log-normalized gene expression value of all genes in the gene set.

[0230] As used herein, the term "GeneSetScore (Up resting vs. Down activated)" of a cell refers to a score that represents the degree to which the cell exhibits a resting T cell phenotype relative to an activated T cell phenotype. A higher GeneSetScore (Up resting vs. Down activated) indicates an increase in the resting T cell phenotype, whereas a lower GeneSetScore (Up resting vs. Down activated) indicates an increase in the activated T cell phenotype. In some embodiments, the GeneSetScore (Up resting vs. Down activated) is determined by the expression of one or more genes that are upregulated in resting T cells and / or downregulated in activated T cells, such as ABCA7, ABCF3, ACAP2, AMT, ANKH, ATF7IP2, ATG14, ATP1A1, ATXN7, ATXN7L3B, BCL7A, BEX4, BSDC1, BTG1, BTG2, BTN3A1, C11orf21, C19orf22, C21orf2, CAMK2G, CARS2, CCNL2, CD248, CD5, CD55, CEP164, CHKB, CLK1, CLK4, CTSL1, DBP, DCUN1D2, DENND1C, DGKD, DLG 1, DUSP1, EAPP, ECE1, ECHDC2, ERBB2IP, FAM117A, FAM134B, FAM134C, FAM169A, FAM190B, FAU, FLJ10038, FOXJ 2, FOXJ3, FOXL1, FOXO1, FXYD5, FYB, HLA-E, HSPA1L, HYAL2, ICAM2, IFIT5, IFITM1, IKBKB, IQSEC1, IRS4, KIAA 0664L3, KIAA0748, KLF3, KLF9, KRT18, LEF1, LINC00342, LIPA, LIPT1, LLGL2, LMBR1L, LPAR2, LTBP3, LYPD3, L ZTFL1, MAMBA, MAP2K6, MAP3K1, MARCH8, MAU2, MGEA5, MMP8, MPO, MSL1, MSL3, MYH3, MYLIP, NAGPA, NDST2, NISC H, NKTR, 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, RNF3 8, RNF44, ROR1, RPL30, RPL32, RPLP1, RPS20, RPS24, RPS27, RPS6, RPS9, RXRA, RYK, SCAND2, SEMA4C , SETD1B, SETD6, SETX, SF3B1, SH2B1, SLC2A4RG, SLC35E2B, SLC46A3, SMAGP, SMARCE1, SMPD1, SNP H, SP140L, SPATA6, SPG7, SREK1IP1, SRSF5, STAT5B, SVIL, SYF2, SYNJ2BP, TAF1C, TBC1D4, TCF20, T The determination is made by measuring the expression of one or more genes selected from the group consisting of ECTA, 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, GeneSetScore (Up resting vs. Down activated) is determined using RNA-seq, e.g., single-cell RNA-seq (scRNA-seq), e.g., as exemplified in Example 10 with respect to Figure 38D for WO 2020 / 047452, which is incorporated by reference in its entirety. In some embodiments, GeneSetScore (Up resting vs. Down activated) is calculated by taking the average log-normalized gene expression value of all genes in the gene set.

[0231] As used herein, the "GeneSetScore (Progressively up in memory differentiation)" of a cell refers to a score representing the stage of the cell in memory differentiation. A higher GeneSetScore (Progressively up in memory differentiation) indicates an increase in late memory T cell phenotype, whereas a lower GeneSetScore (Progressively up in memory differentiation) indicates an increase in early memory T cell phenotype. In some embodiments, the GeneSetScore (UP autophagy) indicates one or more genes upregulated during memory differentiation, such as MTCH2, RAB6C, KIAA0195, SETD2, C2orf24, NRD1, GNA13, COPA, SELT, TNIP1, CBFA2T2, LRP10, PRKCI, BRE, ANKS1A, PNPLA6, ARL6IP1, WDFY1, MAPK1, GPR153, SHKBP1, MAP 1LC3B2, PIP4K2A, HCN3, GTPBP1, TLN1, C4orf34, KIF3B, TCIRG1, PPP3CA, ATG4D, TYMP, TRAF6, C17orf76, WI PF1, FAM108A1, MYL6, NRM, SPCS2, GGT3P, GALK1, CLIP4, ARL4C, YWHAQ, LPCAT4, ATG2A, IDS, TBC1D5, DMPK, S T6GALNAC6, REEP5, ABHD6, KIAA0247, EMB, TSEN54, SPIRE2, PIWIL4, ZSCAN22, ICAM1, CHD9, LPIN2, SETD8, Z C3H12A, ULBP3, IL15RA, HLA-DQB2, LCP1, CHP, RUNX3, TMEM43, REEP4, MEF2D, ABL1, TMEM39A, PCBP4, PLCD1, CHST12, RASGRP1, C1orf58, C11orf63, C6orf129, FHOD1, DKFZp434F142, PIK3CG, ITPR3, BTG3, C4orf50, CN NM3, IFI16, AK1, CDK2AP1, REL, BCL2L1, MVD, TTC39C, PLEKHA2, FKBP11, EML4, FANCA, CDCA4, FUCA2, MFSD10,<h2 style=";text-align:left;direction:ltr">TBCD、CAPN2、IQGAP1、CHST11、PIK3R1、MYO5A、KIR2DL3、DLG3、MXD4、RALGDS 、S1PR5、WSB2、CCR3、TIPARP、SP140、CD151、SOX13、KRTAP5-2、NF1、PEA15、P ARP8、RNF166、UEVLD、LIMK1、CACNB1、TMX4、SLC6A6、LBA1、SV2A、LLGL2、IRF 1、PPP2R5C、CD99、RAPGEF1、PPP4R1、OSBPL7、FOXP4、SLA2、TBC1D2B、ST7、JAZ F1、GGA2、PI4K2A、CD68、LPGAT1、STX11、ZAK、FAM160B1、RORA、C8orf80、APO BEC3F、TGFBI、DNAJC1、GPR114、LRP8、CD69、CMIP、NAT13、TGFB1、FLJ00049、A NTXR2、NR4A3、IL12RB1、NTNG2、RDX、MLLT4、GPRIN3、ADCY9、CD300A、SCD5、A BI3、PTPN22、LGALS1、SYTL3、BMPR1A、TBK1、PMAIP1、RASGEF1A、GCNT1、GABAR APL1、STOM、CALHM2、ABCA2、PPP1R16B、SYNE2、PAM、C12orf75、CLCF1、MXRA7、APOBEC3C、CLSTN3、ACOT9、HIP1、LAG3、TNFAIP3、DCBLD1、KLF6、CACNB3、RNF 19A、RAB27A、FADS3、DLG5、APOBEC3D、TNFRSF1B、ACTN4、TBKBP1、ATXN1、ARA P2、ARHGEF12、FAM53B、MAN1A1、FAM38A、PLXNC1、GRLF1、SRGN、HLA-DRB5、B4G ALT5、WIPI1、PTPRJ、SLFN11、DUSP2、ANXA5、AHNAK、NEO1、CLIC1、EIF2C4、MA P3K5、IL2RB、PLEKHG1、MYO6、GTDC1、EDARADD、GALM、TARP、ADAM8、MSC、HNRPL L、SYT11、ATP2B4、NHSL2、MATK、ARHGAP18、SLFN12L、SPATS2L、RAB27B、PIK3 R3、TP53INP1、MBOAT1、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, A NXA2, EOMES, CADM1, MAF, TPRG1, NBEAL2, PPP2R2B, PELO, SLC4A4, KLRF1, FOSL2, RGS2, TGFBR3, PRF1, MYO1F, GAB3, C17orf66, MICAL2, CYTH3, TOX, HLA -DRA, SYNE1, WEE1, PYHIN1, F2R, PLD1, THBS1, CD58, FAS, NETO2, CXCR6, ST6GALNAC2, DUSP4, AUTS2, C1orf21, KLRG1, TNIP3, GZMA, PRR5L, PRDM1, ST8SIA6, PLXND1, PTPRM, GFPT2, MYBL1, SLAMF7, FLJ16686, GNLY, ZEB2, CST7, IL18RAP, CCL5, KLRD1, and KLRB1. In some embodiments, GeneSetScore (Progressively up in memory differentiation) is determined using RNA-seq, for example, as exemplified in Example 10 with respect to Figure 40B for WO 2020 / 047452, which is incorporated by reference in its entirety. In some embodiments, the GeneSetScore (Progressively up in memory differentiation) is calculated by taking the average log-normalized gene expression value of all of the genes in the gene set.

[0232] As used herein, the term "GeneSetScore (Up TEM vs. Down TN)" of a cell refers to a score that represents the degree to which the cell exhibits an effector memory T cell (TEM) phenotype relative to a naive T cell (TN) phenotype. A higher GeneSetScore (Up TEM vs. Down TN) indicates an increase in the TEM phenotype, whereas a lower GeneSetScore (Up TEM vs. Down TN) indicates an increase in the TN phenotype.In some embodiments, the GeneSetScore (Up TEM vs. Down TN) is calculated based on the expression of one or more genes that are upregulated in TEM cells and / or downregulated in TN cells, such as MYO5A, 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, OS BPL3, CMIP, NAT13, TGFB1, 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, MAN 1A1, 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, NBE It is determined by measuring the expression of one or more genes selected from the group consisting of AL2, 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, GeneSetScore (Up TEM vs. Down TN) is determined using RNA-seq, e.g., single-cell RNA-seq (scRNA-seq), e.g., as exemplified in Example 10 with respect to Figure 40C for WO 2020 / 047452, which is incorporated by reference in its entirety. In some embodiments, GeneSetScore (Up TEM vs. Down TN) is calculated by taking the average log-normalized gene expression value of all genes in a gene set.

[0233] Relative to a GeneSetScore value (e.g., median GeneSetScore), a 100% decrease in a positive GeneSetScore results in a value of 0. A 100% increase in a negative GeneSetScore results in a value of 0. For example, as disclosed in WO 2020 / 047452, the median GeneSetScore for day 1 samples is −0.084; the median GeneSetScore for day 9 samples is 0.035; and the median GeneSetScore for input samples is −0.1. In WO 2020 / 047452, in Figure 39A, a 100% increase in the median GeneSetScore for input samples results in a GeneSetScore value of 0; a 200% increase in the median GeneSetScore for input samples results in a GeneSetScore value of 0.1. In WO 2020 / 047452, in Figure 39A, a 100% decrease in the median GeneSetScore for the day 9 samples results in a GeneSetScore value of 0; a 200% decrease in the median GeneSetScore for the day 9 samples results in a GeneSetScore value of -0.035.

[0234] As used herein, the term "bead" refers to a discrete particle with a solid surface ranging in size from about 0.1 μm to several millimeters in diameter. Beads can be spherical (e.g., microspheres) or irregularly shaped. Beads can comprise a variety of materials, including, but not limited to, paramagnetic materials, ceramic, plastic, glass, polystyrene, methylstyrene, acrylic polymers, titanium, latex, Sepharose™, cellulose, nylon, and the like. In some embodiments, the beads are relatively uniform, approximately 4.5 μm in diameter, spherical, superparamagnetic polystyrene beads, coated with, e.g., coupled to, a mixture of antibodies against CD3 (e.g., CD3ε) and CD28. In some embodiments, the beads are Dynabeads®. In some embodiments, both anti-CD3 and anti-CD28 antibodies are coupled to the same beads to mimic stimulation of T cells by antigen-presenting cells. The properties of Dynabeads® and the use of Dynabeads® for cell isolation and expansion are known in the art, see, e.g., Neurauter et al., Cell isolation and expansion using Dynabeads, Adv Biochem Eng Biotechnol 2007;106:41-73, the entire contents of which are incorporated herein by reference.

[0235] The term "multispecific binding molecule" refers to a molecule that specifically binds to at least two antigens and contains two or more antigen-binding domains, each of which may independently be an antibody fragment (e.g., scFv, Fab, nanobody), a ligand, or a non-antibody-derived binder (e.g., fibronectin, Fynomer, DARPin).

[0236] The term "monovalent," as used herein in reference to a multispecific binding molecule, antibody (e.g., bispecific antibody), or antibody fragment, refers to a multispecific binding molecule, antibody (e.g., bispecific antibody), or antibody fragment that has only one antigen-binding domain for each antigen to which the multispecific binding molecule, antibody (e.g., bispecific antibody), or antibody fragment binds.

[0237] The term "bivalent," as used herein in reference to a multispecific binding molecule, antibody (e.g., bispecific antibody), or antibody fragment, refers to a multispecific binding molecule, antibody (e.g., bispecific antibody), or antibody fragment that has two antigen-binding domains for each antigen to which the multispecific binding molecule, antibody (e.g., bispecific antibody), or antibody fragment binds.

[0238] The term "Fc silent" refers to an Fc domain that has been modified to minimize interaction with effector cells. Silenced effector function can be achieved by mutations in the Fc region of an antibody, including, but not limited to, LALA and N297A (Strohl, W., 2009, Curr. Opin. Biotechnol. vol. 20(6): 685-691); and D265A (Baudino et al., 2008, J. Immunol. 181: 6664-69), as described in the art; see also Heusser et al., WO 2012065950. Examples of Fc silencing mutations include the LALA mutant, which contains L234A and L235A mutations in the IgG1 Fc amino acid sequence, DAPA (D265A, P329A) (see, e.g., U.S. Pat. No. 6,737,056), N297A, DANAPA (D265A, N297A, and P329A), and / or LALADANAPS (L234A, L235A, D265A, N297A, and P331S).

[0239] The term "CD3 / TCR complex" refers to a complex on the surface of a T cell that includes a TCR, which includes a TCRα chain and a TCRβ chain; a CD3, which includes one CD3γ chain, one CD3δ chain, and two CD3ε chains; and a ζ domain. UniProt accession numbers P01848 (TCRα, constant domain), P01850 (TCRβ, constant domain 1), A0A5B9 (TCRβ, constant domain 2), P09693 (CD3γ), P04234 (CD3δ), and P07766 (CD3ε) provide exemplary human sequences for these chains, except that the ζ chain, which is involved in intracellular signaling, is discussed in more detail below. Further relevant accession numbers include A0A075B662 (mouse TCR alpha, constant domain), A0A0A6YWV4 and / or A0A075B5J3 (mouse TCR beta, constant domain 1), A0A075B5J4 (mouse TCR beta, constant domain 2), P11942 (mouse CD3 gamma), P04235 (mouse CD3 delta), P22646 (mouse CD3 epsilon).

[0240] The term "CD28" refers to the T cell-specific glycoprotein CD28, also called Tp44, which functions as a costimulatory molecule, as well as all of its aliases. UniProt accession number P10747 provides an exemplary human CD28 amino acid sequence (see also HGNC:1653, Entrez Gene:940, Ensembl:ENSG00000178562, and OMIM:186760). Additional relevant CD28 sequences include UniProt accession number P21041 (mouse CD28).

[0241] The term "CD2" refers to the T-cell surface antigen T11 / Leu-5 / CD2, lymphocyte function antigen 2, T11, or erythrocyte / rosette / LFA-3 receptor, and its synonyms, which function as a growth factor receptor. UniProt Accession No. P06729 provides an exemplary human CD2 amino acid sequence (see also HGNC:1639, Entrez Gene:914, Ensembl:ENSG00000116824, and OMIM:186990). Additional relevant CD2 sequences include UniProt Accession No. P08920 (mouse CD2).

[0242] As used herein, the term "nanomatrix" refers to a nanostructure comprising a matrix of flexible polymer chains. The nanomatrix is ​​sized between 1 and 500 nm, e.g., between 10 and 200 nm. In some embodiments, the matrix of flexible polymer chains is coupled to one or more agonists, e.g., agonistic anti-CD3 and / or anti-CD28 antibodies, that provide activation signals to T cells. In some embodiments, the nanomatrix comprises a colloidal polymer nanomatrix coupled, e.g., covalently attached, to one or more agonists of stimulatory molecules and / or one or more agonists of costimulatory molecules. In some embodiments, the one or more agonists of stimulatory molecules are CD3 agonists (e.g., anti-CD3 agonist antibodies). In some embodiments, the one or more agonists of stimulatory molecules are CD28 agonists (e.g., anti-CD28 agonist antibodies). In some embodiments, the nanomatrix is ​​characterized by the absence of a solid surface as a binding point for agonists, e.g., anti-CD3 and / or anti-CD28 antibodies. In some embodiments, the nanomatrix is ​​the nanomatrix disclosed in WO 2014 / 048920 A1 or as provided in the MACS® GMP T Cell TransAct™ kit from Miltenyi Biotcc GmbH, the entire contents of which are incorporated herein by reference. MACS® GMP T Cell TransAct™ is composed of a colloidal polymer nanomatrix covalently linked to humanized recombinant agonistic antibodies against human CD3 and CD28.

[0243] Various embodiments of the compositions and methods described herein are described in further detail below. Additional definitions are set forth throughout the specification.

[0244] Provided herein are methods for producing immune effector cells (e.g., T cells or NK cells) engineered to express a CAR, e.g., a CAR described herein, compositions comprising such cells, and methods for treating an autoimmune disease or disorder in a subject, such as lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with severe pulmonary involvement (e.g., as seen in autoHSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-associated myositis, e.g., ILD-associated myositis). Methods of using such cells to treat diseases such as antisynthetase syndrome), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjogren's, severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-associated disease (MOGAD), multiple sclerosis (MS)), severe refractory rheumatoid arthritis, antibody-mediated neuroimmune diseases (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG-associated disease), NMDAR+ encephalitis or antibody-associated paraneoplastic neurological diseases), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis are provided. In some embodiments, the methods disclosed herein can produce immune effector cells engineered to express a CAR in less than 24 hours. Without intending to be bound by theory, the methods described herein 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 described herein comprise a higher percentage of stem cell memory T cells compared to CART cells produced by traditional manufacturing processes, as measured, for example, using scRNA-seq.In some embodiments, CART cells produced by the manufacturing methods described herein comprise a higher percentage of effector T cells compared to CART cells produced by traditional manufacturing processes, e.g., as measured using scRNA-seq. In some embodiments, CART cells produced by the manufacturing methods described herein better preserve T cell stemness compared to CART cells produced by traditional manufacturing processes. In some embodiments, CART cells produced by the manufacturing methods described herein exhibit lower levels of hypoxia compared to CART cells produced by traditional manufacturing processes, e.g., as measured using scRNA-seq. In some embodiments, CART cells produced by the manufacturing methods described herein exhibit lower levels of autophagy compared to CART cells produced by traditional manufacturing processes.

[0245] In some embodiments, the methods disclosed herein do not include the use of beads such as Dynabeads® (e.g., CD3 / CD28 Dynabeads®) and do not include a de-beading step. In some embodiments, CART cells produced by the methods disclosed herein can be administered to a subject with minimal ex vivo expansion, e.g., 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. Thus, the methods described herein provide a high-throughput manufacturing process for making improved CAR-expressing cell products for use in treating diseases in a subject. Furthermore, the present invention relates to the treatment of autoimmune diseases or disorders, such as lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with severe pulmonary involvement (e.g., as seen in autoHSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-associated myositis, e.g., antisynthetase syndrome with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory systemic lupus erythematosus (srSLE), or lupus nephritis, among other diseases. Provided are CAR compositions and uses thereof in medicaments or methods for treating Egren's disease, severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-associated disease (MOGAD), multiple sclerosis (MS)), severe refractory rheumatoid arthritis, antibody-mediated neuroimmune diseases (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG-associated disease), NMDAR+ encephalitis or antibody-related paraneoplastic neurological diseases), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis.

[0246] Activation Process In some embodiments, the disclosure provides a method of generating a population of cells (e.g., T cells) expressing a chimeric antigen receptor (CAR), comprising: (i) generating a cell (e.g., T cells, e.g., a cell or cells associated with an autoimmune disease or disorder, such as lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with severe pulmonary involvement (e.g., as seen in autoHSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-associated myositis, e.g., antisynthetase syndrome with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjogren's disease, severe refractory neuropathies, contacting a population of T cells isolated from frozen or fresh leukapheresis products from a subject with an immune disease (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-associated disease (MOGAD), multiple sclerosis (MS)), severe refractory rheumatoid arthritis, antibody-mediated neuroimmune disease (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG-associated disease), NMDAR+ encephalitis or antibody-associated paraneoplastic neurological disease), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis) with (A) an agent that stimulates the 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 a population of cells (e.g., T cells) with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding a CAR, thereby providing a population of cells (e.g., T cells) comprising the nucleic acid molecule, and (iii) recovering the population of cells (e.g., T cells) for storage (e.g., reformulating the population of cells in cryopreservation medium) or administration, wherein (a) step (ii) is performed together with step (i), or within 20 hours after initiation of step (i), e.g., within 12, 13, 14, 15, 16, 17, or 18 hours after initiation of step (i), e.g., within 18 hours after initiation of step (i), and step (iii) is performed within 26 hours after initiation of step (i), e.g., ... (b) step (ii) is carried out together with step (i) or within 20 hours after the start of step (i), for example within 12, 13, 14, 15, 16, 17 or 18 hours after the start of step (i), for example within 18 hours after the start of step (i), and step (iii) is carried out within 30, 36 or 48 hours after the start of step (ii), for example within 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 start of step (ii);or (c) the population of cells from step (iii) does not expand, or expands by no more than 5, 10, 15, 20, 25, 30, 35, or 40%, e.g., no more than 10%, compared to the population of cells at the start of step (i), e.g., as assessed by viable cell count. In some embodiments, the nucleic acid molecule of step (ii) is a DNA molecule. In some embodiments, the nucleic acid molecule of step (ii) is an RNA molecule. In some embodiments, the nucleic acid molecule of step (ii) is on a viral vector, e.g., a viral vector selected from a lentiviral vector, an adenoviral vector, or a retroviral vector. In some embodiments, the nucleic acid molecule of step (ii) is on a non-viral vector. In some embodiments, the nucleic acid molecule of step (ii) is on a plasmid. In some embodiments, the nucleic acid molecule of step (ii) is not on any vector. In some embodiments, step (ii) comprises transducing a population of cells (e.g., T cells) with a viral vector comprising a nucleic acid molecule encoding a CAR.

[0247] In some embodiments, the population of cells (e.g., T cells) is a T cell population associated with an autoimmune disease or disorder, such as lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with severe pulmonary involvement (e.g., as seen in autoHSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-associated myositis, e.g., antisynthetase syndrome associated with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory shear nephritis, or idiopathic inflammatory myopathy (e.g., inflammatory bowel disease ... The antibody-mediated neuroimmune disease may be collected from an apheresis sample (e.g., a leukapheresis sample) from a subject with a condition such as rheumatoid arthritis, severe refractory neuroimmune disease (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-associated disease (MOGAD), multiple sclerosis (MS)), severe refractory rheumatoid arthritis, antibody-mediated neuroimmune disease (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG-associated disease), NMDAR+ encephalitis or antibody-related paraneoplastic neurological disease), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis.

[0248] In some embodiments, an apheresis sample (e.g., a leukapheresis sample) is collected from a subject and transported as a frozen sample (e.g., a cryopreserved sample) to a cell manufacturing facility. The frozen apheresis sample is then thawed, and T cells (e.g., CD4+ T cells and / or CD8+ T cells) are selected from the apheresis sample, for example, using a cell sorting machine (e.g., a CliniMACS® Prodigy® device). The selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are then inoculated for CART manufacturing using the activation process described herein. In some embodiments, the selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are subjected to one or more rounds of freeze-thawing before inoculation for CART manufacturing.

[0249] In some embodiments, an apheresis sample (e.g., a leukapheresis sample) is collected from a subject and shipped as a fresh product (e.g., an unfrozen product) to a cell manufacturing facility. For example, a cell sorting machine (e.g., a CliniMACS® Prodigy® device) is used to select T cells (e.g., CD4+ T cells and / or CD8+ T cells) from the apheresis sample. The selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are then inoculated for CART manufacturing using the activation process described herein. In some embodiments, the selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are subjected to one or more rounds of freeze-thawing before inoculation for CART manufacturing.

[0250] In some embodiments, an apheresis sample (e.g., a leukapheresis sample) is collected from a subject. T cells (e.g., CD4+ T cells and / or CD8+ T cells) are selected from the apheresis sample, for example, using a cell sorting machine (e.g., a CliniMACS® Prodigy® device). The selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are then transported as frozen samples (e.g., cryopreserved samples) to a cell manufacturing facility. The selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are later thawed and inoculated for CART manufacturing using the activation process described herein.

[0251] In some embodiments, cells (e.g., T cells) are contacted with anti-CD3 and anti-CD28 antibodies for, e.g., 12 hours prior to transduction with a vector (e.g., a lentiviral vector) encoding a CAR. After 24 hours of culture, the cells are washed and formulated for storage or administration.

[0252] Without intending to be bound by theory, brief CD3 and anti-CD28 stimulation may promote efficient transduction of autologous renewing T cells. Compared to traditional CART manufacturing approaches, the activation process provided herein does not involve extended ex vivo expansion. Similar to the cytokine process, the activation process provided herein also preserves undifferentiated T cells during CART manufacturing.

[0253] In some embodiments, the population of cells is contacted with a multispecific binding molecule, eg, as described herein.

[0254] In some embodiments, the population of cells is contacted with (A) an agent that stimulates the CD3 / TCR complex, and / or (B) an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells.

[0255] In some embodiments, the agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3. In some embodiments, the agent that stimulates a costimulatory molecule and / or a growth factor receptor is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof. In some embodiments, the agent that stimulates a costimulatory molecule and / or a growth factor receptor is an agent that stimulates CD28. In some embodiments, the agent that stimulates the CD3 / TCR complex is selected from an antibody (e.g., a single domain antibody (e.g., a heavy chain variable domain antibody), a peptibody, a Fab fragment, or an scFv), a small molecule, or a ligand (e.g., a naturally occurring ligand, a recombinant ligand, or a chimeric ligand). In some embodiments, the agent that stimulates a costimulatory molecule and / or growth factor receptor is selected from an antibody (e.g., a single domain antibody (e.g., a heavy chain variable domain antibody), a peptibody, a Fab fragment, or an scFv), a small molecule, or a ligand (e.g., a naturally occurring ligand, a recombinant ligand, or a chimeric ligand). In some embodiments, the agent that stimulates the CD3 / TCR complex does not comprise a bead. In some embodiments, the agent that stimulates a costimulatory molecule and / or a growth factor receptor does not comprise a bead. In some embodiments, the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody. In some embodiments, the agent that stimulates a costimulatory molecule and / or a growth factor receptor comprises an anti-CD28 antibody. In some embodiments, the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody covalently bound to a colloidal polymer nanomatrix. In some embodiments, the agent that stimulates CD3 comprises one or more of a CD3 or TCR antigen binding domain, such as, but not limited to, an anti-CD3 or anti-TCR antibody or antibody fragment comprising one or more CDRs, a heavy chain, and / or a light chain thereof, such as, for example, an anti-CD3 or anti-TCR antibody provided in Table 27 of WO 2021 / 173985, which is incorporated by reference in its entirety. 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 polymer nanomatrix.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 CD28, ICOS, CD27, CD25, 4-1BB, IL6RB, and / or CD2 antigen binding domains, such as an anti-CD28, anti-ICOS, anti-CD27, anti-CD25, anti-4-1BB, anti-IL6RA, anti-IL6RB, or anti-CD2 antibody or antibody fragments thereof comprising one or more CDRs, heavy chains, and / or light chains, such as the anti-CD28, anti-ICOS, anti-CD27, anti-CD25, anti-4-1BB, anti-IL6RA, anti-IL6RB, or anti-CD2 antibodies provided in Table 27 of WO 2021 / 173985, which is incorporated by reference in its entirety. In some embodiments, the agent that stimulates the 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 the 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. In some embodiments, the multispecific binding molecule comprises one or more heavy chains and / or light chains, such as, but not limited to, the heavy chains and / or light chains provided in Table 28 of WO 2021 / 173985, which is incorporated by reference in its entirety. In some embodiments, the multispecific binding molecule comprises a bispecific antibody. In some embodiments, the bispecific antibody is formed according to any one of the schemes provided in Figure 50A of WO 2021 / 173985, which is incorporated by reference in its entirety. In some embodiments, the bispecific antibody is monovalent or bivalent. In some embodiments, the bispecific antibody comprises an Fc region. In some embodiments, the Fc region of the bispecific antibody is silenced. In some embodiments, the multispecific binding molecule comprises a plurality of bispecific antibodies. In some embodiments, one or more of the plurality of bispecific antibodies is monovalent.In some embodiments, one or more of the plurality of bispecific antibodies comprises an Fc region. In some embodiments, the Fc region of one or more of the plurality of bispecific antibodies is silenced. In some embodiments, one or more of the plurality of bispecific antibodies are conjugated together to form a multimer. In some embodiments, the multimer is formed according to any one of the schemes provided in Figure 50B of WO 2021 / 173985, the entire contents of which are incorporated herein by reference.

[0256] In some embodiments, the matrix comprises or is composed of, for example, a polymer that is non-toxic to cells, such as a biodegradable or biocompatible inert material. In some embodiments, the matrix is ​​composed of hydrophilic polymer chains, which achieve maximum mobility in aqueous solution through chain hydration. In some embodiments, the flexible matrix can be composed of collagen, purified proteins, purified peptides, polysaccharides, glycosaminoglycans, or extracellular matrix compositions. Polysaccharides can include, for example, cellulose ethers, starch, gum arabic, agarose, dextran, chitosan, hyaluronic acid, pectin, xanthan, guar gum, or alginate. Other polymers include polyesters, polyethers, polyacrylates, polyacrylamides, polyamines, polyethyleneimines, polyquaternium polymers, polyphosphazenes, polyvinyl alcohols, polyvinyl acetates, polyvinylpyrrolidones, block copolymers, or polyurethanes. In some embodiments, the flexible matrix is ​​a polymer of dextran.

[0257] 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 DNA encoding a CAR.

[0258] In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs simultaneously with contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells. In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs within 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 initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells as described above. In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs within 20 hours of initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells as described above. In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs within 19 hours of initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells as described above. In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs within 18 hours of initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells as described above. In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs within 17 hours of initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells as described above. In some embodiments, the step of contacting the population of cells with the nucleic acid molecule encoding a CAR occurs within 16 hours of initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or a growth factor receptor on the surface of the cells, as described above.In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs within 15 hours of initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells as described above. In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs within 14 hours of initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells as described above. In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs within 14 hours of initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells as described above. In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs within 13 hours of initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells as described above. In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs within 12 hours of initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells as described above. In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs within 11 hours of initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells as described above. In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs within 10 hours of initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells as described above.In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs within 9 hours of initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an 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 a nucleic acid molecule encoding a CAR occurs within 8 hours of initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an 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 a nucleic acid molecule encoding a CAR occurs within 7 hours of initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an 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 a nucleic acid molecule encoding a CAR occurs within 6 hours of initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an 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 a nucleic acid molecule encoding a CAR occurs within 5 hours of initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells as described above. In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs within 4 hours of initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells as described above. In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs within 3 hours of initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells as described above.In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs within 2 hours of initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells as described above. In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs within 1 hour of initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells as described above. In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs within 30 minutes of initiating contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells.

[0259] In some embodiments, the population of cells is harvested for storage or administration.

[0260] In some embodiments, the population of cells is harvested for storage or administration within 72, 60, 48, 36, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 hours after initiation of contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells. In some embodiments, the population of cells is harvested for storage or administration within 26 hours after initiation of contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells. In some embodiments, the population of cells is harvested for storage or administration within 25 hours after initiation of contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells. In some embodiments, the population of cells is harvested for storage or administration within 24 hours after initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells as described above. In some embodiments, the population of cells is harvested for storage or administration within 23 hours after initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells as described above. In some embodiments, the population of cells is harvested for storage or administration within 22 hours after initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells as described above.

[0261] In some embodiments, the population of cells is not expanded ex vivo.

[0262] 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%, as assessed, e.g., by viable cell count, compared to the population of cells prior to contact with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells as described above. In some embodiments, the population of cells is expanded by no more than 5%, as assessed, e.g., by viable cell count, compared to the population of cells prior to contact with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells as described above. In some embodiments, the population of cells is expanded by no more than 10% compared to the population of cells prior to contact with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells, as described above, e.g., as assessed by viable cell count. In some embodiments, the population of cells is expanded by no more than 15% compared to the population of cells prior to contact with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells, as described above, e.g., as assessed by viable cell count. In some embodiments, the population of cells is expanded by no more than 20% compared to the population of cells prior to contact with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells, as described above, as assessed by viable cell count. In some embodiments, the population of cells is expanded by no more than 25% as assessed by viable cell count compared to the population of cells prior to contact with, for example, an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells. In some embodiments, the population of cells is expanded by no more than 30% as assessed by viable cell count compared to the population of cells prior to contact with, for example, an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells.In some embodiments, the population of cells is expanded by no more than 35% as assessed by viable cell count compared to the population of cells prior to contact with, e.g., an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells. In some embodiments, the population of cells is expanded by no more than 40% as assessed by viable cell count compared to the population of cells prior to contact with, e.g., an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or growth factor receptor on the surface of the cells.

[0263] In some embodiments, the population of cells is expanded by no more than 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, 24, 36, or 48 hours, as assessed by viable cell count, compared to the population of cells prior to contact with, for example, one or more of the cytokines described above.

[0264] In some embodiments, the activation process is carried out in a serum-free cell culture medium. In some embodiments, the activation process is carried out in a cell culture medium containing one or more cytokines selected from IL-2, IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)), or IL-6 (e.g., IL-6 / sIL-6Ra)). In some embodiments, hetIL-15 is [ka] In some embodiments, hetIL-15 comprises an amino acid sequence having at least about 70, 75, 80, 85, 90, 95, or 99% identity to SEQ ID NO: 309. In some embodiments, the activation process is carried out in a cell culture medium comprising an LSD1 inhibitor. In some embodiments, the activation process is carried out in a cell culture medium comprising a MALT1 inhibitor. In some embodiments, the serum-free cell culture medium comprises a blood substitute. In some embodiments, the serum substitute is CTS™ Immune Cell Serum Replacer (ICSR). In some embodiments, the level of ICSR can be, for example, up to 5%, e.g., about 1%, 2%, 3%, 4%, or 5%. Without intending to be bound by theory, the use of a cell culture medium containing ICSR, e.g., 2% ICSR, such as Rapid Media shown in Table 21 or Table 25, can improve cell viability during the manufacturing processes described herein.

[0265] In some embodiments, the disclosure provides a method of generating a population of cells (e.g., T cells) expressing a chimeric antigen receptor (CAR), comprising: (a) administering to a patient suffering from, for example, lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with severe pulmonary involvement (e.g., as seen in autoHSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-associated myositis, e.g., antisynthetase syndrome with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjogren's disease, severe refractory neuroimmune disease (e.g., myasthenia gravis (MG), optic nerve injury), or (b) providing an apheresis sample (e.g., a fresh or cryopreserved leukapheresis sample) collected from a subject with a condition such as neuromyelitis optica (NMO), MOGAD (anti-MOG associated disease), multiple sclerosis (MS), severe refractory rheumatoid arthritis, antibody-mediated neuroimmune disease (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG associated disease), NMDAR+ encephalitis or antibody-related paraneoplastic neurological disease), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis; (b) selecting T cells from the apheresis sample (e.g., using negative selection, positive selection, or bead-less selection); (c) selecting the isolated T cells from a subject, e.g., 1 x 10 6 ~1×10 7(d) contacting the T cells with an agent that stimulates the T cells, e.g., an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule and / or a growth factor receptor on the surface of the cell (e.g., contacting the T cells with an anti-CD3 and / or anti-CD28 antibody, e.g., contacting the T cells with TransAct); (e) contacting the T cells with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding a CAR (e.g., contacting the T cells with a virus comprising a nucleic acid molecule encoding a CAR), e.g., for 6 to 48 hours, e.g., 20 to 28 hours; and (f) washing and recovering the T cells for storage (e.g., reformulating the T cells in cryopreservation medium) or administration. In some embodiments, step (f) is performed within 30, 36, or 48 hours after the initiation of step (d) or (e), such as within 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 initiation of step (d) or (e).

[0266] In some embodiments of the above-described methods, the methods are performed in a closed system. In some embodiments, the isolation, activation, transduction, incubation, and washing of T cells are all performed in a closed system. In some embodiments of the above-described methods, the methods are performed in separate devices. In some embodiments, the isolation, activation and transduction, incubation, and washing of T cells are performed in separate devices.

[0267] In some embodiments of the aforementioned methods, the method further comprises adding an adjuvant or transduction enhancing reagent to the cell culture medium to increase transduction efficiency. In some embodiments, the adjuvant or transduction enhancing reagent comprises a cationic polymer. In some embodiments, the adjuvant or transduction enhancing reagent is selected from LentiBOOST™ (Sirion Biotech), Vectofusin-1, F108 (Poloxamer 338 or Pluronic® F-38), protamine sulfate, hexadimethrine bromide (Polybrene), PEA, Pluronic F68, Pluronic F127, Synperonic, or LentiTrans™. In some embodiments, the transduction enhancing reagent is LentiBOOST™ (Sirion Biotech). In some embodiments, the transduction enhancing reagent is F108 (Poloxamer 338 or Pluronic® F-38).

[0268] In some embodiments of the aforementioned methods, transducing the population of cells (e.g., T cells) with a viral vector comprises subjecting the population of cells and the viral vector to centrifugal force under conditions that enhance transduction efficiency. In one embodiment, the cells are transduced by spinoculation.

[0269] In some embodiments of the aforementioned methods, cells (e.g., T cells) are activated and transduced in cell culture flasks with a gas-permeable membrane at the base and supporting a large medium volume without substantially compromising gas exchange. In some embodiments, cell growth is achieved by providing access, e.g., substantially uninterrupted access, to nutrients via convection.

[0270] Multispecific binding molecules Methods for generating CAR-expressing cells may utilize an agent that stimulates the CD3 / TCR complex and an agent that stimulates a costimulatory molecule and / or a growth factor receptor. In some embodiments, the agent that stimulates the CD3 / TCR complex and the agent that stimulates a costimulatory molecule and / or a growth factor receptor are included in a multispecific binding molecule. In some embodiments, the multispecific binding molecule of the present disclosure is a multispecific binding molecule described in any of WO2021 / 173985 (incorporated by reference in its entirety), WO2022 / 040586 (incorporated by reference in its entirety), and PCT / IB2022 / 057799 (incorporated by reference in its entirety).

[0271] In some embodiments, the multispecific binding molecule comprises a first binding domain and a second binding domain. For example, the first binding domain can be an anti-CD3 binding domain and the second binding domain can be a costimulatory molecule binding domain, or the first binding domain can be a costimulatory molecule binding domain and the second binding domain can be an anti-CD3 binding domain. In some embodiments, the costimulatory molecule binding domain binds to CD2, CD28, CD25, CD27, IL6Rb, ICOS, or 41BB. Non-limiting examples of such binding domains as described above are provided, for example, in Table 27 of WO 2021 / 173985 (the contents of which are incorporated herein by reference in their entireties). In some embodiments, the multispecific binding molecule is configured as any one of the schematic diagrams provided in Figures 50A-50B, 51A-51B, 61A-61B, and 63A-63B of WO 2021 / 173985 (incorporated by reference in its entirety).

[0272] In some embodiments, the multispecific binding molecule comprises a CD3 antigen-binding domain and a CD28 or CD2 antigen-binding domain. In some embodiments, the CD3 antigen-binding domain is an anti-CD3 antibody, optionally anti-CD3(1), anti-CD3(2), anti-CD3(3), or anti-CD3(4), as provided in Table 27 of WO 2021 / 173985 (the contents of which are incorporated by reference in their entirety), or an antibody fragment comprising one or more CDRs, VH, and / or VL thereof. In some embodiments, the CD28 antigen-binding domain is an anti-CD28 antibody, optionally anti-CD28(1) or anti-CD28(2), as provided in Table 27 of WO 2021 / 173985 (the contents of which are incorporated by reference in their entirety), or an antibody fragment comprising one or more CDRs, VH, heavy chain, VL, and / or light chain thereof. In some embodiments, the CD2 antigen-binding domain is an anti-CD2 antibody, optionally an anti-CD2(1) provided in Table 27 of WO 2021 / 173985 (incorporated by reference in its entirety), or an antibody fragment comprising one or more CDRs, VH, heavy chain, VL, and / or light chain thereof. In some embodiments, the multispecific binding molecules described herein comprise a CDR, VH, VL, HC, and / or LC disclosed in Table 27 of WO 2021 / 173985 (incorporated by reference in its entirety), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0273] In some embodiments, the multispecific binding molecule comprises one or more heavy and / or light chains. Non-limiting exemplary heavy and light chain sequences that may be included in the multispecific binding molecules described herein are provided in Table 28 of WO 2021 / 173985 (incorporated by reference in its entirety) or Table 20 of WO 2022 / 040586 (incorporated by reference in its entirety). In some embodiments, the multispecific binding molecule comprises one or more heavy and / or light chain sequences disclosed in Table 20 of WO 2022 / 040586 (incorporated by reference in its entirety), or sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

[0274] In some embodiments, the multispecific binding molecules described herein comprise an Fc region, e.g., the Fc region is Fc-silent, e.g., an Fc region described in WO 2021 / 173985 (incorporated by reference in its entirety) or WO 2022 / 040586 (incorporated by reference in its entirety). In some embodiments, the Fc region comprises a mutation at one or more (e.g., all) of D265, N297, and P329 (numbered according to the EU numbering system). In some embodiments, the Fc region comprises a mutation at one, two, three, or all of positions L234 (e.g., L234A), L235 (e.g., L235A), S267 (e.g., S267K), and P239 (e.g., P329A) (numbered according to the EU numbering system). In some embodiments, the Fc region comprises mutations at L234 (e.g., L234A), L235 (e.g., L235A), S267 (e.g., S267K), and P239 (e.g., P329A) (LALASKPA) (numbered according to the Eu numbering system). In some embodiments, the Fc region comprises one or more mutations, e.g., as described in WO 2021 / 173985 (incorporated by reference in its entirety) or WO 2022 / 040586 (incorporated by reference in its entirety).

[0275] In some embodiments, a multispecific binding molecule comprises (A) an anti-CD3-binding domain and (B) a costimulatory molecule-binding domain (e.g., an anti-CD2-binding domain or an anti-CD28-binding domain). In some embodiments, the anti-CD3-binding domain, e.g., an anti-CD3 scFv, is located N-terminally of the costimulatory molecule-binding domain, e.g., an anti-CD2 Fab or an anti-CD28 Fab. In some embodiments, the anti-CD3-binding domain, e.g., an anti-CD3 scFv, is located C-terminally of the costimulatory molecule-binding domain, e.g., an anti-CD2 Fab or an anti-CD28 Fab.

[0276] In some embodiments, the Fc region is located between the anti-CD3-binding domain and the costimulatory molecule-binding domain. In some embodiments, the anti-CD3-binding domain is located C-terminal to the costimulatory molecule-binding domain, and the Fc region is located between the anti-CD3-binding domain and the costimulatory molecule-binding domain.

[0277] In some embodiments, the multispecific binding molecule comprises a CH2 and the anti-CD3 binding domain is located N-terminal to the CH2. In some embodiments, the anti-CD3 binding domain is linked to the CH2 by a peptide linker, e.g., a glycine-serine linker, e.g., a (G4S)4 linker.

[0278] In some embodiments, the multispecific binding molecule further comprises a CL. In some embodiments, the CL is C-terminal to the VL of the costimulatory molecule-binding domain. In some embodiments, the CL domain is linked to CH1, for example, by a disulfide bridge.

[0279] In some embodiments, a multispecific binding molecule comprises: (i) a first polypeptide comprising, from N- to C-terminus, a VH of a costimulatory molecule-binding domain, a CH1, CH2, CH3, VH of an anti-CD3 binding domain, and a VL of the anti-CD3 binding domain; and (ii) a second polypeptide comprising, from N- to C-terminus, a VL and CL of a costimulatory molecule-binding domain. In some embodiments, the anti-CD3 binding domain comprises an scFv. In some embodiments, the costimulatory molecule-binding domain is a portion of a Fab fragment, e.g., a Fab fragment that is a portion of a polypeptide sequence that includes an Fc domain. In some embodiments, the anti-CD3 binding domain is linked to the CH3 by a peptide linker, e.g., a glycine-serine linker, e.g., a (G4S)4 linker.

[0280] In some embodiments, a multispecific binding molecule comprises: (i) a first polypeptide comprising, from N- to C-terminus, a VH of an anti-CD3 binding domain, a VL of an anti-CD3 binding domain, and a VH, CH1, CH2, and CH3 of a costimulatory molecule binding domain; and (ii) a second polypeptide comprising, from N- to C-terminus, a VL and CL of a costimulatory molecule binding domain. In some embodiments, the anti-CD3 binding domain is linked to the costimulatory molecule binding domain by a peptide linker, e.g., a glycine-serine linker, e.g., a (G4S)4 linker.

[0281] In some embodiments, a multispecific binding molecule comprises: (i) a first polypeptide comprising, from N- to C-terminus, a VH, a CH1 of a costimulatory molecule-binding domain, a VH of an anti-CD3 binding domain, a VL, a CH2, and a CH3 of an anti-CD3 binding domain; and (ii) a second polypeptide comprising, from N- to C-terminus, a VL and a CL of a costimulatory molecule-binding domain. In some embodiments, the anti-CD3 binding domain is linked to the CH1 by a peptide linker, e.g., a glycine-serine linker, e.g., a (GS)2 linker.

[0282] In some embodiments, the multispecific binding molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 726 of WO 2022 / 040586 (incorporated by reference in its entirety), or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or a light chain comprising the amino acid sequence of SEQ ID NO: 728 of WO 2022 / 040586 (incorporated by reference in its entirety), or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0283] It is understood herein that in many embodiments, a multispecific binding molecule comprises two or more polypeptide chains covalently linked to one another, for example, via disulfide bridges, although in some embodiments, the two or more polypeptide chains of a multispecific binding molecule may be non-covalently linked to one another.

[0284] It is also understood that a Fab fragment may exist as part of a larger protein, for example, a Fab fragment may be fused to a CH2 and CH3 and thus be part of a full length antibody.

[0285] Multispecific binding molecules comprising agents that stimulate the CD3 / TCR complex and agents that stimulate costimulatory molecules and / or growth factor receptors disclosed herein are contemplated for use in the manufacturing embodiments disclosed herein, e.g., traditional manufacturing or activated high speed manufacturing.

[0286] Populations of CAR-expressing cells produced by the processes disclosed herein In some embodiments, the disclosure features immune effector cells (e.g., T cells or NK cells) engineered to express a CAR (e.g., a B cell antigen, e.g., CD19), e.g., produced by any of the manufacturing methods described herein, wherein the engineered immune effector cells exhibit immunosuppressive properties. In some embodiments, the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain. An exemplary antigen is a B cell antigen described herein. In some embodiments, a cell (e.g., a T cell or NK cell) is transformed with a CAR, and the CAR is expressed on the cell surface. In some embodiments, the cell (e.g., a T cell or NK cell) is transduced with a viral vector encoding a CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the cell can stably express the CAR. In some embodiments, the cell (e.g., a T cell or NK cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, or DNA, encoding the CAR. In some such embodiments, the cell can transiently express the CAR.

[0287] In some embodiments, a population of cells (e.g., immune effector cells, e.g., T cells or NK cells) engineered to express a CAR is provided, produced by any of the manufacturing methods described herein (e.g., the activation processes described herein).

[0288] In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ T cells, in the population of cells at the end of the manufacturing process (e.g., at the end of an activation process described herein) is (1) the same, (2) differs by, e.g., by no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15%, or (3) is increased by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25%, compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ T cells, in the population of cells at the start of the manufacturing process (e.g., at the start of an activation process described herein). In some embodiments, the population of cells at the end of the manufacturing process (e.g., at the end of an activation process described herein) exhibits a higher percentage (e.g., 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) of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ T cells, compared to cells produced by a similar method but including expanding the population of cells in vitro for more than 26 hours (e.g., more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50% higher).

[0289] In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ T cells, in the population of cells at the end of the manufacturing process (e.g., at the end of the activation process described herein) is 20, 25, 30, 35, 40, 45, 50, 55, or 60% or greater.

[0290] In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells at the end of the manufacturing process (e.g., at the end of an activation process described herein) is (1) the same, (2) differs by, e.g., by no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15%, or (3) is reduced by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25%, compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells at the start of the manufacturing process (e.g., at the start of an activation process described herein). In some embodiments, the population of cells at the end of the manufacturing process (e.g., at the end of an activation process described herein) exhibits a lower percentage (e.g., 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) of naive cells, e.g., central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, compared to cells produced by a similar method but including, e.g., persisting for more than 26 hours (e.g., persisting for more than 5, 6, 7, 8, 9, 10, 11, or 12 days), or expanding the population of cells in vitro for, e.g., more than 3 days (e.g., expanding the population of cells in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days).

[0291] In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells at the end of the manufacturing process (e.g., at the end of the activation process described herein) is 40, 45, 50, 55, 60, 65, 70, 75, or 80% or less.

[0292] In some embodiments, the population of cells at the end of the manufacturing process (e.g., at the end of an activation process described herein) persists for longer than, for example, 26 hours (e.g., persists for more than 5, 6, 7, 8, 9, 10, 11, or 12 days) after administration in vivo, or proliferates for a longer period of time or at a higher level (e.g., at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 85, or 90% higher) than cells produced by a similar method but including a step of expanding the population of cells in vitro for, for example, more than 3 days (e.g., expanding the population of cells in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days).

[0293] In some embodiments, the population of cells is enriched for IL6R-expressing cells (e.g., IL6Rα and / or IL6Rβ) prior to the start of the manufacturing process (e.g., prior to the start of an activation process described herein). In some embodiments, the population of cells comprises, e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80% or less IL6R-expressing cells (e.g., cells positive for IL6Rα and / or IL6Rβ), e.g., at the start of the manufacturing process (e.g., at the start of an activation process described herein).

[0294] Pharmaceutical Composition Furthermore, the present disclosure relates to the use of antibodies against autoimmune diseases involving cells or tissues that express an antigen as described herein, among other diseases (e.g., lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with severe pulmonary involvement (e.g., as seen in autoHSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-associated myositis, e.g., antisynthetase syndrome associated with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjogren's disease, severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-associated diseases, etc.).

[0013] Provided are CAR-expressing cell compositions and their use in medicaments or methods for treating severe refractory rheumatoid arthritis, antibody-mediated neuroimmune diseases (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG associated disease), NMDAR+ encephalitis or antibody-associated paraneoplastic neurological diseases), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis. In some embodiments, provided are pharmaceutical compositions comprising a CAR-expressing cell, e.g., a plurality of CAR-expressing cells, made by a manufacturing process described herein (e.g., an activation process described herein), in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0295] Chimeric antigen receptor (CAR) The present invention provides immune effector cells (e.g., T cells or NK cells) engineered to contain one or more CARs that direct the immune effector cells to cells associated with an autoimmune disorder. This is achieved through an antigen-binding domain on the CAR that is specific for a B-cell-associated antigen. There are two classes of B-cell antigens that can be targeted by the CARs described herein: (1) B-cell antigens that are expressed on the surface of B cells; and (2) B-cell antigens that are themselves intracellular, but where fragments (peptides) of such antigens are presented on the surface of B cells by the MHC (major histocompatibility complex).

[0296] Thus, for example, immune effector cells obtained by the methods described herein can be engineered to contain a CAR that targets one or more of the following B cell antigens: CD19.

[0297] Non-limiting example sequences of various components that may be part of the CAR molecules described herein are listed in Table 1, where "aa" represents an amino acid and "na" represents a nucleic acid that encodes the corresponding peptide.

[0298] [Table 1]

[0299] [Table 2]

[0300] [Table 3]

[0301] [Table 4]

[0302] Bispecific CAR In some embodiments, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In some embodiments, the first and second epitopes are the same antigen, e.g., the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes overlap. In some embodiments, the first and second epitopes do not overlap. In some embodiments, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In some embodiments, a bispecific antibody molecule comprises heavy and light chain variable domain sequences that have binding specificity for a first epitope and heavy and light chain variable domain sequences that have binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises a half antibody that has binding specificity for a first epitope and a half antibody that has binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises a half antibody or fragment thereof that has binding specificity for a first epitope and a half antibody or fragment thereof that has binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises an scFv or fragment thereof that has binding specificity for a first epitope and an scFv or fragment thereof that has binding specificity for a second epitope.

[0303] In certain embodiments, the antibody molecule is a multispecific (e.g., bispecific or trispecific) antibody molecule. Protocols for producing bispecific or heterodimeric antibody molecules and various configurations of bispecific antibody molecules are described, for example, in paragraphs 455-458 of WO 2015 / 142675, filed March 13, 2015, which is incorporated by reference in its entirety.

[0304] In some embodiments, the bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence, e.g., an scFv, that has binding specificity for CD19, e.g., comprises an scFv as described herein, or comprises light chain CDRs and / or heavy chain CDRs from an scFv described herein and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope for a different antigen.

[0305] Chimeric TCR In some embodiments, antigens and antigen fragments of the present invention (e.g., CD19 antibodies and fragments) can be grafted into one or more constant domains of a T cell receptor ("TCR") chain, such as a TCR alpha or TCR beta chain, to create a chimeric TCR. Without being bound by theory, it is believed that the chimeric TCR signals through the TCR complex upon antigen binding. For example, an scFv as described herein can be grafted into the constant domains of a TCR chain, such as the TCR alpha chain and / or the TCR beta chain, e.g., at least a portion of the extracellular constant domain, transmembrane domain, and cytoplasmic domain. As another example, an antibody fragment, such as a VL domain as described herein, can be grafted into the constant domain of a TCR alpha chain, and an antibody fragment, such as a VH domain as described herein, can be grafted into the constant domain of a TCR beta chain (or alternatively, the VL domain can be grafted into the constant domain of a TCR beta chain, and the VH domain can be grafted into the TCR alpha chain). As another example, the CDRs of an antibody or antibody fragment can be grafted into the TCR alpha chain and / or beta chain to create a chimeric TCR. For example, an LCDR described herein may be grafted into the variable domain of a TCR alpha chain, and an HCDR described herein may be grafted into the variable domain of a TCR beta chain, or vice versa. Such chimeric TCRs may be produced, for example, by methods known in the art (e.g., Willemsen RA et al., Gene Therapy 2000;7:1369-1377; Zhang T et al., Cancer Gene Ther 2004;11:487-496; Aggen et al., Gene Ther. 2012 Apr;19(4):365-74).

[0306] Non-antibody scaffolds In embodiments, the antigen-binding domain comprises a non-antibody scaffold, such as fibronectin, ankyrin, domain antibody, lipocalin, small modular immunopharmaceutical, maxibody, protein A, or affilin. The non-antibody scaffold has the ability to bind to a target antigen on a cell. In embodiments, the antigen-binding domain is a polypeptide or fragment thereof of a naturally occurring protein expressed on a cell. In some embodiments, the antigen-binding domain comprises a non-antibody scaffold. A variety of non-antibody scaffolds can be used, so long as the resulting polypeptide contains at least one binding region that specifically binds to a target antigen on a target cell.

[0307] Non-antibody scaffolds include fibronectin (Novartis, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd., Cambridge, MA and Ablynx nv, Zwijnaarde, Belgium), lipocalins (Pieris Proteolab AG, Freising, Germany), small modular immunopharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA), maxybodies (Avidia, Inc., Mountain View, CA), protein A (Affibody AG, Sweden), and affilin (gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany).

[0308] In some embodiments, the antigen-binding domain comprises the extracellular domain of a molecule that binds to a counter-ligand on the surface of a target cell, or a counter-ligand-binding fragment thereof.

[0309] The immune effector cells can comprise a recombinant DNA construct comprising sequences encoding a CAR, where the CAR comprises an antigen binding domain (e.g., an antibody or antibody fragment, a TCR or a TCR fragment) that specifically binds to a B cell antigen, e.g., a B cell antigen described herein, and an intracellular signaling domain. The intracellular signaling domain can comprise a costimulatory signaling domain and / or a primary signaling domain, e.g., a zeta chain. As described elsewhere, the methods described herein can include transducing cells (e.g., from a population of T regulatory-depleted cells) with a nucleic acid encoding a CAR, e.g., a CAR described herein.

[0310] In some embodiments, the CAR comprises an scFv domain, where the scFv can be preceded by an optional leader sequence such as provided in SEQ ID NO: 1 and can be followed by an optional hinge sequence such as provided in SEQ ID NO: 2 or SEQ ID NO: 36 or SEQ ID NO: 38, a transmembrane region such as provided in SEQ ID NO: 6, an intracellular signaling domain comprising SEQ ID NO: 7 or SEQ ID NO: 16, and a CD3ζ sequence comprising SEQ ID NO: 9 or SEQ ID NO: 10, e.g., where these domains are contiguous and in the same reading frame to form a single fusion protein.

[0311] In some embodiments, exemplary CAR constructs comprise an optional leader sequence (e.g., a leader sequence described herein), an extracellular antigen binding domain (e.g., an antigen binding domain described herein), a hinge (e.g., a hinge region described herein), a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular stimulatory domain (e.g., an intracellular stimulatory domain described herein). In some embodiments, exemplary CAR constructs comprise an optional leader sequence (e.g., a leader sequence described herein), an extracellular antigen binding domain (e.g., an antigen binding domain described herein), a hinge (e.g., a hinge region described herein), a transmembrane domain (e.g., a transmembrane domain described herein), an intracellular costimulatory signaling domain (e.g., a costimulatory signaling domain described herein), and / or an intracellular primary signaling domain (e.g., a primary signaling domain described herein).

[0312] An exemplary leader sequence is provided as SEQ ID NO: 1. An exemplary hinge / spacer sequence is provided as SEQ ID NO: 2 or SEQ ID NO: 36 or SEQ ID NO: 38. An exemplary transmembrane domain sequence is provided as SEQ ID NO: 6. An exemplary 4-1BB protein intracellular signaling domain sequence is provided as SEQ ID NO: 7. An exemplary CD27 intracellular signaling domain sequence is provided as SEQ ID NO: 16. An exemplary CD3 zeta domain sequence is provided as SEQ ID NO: 9 or SEQ ID NO: 10.

[0313] In some embodiments, the immune effector cell comprises a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding an antigen-binding domain, which sequence is contiguous to and in the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. Exemplary intracellular signaling domains that can be used in a CAR include, but are not limited to, one or more intracellular signaling domains such as CD3-zeta, CD28, 4-1BB, etc. In some examples, a CAR can comprise any combination of CD3-zeta, CD28, CD27, 4-1BB, etc.

[0314] Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, such as by screening libraries from cells which express the nucleic acid molecule, by deriving the nucleic acid molecule from a vector known to contain it, or by isolating it directly from cells and tissues which contain it, using standard techniques. Alternatively, the nucleic acid of interest can be produced synthetically rather than cloned.

[0315] Nucleic acids encoding CARs can be introduced into immune effector cells, for example, using retroviral or lentiviral vector constructs.

[0316] Nucleic acids encoding CARs can also be introduced into immune effector cells using, for example, RNA constructs that can be directly transfected into cells. Methods for generating mRNA for use in transfection include in vitro transcription (IVT) of a template with specifically designed primers, followed by poly(A) addition, to produce a construct, typically 50-2000 bases long (e.g., SEQ ID NO: 35 herein), that includes 3' and 5' untranslated sequences ("UTRs") (e.g., 3' and / or 5' UTRs described herein), a 5' cap (e.g., 5' caps described herein), and / or an internal ribosome entry site (IRES) (e.g., IRES described herein), the nucleic acid to be expressed, and a poly(A) tail. The RNA thus produced can be efficiently transfected into cells of different species. In some embodiments, the template includes the sequence of the CAR. In some embodiments, the RNA CAR vector is transduced into cells, e.g., T cells, by electroporation.

[0317] antigen-binding domain In some embodiments, a population of immune effector cells, e.g., T regulatory depleted cells, comprises a nucleic acid encoding a CAR comprising a target-specific binding element, also referred to as an antigen-binding domain. The selection of the binding element depends on the type and number of ligands that define the surface of the target cell. For example, the antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular pathology. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain in the CARs described herein include those associated with autoimmune diseases.

[0318] In some embodiments, the portion of the CAR that comprises the antigen binding domain comprises an antigen binding domain that targets a B cell antigen, e.g., a B cell antigen described herein.

[0319] The antigen-binding domain may be any domain that binds to an antigen, including, but not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and functional fragments thereof, such as, but not limited to, single-domain antibodies, such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains (VHH) of camelid-derived nanobodies, and recombinant fibronectin domains, T cell receptors (TCRs) or fragments thereof, e.g., single-chain TCRs, and alternative scaffolds known in the art to function as antigen-binding domains. In some cases, it is beneficial for the antigen-binding domain to be derived from the same species in which the CAR will ultimately be used. For example, for human use, it may be beneficial for the antigen-binding domain of the CAR to comprise human or humanized residues in the antigen-binding domain of an antibody or antibody fragment.

[0320] CD19 CAR In some embodiments, a CAR-expressing cell described herein is a CD19 CAR-expressing cell (e.g., a cell that expresses a CAR that binds to human CD19).

[0321] In some embodiments, the antigen binding domain of the CD19 CAR has the same or 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 comprises the scFv fragment described in Nicholson et al. Mol. Immun. 34(16-17):1157-1165 (1997).

[0322] In some embodiments, the CD19 CAR comprises an antigen-binding domain (e.g., a humanized antigen-binding domain) according to Table 3 of WO 2014 / 153270 (incorporated herein by reference). WO 2014 / 153270 also describes methods for assaying the binding and efficacy of various CAR constructs.

[0323] In some embodiments, the parental murine scFv sequence is the CAR19 construct described in WO 2012 / 079000 (incorporated herein by reference). In some embodiments, the anti-CD19 binding domain is an scFv described in WO 2012 / 079000.

[0324] In some embodiments, the CAR molecule comprises a fusion polypeptide sequence set forth in SEQ ID NO: 12 of WO 2012 / 079000, which provides a murine-derived scFv fragment that specifically binds to human CD19.

[0325] In some embodiments, the CD19 CAR comprises the amino acid sequence set forth in SEQ ID NO: 12 of WO 2012 / 079000.

[0326] In some embodiments, the amino acid sequence is [ka] or a substantially compatible sequence thereto.

[0327] In some embodiments, the CD19 CAR has the USAN name TISAGENLECLEUCEL-T. In embodiments, CTL019 is generated by genetic modification of T cells, the modification being mediated by stable insertion by transduction with a self-inactivating, replication-deficient lentiviral (LV) vector containing the CTL019 transgene under the control of the EF-1α promoter. CTL019 can be a mixture of transgene-positive and transgene-negative T cells delivered to a subject based on percent transgene-positive T cells.

[0328] In some embodiments, the population of CAR T cells that specifically bind to CD19 comprises lapcaptagene autoleucel. Lapcaptagene autoleucel is generated using autologous T cells obtained by leukapheresis from peripheral blood mononuclear cells (e.g., from a subject with an autoimmune disease or disorder) and subsequently transduced with a lentiviral vector encoding an autoinactivated, non-replicating T cell chimeric antigen receptor that targets CD19. The expressed transgene contains a CD8α leader sequence, a murine anti-CD19 single-chain variable fragment (scFv) derived from the murine hybridoma FMC63, a CD8α hinge and transmembrane region, and 4-1BB (CD137) and CD3ζ (TCRζ) signaling domains, and is under the control of the elongation factor 1α (EF1α) promoter. This construct is flanked by 5' and 3' long terminal repeats (LTRs) and also contains a psi packaging signal, a Rev response element (RRE), a central polypurine tract (cPPT) sequence, and an optimized woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Leukapheresis material is enriched for CD4 / CD8 T cells by positive immunoselection, activated with CD3 and CD28 agonists, and transduced with vectors. Once no further cell proliferation is observed, T cells are washed, formulated for injection, and cryopreserved. Lapcaptagene autoleucel is composed of >80% T cells and <1% B cells, representing 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 preserved.

[0329] In some embodiments, the CAR-expressing or CAR-positive cells described herein (e.g., CD19 CAR-expressing or CD19 CAR-positive cells) are lapcaptagene autoleucel. In some embodiments, the population of ARM-CD19 CAR T cells is lapcaptagene autoleucel.

[0330] In some embodiments, lapcavtagene autoleucel is used to treat an autoimmune disease or disorder, such as lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with severe pulmonary involvement (e.g., as seen in autoHSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-associated myositis, e.g., antisynthetase syndrome associated with ILD), vasculitis (e.g., ANCA-associated vasculitis), ), severe refractory Sjogren's disease, severe refractory neuroimmune disease (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-associated disease (MOGAD), multiple sclerosis (MS)), severe refractory rheumatoid arthritis, antibody-mediated neuroimmune disease (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG-associated disease), NMDAR+ encephalitis or antibody-related paraneoplastic neurological disease), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis.

[0331] In other embodiments, the CD19 CAR comprises an antigen-binding domain (e.g., a humanized antigen-binding domain) described in Table 3 of WO 2014 / 153270, which is incorporated herein by reference.

[0332] Humanization of murine CD19 antibodies is desirable in clinical settings, where murine-specific residues may induce a human-anti-mouse antigen (HAMA) response in patients undergoing CART19 treatment, i.e., treatment with T cells transduced with a CAR19 construct. The generation, characterization, and efficacy of humanized CD19 CAR sequences are described in WO 2014 / 153270, the contents of which are incorporated herein by reference in their entirety, including Examples 1-5 (pp. 115-159).

[0333] In some embodiments, the CAR molecule is [ka] It is a humanized CD19 CAR comprising the amino acid sequence:

[0334] In some embodiments, the CAR molecule is [ka] It is a humanized CD19 CAR comprising the amino acid sequence:

[0335] Any known CD19 CAR, for example, the CD19 antigen binding domain of any known CD19 CAR in the art, can be used in accordance with the present disclosure. For example, LG-740; US Patent No. 8,399,645; US Patent No. 7,446,190; al.,Blood,118(18):4817-4828(2011);Kochenderfer et al.,Blood 116(20):4099-102(2010);Kochenderfer et al.,Blood 122(25):4129-39(2013);and 16th Annu Meet Am Soc Gen Cell CD19 described in Ther(ASGCT)(May 15-18,Salt Lake City)2013,Abst 10 It's a CAR.

[0336] Exemplary CD19 CARs include the CD19 CARs described herein or those 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, N CT02546739, NCT02656147, NCT02772198, NCT00709033, NCT02081937, NCT00924326, NCT02735083, NCT02794246, NCT02746952, NCT01593 696, NCT02134262, NCT01853631, NCT02443831, NCT02277522, NCT02348216, NCT02614066, NCT02030834, NCT02624258, NCT02625480, NC T02030847, NCT02644655, NCT02349698, NCT02813837, NCT02050347, NCT01683279, NCT02529813, NCT02537977, NCT02799550, NCT026725 01, NCT02819583, NCT02028455, NCT01840566, NCT01318317, NCT01864889, NCT02706405, NCT01475058, NCT01430390, NCT02146924, NCT 02051257, NCT02431988, NCT01815749, NCT02153580, NCT01865617, NCT02208362, NCT02685670, NCT02535364, NCT02631044, NCT0272888 2, NCT02735291, NCT01860937, NCT02822326, NCT02737085, NCT02465983, NCT02132624, NCT02782351, NCT01493453, NCT02652910, NCT02247609, NCT01029366, NCT01626495, NCT02721407, NCT01044069, NCT00422383, NCT01680991, NCT02794961, or NCT02456207, each of which is incorporated herein by reference in its entirety.

[0337] In some embodiments, the CD19 CAR comprises, for example, a CDR, VH, VL, scFv or complete CAR sequence disclosed in Table 2, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto.

[0338] [Table 5]

[0339] [Table 6]

[0340] [Table 7]

[0341] [Table 8]

[0342] [Table 9]

[0343] [Table 10]

[0344] Other exemplary CAR properties In some embodiments, the B cell antigen binding domain is a fragment, e.g., a single-chain variable fragment (scFv). In some embodiments, the B cell antigen binding domain is an Fv, Fab, (Fab')2, or a bifunctional (e.g., bispecific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In some embodiments, the antibodies and fragments thereof of the present invention bind to a B cell antigen protein as described herein with wild-type or enhanced affinity.

[0345] In some instances, scFvs can be produced by methods known in the art (see, e.g., Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFv molecules can be produced by linking VH and VL domains together using a flexible polypeptide linker. The scFv molecule contains a linker (e.g., a Ser-Gly linker) of optimal length and / or amino acid composition. Linker length can significantly affect how the variable regions of an scFv fold and interact. Indeed, when short polypeptide linkers are used (e.g., 5-10 amino acids), intrachain folding is prevented. Intrachain folding is also required for the two variable regions to join together to form a functional epitope-binding site. For examples of linker orientations and sizes, see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. USA 90:6444-6448, U.S. Patent Application Publication Nos. 2005 / 0100543, 2005 / 0175606, 2007 / 0014794, and WO 2006 / 020258 and WO 2007 / 024715, which are incorporated herein by reference.

[0346] An scFv may comprise a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acid residues between its VL and VH regions. The linker sequence may comprise any naturally occurring amino acid. In one embodiment, the linker sequence comprises the amino acids glycine and serine. In some embodiments, the linker sequence comprises a series of glycine and serine repeats, such as (Gly4Ser)n, where n is a positive integer of 1 or more (SEQ ID NO: 25). In some embodiments, the linker may be (Gly4Ser)4 (SEQ ID NO: 27) or (Gly4Ser)3 (SEQ ID NO: 28). Varying the linker length may maintain or enhance activity, resulting in superior efficacy in activity tests.

[0347] In some embodiments, the antigen-binding domain is a T cell receptor ("TCR") or a fragment thereof, such as a single-chain TCR (scTCR). Methods for generating such TCRs are known in the art. See, e.g., Illemsen RA et al., Gene Therapy 7:1369-1377 (2000); Zhang T et al., Cancer Gene Ther 11:487-496 (2004); Aggen et al., Gene Ther. 19(4):365-74 (2012) (references incorporated herein in their entireties). For example, scTCRs can be engineered that contain Vα and Vβ genes from a T cell clone linked by a linker (e.g., a flexible peptide).

[0348] Transmembrane domain With regard to the transmembrane domain, in various embodiments, a CAR can be designed to include a transmembrane domain that binds to the extracellular domain of the CAR. The transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, such as one or more amino acids associated with the extracellular region of the protein from which the transmembrane is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and / or one or more amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region). In some embodiments, the transmembrane domain is one that is used in conjunction with one of the other domains of the CAR. In certain examples, the transmembrane domain can be selected or modified by amino acid substitution to avoid such domain binding to the transmembrane domain of the same or a different surface membrane protein, e.g., to minimize interaction with other members of the receptor complex. In some embodiments, the transmembrane domain can homodimerize with another CAR on the surface of a CAR-expressing cell, e.g., a CART cell. In some embodiments, the amino acid sequence of the transmembrane domain may be modified or substituted to minimize interaction with the binding domain of a natural binding partner present in the same CAR-expressing cell, e.g., a CART.

[0349] The transmembrane domain can be derived from natural or recombinant sources. When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. In some embodiments, the transmembrane domain can transmit a signal to the intracellular domain whenever the CAR binds to the target. Transmembrane domains particularly useful in the present invention can include at least the transmembrane region of, for example, the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8 (e.g., CD8α, CD8β), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.In some embodiments, the transmembrane domain is selected from the group consisting of at least one of a costimulatory molecule, e.g., an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), an activating NK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB(CD137), B7-H3, CDS, ICAM-1, ICOS(CD278), GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB 2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and the transmembrane region of a ligand that specifically binds to CD83.

[0350] In some cases, the transmembrane domain can be attached to the extracellular region of the CAR, e.g., the antigen-binding domain of the CAR, via a hinge, e.g., a hinge from a human protein. For example, in some embodiments, the hinge can be a human Ig (immunoglobulin) hinge, e.g., an IgG4 hinge or a CD8a hinge. In some embodiments, the hinge or spacer comprises (e.g., consists of) the amino acid sequence of SEQ ID NO: 2. In some embodiments, the transmembrane domain comprises (e.g., consists of) the transmembrane domain of SEQ ID NO: 6.

[0351] In some embodiments, the hinge or spacer comprises an IgG4 hinge. For example, in some embodiments, the hinge or spacer comprises the hinge of SEQ ID NO: 3. In some embodiments, the hinge or spacer comprises a hinge encoded by the nucleotide sequence of SEQ ID NO: 14.

[0352] In some embodiments, the hinge or spacer comprises an IgD hinge. For example, in some embodiments, the hinge or spacer comprises a hinge of the amino acid sequence of SEQ ID NO: 4. In some embodiments, the hinge or spacer comprises a hinge encoded by the nucleotide sequence of SEQ ID NO: 15.

[0353] In some embodiments, the transmembrane domain may be recombinant, and if so, comprises predominantly hydrophobic residues such as leucine and valine, hi some embodiments, triplets of phenylalanine, tryptophan, and valine can be found at each end of the recombinant transmembrane domain.

[0354] Optionally, a short oligo- or polypeptide linker, 2-10 amino acids in length, can form the bond between the transmembrane domain and the cytoplasmic region of the CAR. A glycine-serine doublet provides a particularly suitable linker. For example, in some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 5. In one embodiment, the linker is encoded by the nucleotide sequence of SEQ ID NO: 16.

[0355] In some embodiments, the hinge or spacer comprises a KIR2DS2 hinge.

[0356] Cytoplasmic domain The cytoplasmic domain or region of the CAR of the invention comprises an intracellular signaling domain, which is generally responsible for activating at least one normal effector function of the immune cell into which the CAR is introduced.

[0357] Examples of intracellular signaling domains for use in the CARs of the invention include the cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that function in concert to initiate signal transduction following antigen receptor binding, as well as any derivatives or variants thereof and any recombinant sequences that have the same functional capability.

[0358] It is known that signals generated by the TCR alone are insufficient for full activation of T cells; secondary and / or costimulatory signals are also required. Therefore, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary intracellular signaling domain) and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic domains, e.g., costimulatory domains).

[0359] Primary signaling domains control the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0360] Examples of ITAM-containing primary intracellular signaling domains that are particularly useful in the present invention include those of TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, DAP10, DAP12, and CD66d. In some embodiments, a CAR of the invention comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3ζ.

[0361] In some embodiments, the primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain, that has altered (e.g., increased or decreased) activity compared to the native ITAM domain. In some embodiments, the primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and / or truncated ITAM-containing primary intracellular signaling domain. In some embodiments, the primary signaling domain comprises one, two, three, four, or more ITAM motifs.

[0362] Further examples of molecules containing a primary intracellular signaling domain that are particularly useful in the present invention include the molecules DAP10, DAP12 and CD32.

[0363] The intracellular signaling domain of a CAR may comprise a primary signaling domain, such as a CD3-ζ signaling domain, by itself, or may be combined with any other desired intracellular signaling domain useful in connection with the CAR of the present invention. For example, the intracellular signaling domain of a CAR may comprise a primary signaling domain, such as a CD3ζ chain portion, as well as a costimulatory molecule transduction domain. A costimulatory molecule signaling domain refers to a portion of a CAR that comprises the intracellular signaling domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is necessary for an efficient response of lymphocytes to antigens. Examples of such molecules include MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, N KG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, L These include ligands that specifically bind to y9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83.For example, CD27 costimulation has been demonstrated to increase the expansion, effector function, and survival of human CAR cells in vitro and to enhance human T cell persistence and anti-tumor activity in vivo (Song et al. Blood. 2012;119(3):696-706). The intracellular signaling sequences within the cytoplasmic portion of the CAR of the present invention can also be linked to each other randomly or in a specified order. Optionally, a short oligo- or polypeptide linker between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length can form the link between the intracellular signaling sequences. In some embodiments, a glycine-serine doublet can be used as a suitable linker. In some embodiments, a single amino acid, e.g., alanine, glycine, can be used as a suitable linker.

[0364] In some embodiments, the intracellular signaling domain is designed to include two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains. In some embodiments, the two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains are separated by a linker molecule, e.g., a linker molecule described herein. In some embodiments, the intracellular signaling domain includes two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

[0365] In some embodiments, the intracellular signaling domain is designed to comprise the signaling domain of CD3ζ and the signaling domain of CD28. In some embodiments, the intracellular signaling domain is designed to comprise the signaling domain of CD3ζ and the signaling domain of 4-1BB. In some embodiments, the signaling domain of 4-1BB is the signaling domain of SEQ ID NO: 7. In some embodiments, the signaling domain of CD3ζ is the signaling domain of SEQ ID NO: 9 (mutant CD3ζ) or SEQ ID NO: 10 (wild-type human CD3ζ).

[0366] In some embodiments, the intracellular signaling domain is designed to include the signaling domain of CD3ζ and the signaling domain of CD27. In some embodiments, the signaling domain of CD27 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the signaling domain of CD27 is encoded by the nucleic acid sequence of SEQ ID NO: 19.

[0367] In some embodiments, the intracellular signaling domain is designed to comprise the signaling domain of CD3ζ and the signaling domain of CD28. In some embodiments, the signaling domain of CD28 comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the signaling domain of CD28 is encoded by the nucleic acid sequence of SEQ ID NO: 37. In some embodiments, the intracellular signaling domain is designed to comprise the signaling domain of CD3ζ and the signaling domain of ICOS. In some embodiments, the signaling domain of ICOS comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the signaling domain of ICOS is encoded by the nucleic acid sequence of SEQ ID NO: 39.

[0368] Co-expression of CAR with other molecules or drugs Co-expression of a second CAR In some embodiments, a CAR-expressing cell described herein may further comprise a second CAR, e.g., a different antigen binding domain of the second CAR, e.g., against the same target (e.g., CD19) or a different target (e.g., a target other than CD19, e.g., a target described herein).

[0369] In some embodiments, a CAR-expressing cell comprises a first CAR that targets a first antigen and comprises an intracellular signaling domain with a costimulatory signaling domain but no primary signaling domain, and a second CAR that targets a second, different antigen and comprises an intracellular signaling domain with a primary signaling domain but no costimulatory signaling domain. Placing a costimulatory signaling domain, e.g., 4-1BB, CD28, CD27, OX-40, or ICOS, on the first CAR and a primary signaling domain, e.g., CD3ζ, on the second CAR restricts CAR activity to cells in which both targets are expressed. In some embodiments, a CAR-expressing cell comprises a first CAR that comprises an antigen-binding domain, a transmembrane domain, and a costimulatory domain, and a second CAR that targets another antigen and comprises an antigen-binding domain, a transmembrane domain, and a primary signaling domain. In some embodiments, a CAR-expressing cell comprises a first CAR that comprises an antigen-binding domain, a transmembrane domain, and a primary signaling domain, and a second CAR that targets another antigen and comprises an antigen-binding domain for the antigen, a transmembrane domain, and a costimulatory signaling domain.

[0370] In some embodiments, when a CAR-expressing cell comprises two or more different CARs, the antigen-binding domains of the different CARs are such that the antigen-binding domains do not interact with each other. For example, a cell expressing a first and a second CAR can have the antigen-binding domain of the first CAR, e.g., as a fragment, e.g., an scFv, that does not form a bond with the antigen-binding domain of the second CAR, e.g., a VHH.

[0371] In some embodiments, the antigen-binding domain comprises a single-domain antigen-binding (SDAB) molecule, including molecules in which the complementary determining regions are part of a single-domain polypeptide. Examples include, but are not limited to, binding molecules that naturally lack a heavy chain variable domain, a light chain, single domains derived from conventional four-chain antibodies, engineered domains, and single-domain scaffolds other than those derived from antibodies. SDAB molecules can be any current or future single-domain molecule in the art. SDAB molecules can be derived from any species, including, but not limited to, mouse, human, camel, llama, lamprey, fish, shark, goat, rabbit, and cow. The term also includes naturally occurring single-domain antibody molecules from species other than Camelidae and sharks.

[0372] In some embodiments, SDAB molecules may be derived from the variable regions of immunoglobulins found in fish, such as from the immunoglobulin isotype known as the novel antigen receptor (NAR) found in the serum of sharks. Methods for producing single domain molecules derived from the variable regions of NARs ("IgNARs") are described in WO 03 / 014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

[0373] In some embodiments, the SDAB molecule is a naturally occurring single-domain antigen-binding molecule known as a heavy chain devoid of light chains. Such single-domain molecules are described, for example, in WO 9404678 and Hamers-Casterman, C. et al. (1993) Nature 363:446-448. For clarity, this variable domain from a heavy chain molecule naturally devoid of light chains is known herein as a VHH or nanobody to distinguish it from the conventional VH of four-chain immunoglobulins. Such VHH molecules can be derived from Camelidae species, such as camel, llama, dromedary, alpaca, and guanaco. Other species outside of Camelidae can produce heavy chain molecules naturally devoid of light chains, and such VHHs are within the scope of the present invention.

[0374] SDAB molecules may be recombinant, CDR-grafted, humanized, camelized, deimmunized and / or produced in vitro (eg, selected by phage display).

[0375] It has also been found that cells having multiple chimeric membrane-embedded receptors comprising antigen-binding domains that interact with each other may be undesirable, for example, because they prevent one or more of the antigen-binding domains from binding to their cognate antigen. Accordingly, disclosed herein are cells having first and second non-naturally occurring chimeric membrane-embedded receptors comprising antigen-binding domains that minimize such interactions. Also disclosed herein are nucleic acids encoding first and second non-naturally occurring chimeric membrane-embedded receptors comprising antigen-binding domains that minimize such interactions, as well as methods for making and using such cells and nucleic acids. In some embodiments, one of the antigen-binding domains of the first and second non-naturally occurring chimeric membrane-embedded receptors comprises an scFv, and the other comprises a single VH domain, e.g., a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence.

[0376] In some embodiments, the compositions herein comprise a first and a second CAR, wherein the antigen-binding domain of one of the first and second CARs does not comprise a variable light domain and a variable heavy domain. In one embodiment, the antigen-binding domain of one of the first and second CARs is an scFv, and the other is not an scFv. In one embodiment, the antigen-binding domain of one of the first and second CARs comprises a single VH domain, such as a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence. In one embodiment, the antigen-binding domain of one of the first and second CARs comprises a nanobody. In one embodiment, the antigen-binding domain of one of the first and second CARs comprises a camelid VHH domain.

[0377] In one embodiment, the antigen-binding domain of one of the first and second CARs comprises an scFv, and the other comprises a single VH domain, such as a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence. In one embodiment, the antigen-binding domain of one of the first and second CARs comprises an scFv, and the other comprises a nanobody. In one embodiment, the antigen-binding domain of one of the first and second CARs comprises an scFv, and the other comprises a camelid VHH domain.

[0378] In one embodiment, when present on the surface of a cell, binding of the antigen-binding domain of the first CAR to its cognate antigen is not substantially reduced by the presence of the second CAR, hi one embodiment, binding of the antigen-binding domain of the first CAR to its cognate antigen in the presence of the second CAR is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%, for example 85%, 90%, 95%, 96%, 97%, 98%, or 99%, of the binding of the antigen-binding domain of the first CAR to its cognate antigen in the absence of the second CAR.

[0379] In one embodiment, when present on the surface of a cell, the antigen binding domains of the first and second CARs bind to each other less than when both are scFv antigen binding domains, hi one embodiment, the antigen binding domains of the first and second CARs bind to each other at least 85%, 90%, 95%, 96%, 97%, 98% or 99% less than when both are scFv antigen binding domains, for example 85%, 90%, 95%, 96%, 97%, 98% or 99% less.

[0380] Co-expression of drugs that enhance CAR activity In some embodiments, the CAR-expressing cells described herein can further express another agent, for example, an agent that enhances the activity or fitness of the CAR-expressing cell.

[0381] For example, in some embodiments, the agent can be an agent that inhibits a molecule that modulates or regulates, e.g., inhibits, T cell function. In some embodiments, the molecule that modulates or regulates T cell function is an inhibitory molecule. Inhibitory molecules, e.g., PD1, can, in some embodiments, reduce the ability of CAR-expressing cells to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, or TGFβ.

[0382] In some embodiments, an agent, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA; or, e.g., an inhibitory protein or system, e.g., a clustered regularly interspaced short palindromic repeats (CRISPR), a transcription-activator-like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN), e.g., as described herein, can be used to inhibit expression of a molecule that modulates or regulates, e.g., inhibits, T cell function in a CAR-expressing cell. In some embodiments, the agent is an shRNA, e.g., an shRNA described herein. In some embodiments, an agent that modulates or regulates, e.g., inhibits, T cell function is inhibited in a CAR-expressing cell. For example, a dsRNA molecule that inhibits expression of a molecule that modulates or regulates, e.g., inhibits, T cell function is linked to a nucleic acid encoding a component, e.g., all of the components, of the CAR.

[0383] In some embodiments, the agent comprises a first polypeptide of an inhibitory molecule such as, for example, PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, or TGFβ, or a fragment of any of these (e.g., at least a portion of the extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain as described herein (e.g., a costimulatory domain (e.g., a co-stim ... In some embodiments, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1) and a second polypeptide of an intracellular signaling domain described herein (e.g., the CD28 signaling domain described herein and / or the CD3ζ signaling domain described herein). PD1 is an inhibitory member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells (Agata (Et al. 1996 Int. Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD-L2, have been shown to downregulate T cell activation by binding to PD1 (Freeman et al. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43).

[0384] In some embodiments, the agent comprises the extracellular domain (ECD) of an inhibitory molecule, such as programmed cell death 1 (PD1) (referred to herein as PD1 CAR), which may be fused to a transmembrane domain and an intracellular signaling domain such as 41BB and CD3ζ. In some embodiments, the PD1 CAR improves T cell persistence when used in combination with an XCAR described herein. In some embodiments, the CAR is a PD1 CAR comprising the extracellular domain of PD1 shown underlined in SEQ ID NO: 24. In some embodiments, the PD1 CAR comprises the amino acid sequence of SEQ ID NO: 24.

[0385] In some embodiments, the PD1 CAR comprises the amino acid sequence of SEQ ID NO: 22.

[0386] In some embodiments, the agent comprises a nucleic acid sequence encoding a PD1 CAR, such as a PD1 CAR described herein. In some embodiments, the nucleic acid sequence of the PD1 CAR is presented as SEQ ID NO: 23, with the PD1 ECD underlined.

[0387] In another example, in some embodiments, the agent that enhances the activity of a CAR-expressing cell can be a costimulatory molecule or costimulatory molecule ligand. Examples of costimulatory molecules include MHC class I molecules, BTLA, and Toll ligand receptors, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG Ligands that specifically bind to 2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83, including, for example, those described herein. Examples of costimulatory molecule ligands include CD80, CD86, CD40L, ICOSL, CD70, OX40L, 4-1BBL, GITRL, and LIGHT. In embodiments, the costimulatory molecule ligand is a ligand of a costimulatory molecule that is different from the costimulatory molecule domain of the CAR. In embodiments, the costimulatory molecule ligand is a ligand of a costimulatory molecule that is the same as the costimulatory molecule domain of the CAR. In some embodiments, the costimulatory molecule ligand is 4-1BBL. In some embodiments, the costimulatory ligand is CD80 or CD86.In some embodiments, the costimulatory molecule ligand is CD70. In embodiments, the CAR-expressing immune effector cells described herein can be further engineered to express one or more additional costimulatory molecules or costimulatory molecule ligands.

[0388] Nucleic acid construct encoding a CAR The present invention also provides immune effector cells, for example, those prepared by the methods described herein, that comprise the nucleic acid molecule encoding one or more CAR constructs described herein.In some embodiments, nucleic acid molecules are provided as messenger RNA transcripts.In some embodiments, nucleic acid molecules are provided as DNA constructs.

[0389] The nucleic acid molecules described herein can be DNA molecules, RNA molecules, or a combination thereof. In some embodiments, the nucleic acid molecule is an mRNA encoding a CAR polypeptide as described herein. In other embodiments, the nucleic acid molecule is a vector comprising any of the aforementioned nucleic acid molecules.

[0390] In some embodiments, the antigen-binding dom...

Claims

1. autoimmune diseases or disorders such as lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with severe pulmonary involvement (e.g., as seen in autoHSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-associated myositis, e.g., antisynthetase syndrome associated with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjogren's disease, severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), 1. A method of treating a subject having MOGAD (Modular Ovarian Glandular Associated Disorder), multiple sclerosis (MS), severe refractory rheumatoid arthritis, an antibody-mediated neuroimmune disorder (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG associated disorder), NMDAR+ encephalitis or antibody-related paraneoplastic neurological disorder), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis, comprising administering to the subject a population of cells (e.g., T cells) that express, or comprise a nucleic acid configured to express, a CD19 chimeric antigen receptor (CAR), wherein the population of cells (i) contacting (e.g., binding) a population of cells (e.g., T cells, e.g., T cells isolated from frozen or fresh leukapheresis products) with an agent that stimulates the CD3 / TCR complex and / or a costimulatory molecule on the surface of the cells; (ii) contacting the population of cells (e.g., T cells) with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding the CAR, thereby providing a population of cells (e.g., T cells) comprising the nucleic acid molecule, wherein the CAR comprises a CD19 antigen-binding domain ("CD19 CAR"); and (iii) recovering the population of cells (e.g., T cells) for storage (e.g., reformulating the population of cells in cryopreservation medium) or administration. and (a) step (ii) is carried out together with step (i) or within 20 hours after the start of step (i), such as within 12, 13, 14, 15, 16, 17 or 18 hours after the start of step (i), for example within 18 hours after the start of step (i); and step (iii) is carried out within 30 (e.g. 26) hours after the start of step (i), such as within 22, 23, 24, 25, 26, 27, 28, 29 or 30 hours after the start of step (i), for example within 24 hours after the start of step (i); (b) step (ii) is carried out together with step (i) or within 20 hours after the start of step (i), such as within 12, 13, 14, 15, 16, 17 or 18 hours after the start of step (i), for example within 18 hours after the start of step (i); and Step (iii) is carried out within 30 hours after the start of step (ii), for example within 22, 23, 24, 25, 26, 27, 28, 29 or 30 hours after the start of step (ii); or (c) the population of cells from step (iii) is not expanded, or is expanded by no more than 5, 10, 15, 20, 25, 30, 35 or 40%, e.g., no more than 10%, compared to the population of cells at the start of step (i), e.g., as assessed by viable cell count; Optionally, the nucleic acid molecule of step (ii) is on a viral vector; optionally, the nucleic acid molecule of step (ii) is an RNA molecule on a viral vector; and optionally, step (ii) comprises transducing the population of cells (e.g., T cells) with a viral vector comprising a nucleic acid molecule encoding the CAR.

2. The agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3 (e.g., an anti-CD3 antibody), and the agent that stimulates a costimulatory molecule is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof; optionally, the agent that stimulates the CD3 / TCR complex or the agent that stimulates a costimulatory molecule is an antibody (e.g., a single domain antibody (e.g., a heavy chain variable domain antibody), a peptibody, a Fab fragment, or an scFv), a small molecule, or a ligand (e.g., a naturally occurring ligand, a recombinant ligand, or a 10. The method of claim 1, wherein the agent that stimulates the CD3 / TCR complex or the agent that stimulates a costimulatory molecule is selected from a chimeric ligand (or a chimeric ligand), optionally wherein the agent that stimulates the CD3 / TCR complex or the agent that stimulates a costimulatory molecule does not comprise a bead, optionally wherein the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody, and the agent that stimulates a costimulatory molecule comprises an anti-CD28 antibody, optionally wherein the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody covalently bound to a colloidal polymer nanomatrix, and the agent that stimulates a costimulatory molecule comprises an anti-CD28 antibody covalently bound to a colloidal polymer nanomatrix, and optionally wherein the agent that stimulates the CD3 / TCR complex and the agent that stimulates a costimulatory molecule comprise T Cell TransAct™.

3. 3. The method of claim 1 or 2, wherein step (i) increases the percentage of CAR-expressing cells in the population of cells from step (iii), e.g., the population of cells from step (iii) exhibits a higher percentage of CAR-expressing cells (e.g., at least 10, 20, 30, 40, 50, or 60% higher) compared to cells made by an otherwise similar method without step (i).

4. (a) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ T cells, in said 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, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ cells, in said population of cells at the start of step (i); (b) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ T cells, in the population of cells from step (iii) is increased, e.g., by at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3-fold, compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ cells, in the population of cells at the start of step (i); (c) the percentage of CAR-expressing naive T cells, e.g., CAR-expressing CD45RA+ CD45RO- CCR7+ T cells, in said population of cells increases during step (ii), e.g., increases by at least 30, 35, 40, 45, 50, 55, or 60% between 18 and 24 hours after initiation of step (ii); or 4. The method of any one of claims 1 to 3, wherein (d) the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO-CCR7+ T cells, in the population of cells from step (iii) is not reduced, or is reduced by no more than 5 or 10%, compared to the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO-CCR7+ cells, in the population of cells at the start of step (i).

5. (a) the population of cells from step (iii) exhibits a higher percentage (e.g., at least 10, 20, 30, or 40% higher) of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ T cells, compared to cells produced by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (b) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ T cells, in said population of cells from step (iii) is higher (e.g., 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, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ T cells, in cells made by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (c) the percentage of CAR-expressing naive T cells, e.g., CAR-expressing CD45RA+ CD45RO-CCR7+ T cells, in the population of cells from step (iii) is higher (e.g., at least 4, 6, 8, 10, or 12-fold higher) than the percentage of CAR-expressing naive T cells, e.g., CAR-expressing CD45RA+ CD45RO-CCR7+ T cells, in cells made by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (d) the population of cells from step (iii) exhibits a higher percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ T cells (e.g., at least 10, 20, 30, or 40% higher), compared to cells produced by a similar method but further comprising, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days; (e) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ T cells, in the population of cells from step (iii) is higher (e.g., at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3 times higher) than the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ T cells, in cells made by a similar method but further comprising the step of expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii); or (f) the percentage of CAR-expressing naive T cells, e.g., CAR-expressing CD45RA+ CD45RO-CCR7+ T cells, in the population of cells from step (iii) is higher (e.g., at least 4, 6, 8, 10, or 12 fold) than the percentage of CAR-expressing naive T cells, e.g., CAR-expressing CD45RA+ CD45RO-CCR7+ T cells, in cells made by a similar method but further comprising the step of expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii).

6. (a) the percentage of central memory cells, e.g., central memory T cells, e.g., 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, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells at the start of step (i); (b) the percentage of central memory cells, e.g., central memory T cells, e.g., 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% compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in the population of cells at the start of step (i); (c) the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+ CD45RO+ cells, is decreased during step (ii), e.g., by at least 8, 10, 12, 14, 16, 18, or 20% between 18 and 24 hours after initiation of step (ii); or 6. The method of any one of claims 1 to 5, wherein (d) the percentage of central memory cells, such as central memory T cells, such as CCR7+ CD45RO+ T cells, in the population of cells from step (iii) is not increased, or is increased by no more than 5 or 10%, compared to the percentage of central memory cells, such as central memory T cells, such as CCR7+ CD45RO+ T cells, in the population of cells at the start of step (i).

7. (a) the population of cells from step (iii) exhibits a lower percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells (e.g., at least 10, 20, 30, or 40% lower), compared to cells produced by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (b) the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in the population of cells from step (iii) is lower (e.g., at least 20, 30, 40, or 50% lower) than the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in cells made by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (c) the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+ CD45RO+ T cells, in the population of cells from step (iii) is lower (e.g., at least 10, 20, 30, or 40% lower) than the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+ CD45RO+ T cells, in cells generated by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (d) the population of cells from step (iii) exhibits a lower percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells (e.g., at least 10, 20, 30, or 40% lower), compared to cells produced by a similar method but further comprising, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days; (e) the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in the population of cells from step (iii) is lower (e.g., at least 20, 30, 40, or 50% lower) than the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in cells made by a similar method but further comprising the step of expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii); or (f) the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+ CD45RO+ T cells, in the population of cells from step (iii) is lower (e.g., at least 10, 20, 30, or 40% lower) than the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+ CD45RO+ T cells, in cells made by a similar method but further comprising the step of expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii).

8. (a) is the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) increased compared to the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells at the start of step (i); (b) is the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) increased compared to the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells at the start of step (i); (c) the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) is greater than the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in cells made by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); or (d) is the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) higher than the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in cells generated by a similar method except that step (iii) is performed more than 26 hours after the initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the initiation of step (i); (e) the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) is higher than the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in cells made by a similar method, but further comprising, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days; or (f) the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) is higher than the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in cells made by a similar method but further comprising the step of expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii).

9. (a) the median GeneSetScore (Up TEM vs. Down TSCM) of the population of cells from step (iii) is about the same as, or differs by (e.g., increases by) no more than about 25, 50, 75, 100, or 125% from, the median GeneSetScore (Up TEM vs. Down TSCM) of the population of cells at the start of step (i); (b) the median GeneSetScore (Up TEM vs. Down TSCM) of the population of cells from step (iii) is Cells produced by a similar method, except that step (iii) is carried out more than 26 hours after the start of step (i), for example more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i); or Cells made by a similar method, but further comprising, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days. lower (e.g., at least about 100, 150, 200, 250, or 300% lower) than the median GeneSetScore (Up TEM vs. Down TSCM) of (c) the median GeneSetScore (Up Treg vs. Down Teff) of the population of cells from step (iii) is about the same as, or differs by (e.g., increases 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 at the start of step (i); (d) the median GeneSetScore (Up Treg vs. Down Teff) of the population of cells from step (iii) is Cells produced by a similar method, except that step (iii) is carried out more than 26 hours after the start of step (i), for example more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i); or Cells made by a similar method, but further comprising, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days. lower (e.g., at least about 50, 100, 125, 150, or 175% lower) than the median GeneSetScore (Up Treg vs. Down Teff) of (e) the median GeneSetScore (Down stemness) of the population of cells from step (iii) is about the same as or differs from (e.g., increases by) no more than about 25, 50, 100, 150, 200, or 250% of the median GeneSetScore (Down stemness) of the population of cells at the start of step (i); (f) the median GeneSetScore (Down stemness) of the population of cells from step (iii) is Cells produced by a similar method, except that step (iii) is carried out more than 26 hours after the start of step (i), for example more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i); or Cells made by a similar method, but further comprising, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days. lower (e.g., at least about 50, 100, or 125% lower) than the median GeneSetScore (Down stemness) of (g) the median GeneSetScore (Up hypothesis) of the population of cells from step (iii) is about the same as or differs by (e.g., increases by) no more than about 125, 150, 175, or 200% from the median GeneSetScore (Up hypothesis) of the population of cells at the start of step (i); (h) the median GeneSetScore (Up hypoxia) of the population of cells from step (iii) is Cells produced by a similar method, except that step (iii) is carried out more than 26 hours after the start of step (i), for example more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i); or Cells made by a similar method, but further comprising, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days. lower (e.g., at least about 40, 50, 60, 70, or 80% lower) than the median GeneSetScore (Up hypoxia); (j) the median GeneSetScore (Up autophagy) of the population of cells from step (iii) is about the same as or differs from (e.g., increases by) no more than about 180, 190, 200, or 210% of the median GeneSetScore (Up autophagy) of the population of cells at the start of step (i); or (k) the median GeneSetScore (Up autophagey) of the population of cells from step (iii) is Cells produced by a similar method, except that step (iii) is carried out more than 26 hours after the start of step (i), for example more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i); or Cells made by a similar method, but further comprising, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days. The method of any one of claims 1 to 8, wherein the median GeneSetScore (Up autophagy) is lower (for example at least 20, 30 or 40% lower).

10. 10. The method of any one of claims 1 to 9, wherein the population of cells from step (iii), after incubation with cells expressing the antigen recognized by the CAR, secrete IL-2 at a higher (e.g., at least 2, 4, 6, 8, 10, 12 or 14 fold) level than cells produced by a similar method except that step (iii) is carried out more than 26 hours after the start of step (i), such as more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i), or than cells produced by a similar method except that the method further comprises expanding the population of cells (e.g., T cells) in vitro for more than 3 days, such as 5, 6, 7, 8 or 9 days, after step (ii) and before step (iii).

11. 11. The method of any one of claims 1 to 10, wherein the population of cells from step (iii), after administration in vivo to the subject, persists longer or proliferates to a higher level than cells made by a similar method except that step (iii) is carried out more than 26 hours after the start of step (i), such as more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i), or compared to cells made by a similar method except that the method further comprises expanding the population of cells (e.g., T cells) in vitro for more than 3 days, such as 5, 6, 7, 8 or 9 days after step (ii) and before step (iii).

12. The population of cells from step (iii), after administration in vivo to the subject, exhibits a stronger activity (e.g., a lower dose, e.g., 0.15×10) than cells produced by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i), or than cells produced by a similar method except that step (iii) further comprises expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). ６ , 0.2 × 10 ６ , 0.25×10 ６ or 0.3 x 10 ６ The method of any one of claims 1 to 11, wherein the antibody exhibits a potent activity at a dose of 100 or less viable CAR-expressing cells.

13. 13. The method of any one of claims 1 to 12, wherein the population of cells from step (iii) is not expanded, or is expanded by no more than 5, 10, 15, 20, 25, 30, 35 or 40%, such as no more than 10%, compared to the population of cells at the start of step (i), e.g. as assessed by viable cell count, and optionally the number of viable cells in the population of cells from step (iii) is reduced from the number of viable cells in the population of cells at the start of step (i).

14. 14. The method of any one of claims 1 to 13, wherein the population of cells from step (iii) is not expanded or is expanded for less than 2 hours, such as less than 1 or 1.5 hours, compared to the population of cells at the start of step (i).

15. The method of any one of claims 1 to 14, wherein steps (i) and / or (ii) are carried out in a cell culture medium (e.g., serum-free medium) comprising IL-2, IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)), IL-7, IL-21, IL-6 (e.g., IL-6 / sIL-6Ra), an LSD1 inhibitor, a MALT1 inhibitor, or a combination thereof.

16. The method of any one of claims 1 to 15, wherein steps (i) and / or (ii) are carried out in a serum-free cell culture medium comprising a serum replacement.

17. 17. The method of claim 16, wherein the serum replacement is CTS™ Immune Cell Serum Replacement (ICSR).

18. Before step (i), (iv) (optionally) receiving a fresh leukapheresis product (or an alternative source of hematopoietic tissue, such as a fresh whole blood product, a fresh bone marrow product, or a fresh organ biopsy or resection (e.g., a fresh product from a thymus removal)) from an entity, e.g., a laboratory, hospital, or healthcare provider; and (v) isolating the population of cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells) contacted in step (i) from a fresh leukapheresis product (or an alternative source of hematopoietic tissue, such as a fresh whole blood product, a fresh bone marrow product, or a fresh organ biopsy or resection (e.g., a fresh product from a thymus removal)). and optionally further comprising: Step (iii) is carried out within 35 hours after the start of step (v), such as within 27, 28, 29, 30, 31, 32, 33, 34 or 35 hours after the start of step (v), for example within 30 hours after the start of step (v); or 18. A method according to any one of claims 1 to 17, wherein the population of cells from step (iii) is not expanded, or is expanded by no more than 5, 10, 15, 20, 25, 30, 35 or 40%, such as no more than 10%, compared to the population of cells at the end of step (v), for example as assessed by viable cell count.

19. 18. The method of any one of claims 1 to 17, further comprising receiving cryopreserved T cells isolated from a leukapheresis product (or an alternative source of hematopoietic tissue such as whole blood, bone marrow or cryopreserved T cells isolated from an organ biopsy or resection (e.g., thymus removal)) from an entity, e.g., a laboratory, hospital or healthcare provider, prior to step (i).

20. Before step (i), (iv) (optionally) receiving a cryopreserved leukapheresis product (or an alternative source of hematopoietic tissue, such as a cryopreserved whole blood product, a cryopreserved bone marrow product, or a cryopreserved organ biopsy or resection (e.g., a cryopreserved product from a thymus removal)) from an entity, e.g., a laboratory, hospital, or healthcare provider; and (v) isolating the population of cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells) contacted in step (i) from a cryopreserved leukapheresis product (or an alternative source of hematopoietic tissue, such as a cryopreserved whole blood product, a cryopreserved bone marrow product, or a cryopreserved organ biopsy or resection (e.g., a cryopreserved product from a thymus removal)). and optionally further comprising: Step (iii) is carried out within 35 hours after the start of step (v), such as within 27, 28, 29, 30, 31, 32, 33, 34 or 35 hours after the start of step (v), for example within 30 hours after the start of step (v); or 18. A method according to any one of claims 1 to 17, wherein the population of cells from step (iii) is not expanded, or is expanded by no more than 5, 10, 15, 20, 25, 30, 35 or 40%, such as no more than 10%, compared to the population of cells at the end of step (v), for example as assessed by viable cell count.

21. Step (vi): culturing a portion of the population of cells from step (iii) for at least 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7 days, e.g., at least 2 days and no more than 7 days, and measuring the level of CAR expression in said portion (e.g., measuring the percentage of surviving CAR-expressing cells in said portion). and optionally further comprising:

21. The method of any one of claims 1 to 20, wherein step (iii) comprises harvesting and freezing the population of cells (e.g., T cells), and step (vi) comprises thawing a portion of the population of cells from step (iii), culturing the portion for at least 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5 or 7 days, such as at least 2 days and not more than 7 days, and measuring the level of CAR expression in the portion (e.g., measuring the percentage of surviving CAR-expressing cells in the portion).

22. 22. The method of any one of claims 1 to 21, wherein the population of cells at the start of step (i) or step (1) is enriched for IL6R-expressing cells (e.g., cells positive for IL6Rα and / or IL6Rβ).

23. 23. The method of any one of claims 1 to 22, wherein the population of cells at the start of step (i) or step (1) comprises 50, 60, or 70% or more IL6R-expressing cells (e.g., cells positive for IL6Rα and / or IL6Rβ).

24. 24. The method of any one of claims 1 to 23, wherein steps (i) and (ii) or steps (1) and (2) are carried out in a cell culture medium containing IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)).

25. 25. The method of claim 24, wherein IL-15 increases the ability of the population of cells to proliferate, for example after 10, 15, 20 or 25 days.

26. The method of claim 24, wherein IL-15 increases the percentage of IL6Rβ-expressing cells in the population of cells.

27. Autoimmune diseases or disorders, such as lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with severe pulmonary involvement (e.g., as seen in autoHSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-associated myositis, e.g., antisynthetase syndrome associated with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjögren's disease 1. A method of treating a subject having rheumatoid arthritis, a severe refractory neuroimmune disease (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOGAD (anti-MOG associated disease), multiple sclerosis (MS)), severe refractory rheumatoid arthritis, an antibody-mediated neuroimmune disease (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG associated disease), NMDAR+ encephalitis or antibody-associated paraneoplastic neurological disease), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis, comprising administering to the subject a population of cells engineered to express a CD19 CAR (a "population of CAR-expressing cells"), wherein the population comprises: (a) about the same percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ T cells, as compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ cells, in the same population of cells before they are engineered to express the CAR; (b) a change within about 5% to about 10% of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ T cells, as compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ cells, in the same population of cells before they are engineered to express the CAR; (c) an increased percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ T cells, as compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ cells, in the same population of cells before they are engineered to express the CAR, e.g., by at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3-fold; (d) about the same percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, as compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in the same population of cells before they were engineered to express said CAR; (e) a change of within about 5% to about 10% in central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in the same population of cells before they were engineered to express said CAR; (f) a reduced percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in the same population of cells before they were engineered to express the CAR; (g) approximately the same percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, as compared to the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the same population of cells prior to being engineered to express the CAR; (h) a change within about 5% to about 10% of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, compared to the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the same population of cells before they were engineered to express the CAR; or (i) an increased percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, compared to the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the same population of cells prior to being engineered to express the CAR. A method comprising:

28. Autoimmune diseases or disorders, such as lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with severe pulmonary involvement (e.g., as seen in autoHSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-associated myositis, e.g., antisynthetase syndrome associated with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjögren's disease 1. A method of treating a subject having rheumatoid arthritis, a severe refractory neuroimmune disease (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOGAD (anti-MOG associated disease), multiple sclerosis (MS)), severe refractory rheumatoid arthritis, an antibody-mediated neuroimmune disease (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG associated disease), NMDAR+ encephalitis or antibody-associated paraneoplastic neurological disease), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis, comprising administering to the subject a population of cells engineered to express a CD19 CAR (a "population of CAR-expressing cells"); (a) the median GeneSetScore (Up TEM vs. Down TSCM) of the population of cells is about the same as, or differs by (e.g., is increased by) no more than about 25, 50, 75, 100, or 125% compared to, the median GeneSetScore (Up TEM vs. Down TSCM) of the same population of cells before they were engineered to express the CAR; (b) the median GeneSetScore (Up Treg vs. Down Teff) of the population of cells is about the same as, or differs by (e.g., is increased by) no more than about 25, 50, 100, 150, or 200% compared to, the median GeneSetScore (Up Treg vs. Down Teff) of the population of cells before they were engineered to express the CAR; (c) the median GeneSetScore (Down stemness) of the population of cells is about the same as, or differs by (e.g., is increased by) no more than about 25, 50, 100, 150, 200, or 250% compared to, the median GeneSetScore (Down stemness) of the population of cells before they were engineered to express the CAR; (d) the median GeneSetScore (Up hypoxia) of the population of cells is about the same as, or differs from (e.g., is increased by) no more than about 125, 150, 175, or 200% compared to the median GeneSetScore (Up hypoxia) of the population of cells before they were engineered to express the CAR; or (e) the median GeneSetScore (Up autophagy) of the population of cells is about the same as, or differs by (e.g., is increased by) no more than about 180, 190, 200, or 210% compared to, the median GeneSetScore (Up autophagy) of the population of cells before they were engineered to express the CAR.

29. autoimmune diseases or disorders, such as lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with severe pulmonary involvement (e.g., as seen in autoHSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-associated myositis, e.g., antisynthetase syndrome associated with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjogren's disease, severe refractory neuroimmune diseases (e.g., For example, myasthenia gravis (MG), neuromyelitis optica (NMO), MOGAD (anti-MOG associated disease), multiple sclerosis (MS), severe refractory rheumatoid arthritis, antibody-mediated neuroimmune disease (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG associated disease), NMDAR+ encephalitis or antibody-related paraneoplastic neurological disease), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis, the method comprising the step of administering lapcaptagene autoleucel to the subject.

30. 1. A method of treating a subject having a severe, intractable autoimmune disease, comprising administering to said subject rapcaptagene autoleucel.

31. 31. The method of claim 30, wherein the severe intractable autoimmune disease is selected from systemic lupus erythematosus, lupus nephritis, idiopathic inflammatory myopathy, systemic sclerosis, and ANCA-associated vasculitis.

32. 30. The method of any one of claims 1 to 29, wherein the lupus is systemic lupus erythematosus.

33. 33. The method of claim 32, wherein the SLE is severe refractory SLE (srSLE).

34. The method of any one of claims 1 to 28, wherein the CD19 CAR comprises a CD19-binding domain, a transmembrane domain, and an intracellular signaling domain.

35. (a) the transmembrane domain comprises a transmembrane domain of a protein selected from the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154; or (b) the transmembrane domain comprises the transmembrane domain of CD8; or (c) the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; or 35. The method of Claim 34, wherein (d) the nucleic acid molecule comprises a nucleic acid sequence encoding the transmembrane domain, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 17, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereof.

36. 1. A method of treating a subject with severe refractory systemic lupus erythematosus (srSLE), comprising administering to the subject a population of cells comprising a CD19 chimeric antigen receptor (CD19 CAR) or a nucleic acid encoding said CD19 CAR, The CAR comprises a CD19-binding domain, a transmembrane domain, and an intracellular signaling domain, and the transmembrane domain is a transmembrane domain of a CD8 protein. an amount sufficient to treat said srSLE wherein the method comprises the steps of:

37. (a) the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; or 37. The method of Claim 36, wherein (a) the nucleic acid molecule comprises a nucleic acid sequence encoding the transmembrane domain, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 17, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereof.

38. The population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is about 0.5 x 10 ６ ~50 x 10 ６ viable CAR-expressing cells, e.g., about 5 x 10 ６ and optionally, the population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is administered at a dose of 5×10 ６ The method of any one of claims 1 to 28, 36, or 37, wherein the patient is administered in a dose of viable CAR-expressing cells.

39. The population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is about 2.5 x 10 ６ ~2.5 x 10 ８ viable CAR-expressing cells, e.g., about 1.25 x 10 ７ and optionally, the population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is administered at a dose of 1.25 x 10 ７ The method of any one of claims 1 to 28, 36, or 37, wherein the patient is administered in a dose of viable CAR-expressing cells.

40. The population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is about 1.25 x 10 ７ ~1.25 x 10 ９ viable CAR-expressing cells, e.g., about 1.25 x 10 ８ and optionally, the population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is administered at a dose of 1.25 x 10 ８ The method of any one of claims 1 to 28, 36, or 37, wherein the patient is administered in a dose of viable CAR-expressing cells.

41. The population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is about 2.5 x 10 ６ ~2.5 x 10 ８ viable CAR-expressing cells, e.g., about 1 x 10 ７ or 5 x 10 ７ The method of any one of claims 1 to 28, 36, or 37, wherein the patient is administered in a dose of viable CAR-expressing cells.

42. 1. A method of treating a subject with severe refractory systemic lupus erythematosus (srSLE), comprising administering to the subject rapcavtagene autoleucel; an amount sufficient to treat said srSLE thereby treating said srSLE.

43. Lapcaptagene autoleucel is approximately 0.5 x 10 ６ ~50 x 10 ６ viable CAR-positive cells, e.g., about 5 x 10 ６ viable CAR-positive cells, and optionally rapcaptagen autoleucel is administered at a dose of 5 x 10 ６ The method of any one of claims 29-33 or 42, wherein the patient is administered at a dose of viable CAR-positive cells.

44. Lapcaptagene autoleucel is approximately 2.5 x 10 ６ ~2.5 x 10 ８ viable CAR-expressing cells, e.g., about 1.25 x 10 ７ viable CAR-positive cells, and optionally rapcavtagene autoleucel is administered at a dose of 1.25 x 10 ７ The method of any one of claims 29-33 or 42, wherein the patient is administered at a dose of viable CAR-positive cells.

45. Lapcaptagene autoleucel is approximately 1.25 x 10 ７ ~1.25 x 10 ９ viable CAR-expressing cells, e.g., about 1.25 x 10 ８ viable CAR-positive cells, and optionally rapcavtagene autoleucel is administered at a dose of 1.25 x 10 ８ The method of any one of claims 29-33 or 42, wherein the patient is administered at a dose of viable CAR-positive cells.

46. Lapcaptagene autoleucel is approximately 2.5 x 10 ６ ~2.5 x 10 ８ viable CAR-positive cells, e.g., about 1 x 10 ７ or 5 x 10 ７ The method of any one of claims 29-33 or 42, wherein the patient is administered at a dose of viable CAR-positive cells.

47. 1. A method of treating a subject having lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), comprising administering to the subject a population of cells that express, or comprise a nucleic acid configured to express, a CD19 chimeric antigen receptor (CD19 CAR), wherein the cells are between 0.5 and 50 x 10 ６ viable CAR+ T cells (e.g., 5-12.5 x 10 ６ The method comprises administering a dose of up to 1000 viable CAR+ T cells to a subject.

48. 1. A method of treating a subject with lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), comprising administering to the subject lapcavtagene autoleucel, wherein the lapcavtagene autoleucel is administered in an amount of 0.5 to 50×10 ６ viable CAR+ T cells (e.g., 5-12.5 x 10 ６ The method comprises administering a dose of up to 1000 viable CAR+ T cells to a subject.

49. 49. The method of claim 47 or 48, wherein the lupus is systemic lupus erythematosus.

50. 50. The method of claim 49, wherein the SLE is severe refractory SLE (srSLE), and optionally the subject has renal involvement.

51. 51. The method of any one of claims 47, 49, or 50, wherein the CAR comprises a CD19 binding domain, a transmembrane domain, and an intracellular signaling domain.

52. (a) the transmembrane domain comprises a transmembrane domain of a protein selected from the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154; or (b) the transmembrane domain comprises the transmembrane domain of CD8; or (c) the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; or (d) the nucleic acid molecule comprises a nucleic acid sequence encoding the transmembrane domain, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 17, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereof.

53. the CD19 binding domain comprises a heavy chain complementarity determining region 1 (HC CDR1), a HC CDR2, a HC CDR3, a light chain complementarity determining region 1 (LC CDR1), a LC CDR2, and a LC CDR3; (a) the HC CDR1 comprises the amino acid sequence of SEQ ID NO:295; (b) the HC CDR2 comprises the amino acid sequence of SEQ ID NO: 296; (c) the HC CDR3 comprises the amino acid sequence of SEQ ID NO: 297; (d) the LC CDR1 comprises the amino acid sequence of SEQ ID NO: 298; (e) the LC CDR2 comprises the amino acid sequence of SEQ ID NO: 299; and (f) the LC CDR3 comprises the amino acid sequence of SEQ ID NO:

300.

54. 54. The method of any one of claims 1-26, 34-41, 47, or 49-53, wherein the CD19 binding domain comprises a VH and a VL, wherein the VH and VL are linked by a linker, and optionally the linker comprises the amino acid sequence of SEQ ID NO: 63 or 104.

55. the CD19 binding domain is linked to the transmembrane domain by a hinge region, and optionally (a) the hinge region comprises the amino acid sequence of SEQ ID NO: 2, 3, or 4, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; or (b) the nucleic acid molecule comprises a nucleic acid sequence encoding the hinge region, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 13, 14, or 15, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereof.

56. the intracellular signaling domain comprises a primary signaling domain, and optionally the primary signaling domain comprises a functional signaling domain derived from CD3ζ, TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (ICOS), FcεRI, DAP10, DAP12, or CD66d, and optionally (a) the primary signaling domain comprises a functional signaling domain derived from CD3ζ; or (b) the primary signaling domain comprises the amino acid sequence of SEQ ID NO: 9 or 10, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereof; or (c) the nucleic acid molecule comprises a nucleic acid sequence encoding the primary signaling domain, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 20 or 21, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereof.

57. The intracellular signaling domain comprises a costimulatory signaling domain, and optionally the costimulatory signaling domain is selected from the group consisting of an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), an activating NK cell receptor, BTLA, a Toll Ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, I CAM-1, 4-1BB (CD137), B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NK p30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGA D, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY 55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD28-OX40, CD28-4-1BB, or a ligand that specifically binds CD83; and optionally (a) the costimulatory signaling domain comprises a functional signaling domain derived from 4-1BB; (b) the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereof; or (c) the nucleic acid molecule comprises a nucleic acid sequence encoding the costimulatory signaling domain, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 18, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereof.

58. 58. The method of any one of claims 34-41, 47, or 49-57, wherein the intracellular signaling domain comprises a functional signaling domain derived from 4-1BB and a functional signaling domain derived from CD3ζ, and optionally the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7 (or an amino acid sequence with at least about 85%, 90%, 95%, or 99% sequence identity thereof) and the amino acid sequence of SEQ ID NO: 9 or 10 (or an amino acid sequence with at least about 85%, 90%, 95%, or 99% sequence identity thereof), and optionally the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 9 or 10.

59. The method of any one of claims 1 to 28, 32 to 41, 47, or 49 to 58, wherein the CAR further comprises a leader sequence comprising the amino acid sequence of SEQ ID NO:

1.

60. 60. The method of any one of claims 1-28, 32-41, 47, or 49-59, wherein the CD19 CAR comprises the amino acid sequence of SEQ ID NO: 301, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto.

61. 61. The method of any one of claims 1-28, 32-41, 47, or 49-60, wherein the nucleic acid molecule encoding the CD19 CAR comprises the nucleotide sequence of SEQ ID NO: 302, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereof.

62. 62. The method of any one of claims 1-61, wherein the subject has been previously treated or is concurrently being treated with one or more of: an antimalarial drug (e.g., hydroxychloroquine or quinacrine), a glucocorticoid (e.g., prednisone), a calcineurin inhibitor, an immunomodulatory agent (e.g., methotrexate, azathioprine, mycophenolate mofetil, cyclophosphamide, or tacrolimus), a biologic agent (e.g., belimumab, rituximab, a disease-modifying antirheumatic drug (DMARD) (e.g., leflunomide).

63. 63. The method of any one of claims 1-62, wherein the subject has been identified as having failed a treatment including two or more immunosuppressive therapies (e.g., a combination of mycophenolate or cyclophosphamide with a glucocorticoid) and one biologic agent.

64. 64. The method of any one of claims 1-63, wherein the subject has not previously received a therapy comprising a CD19 CAR (e.g., rapcaptagen autoleucel), adoptive T cell therapy, or gene therapy product.

65. 65. The method of any one of claims 1-64, wherein prior to administration of the CD19 CAR (e.g., rapcaptagen autoleucel), the subject undergoes lymphodepleting therapy.

66. 66. The method of claim 65, wherein the subject undergoes lymphodepletion therapy about two weeks prior to administration of the CD19 CAR (e.g., rapcaptagen autoleucel).

67. The lymphodepleting therapy may be fludarabine (e.g., 25 mg / m ２ IV daily for three doses) and cyclophosphamide (e.g., 250 mg / m ２ 67. The method of claim 65 or 66, comprising administering IV three doses daily.

68. 68. The method of any one of claims 1 to 67, further comprising administering a second therapeutic agent to the subject.

69. 69. The method of claim 68, wherein the second therapeutic agent is administered prior to, concurrently with, or following administration of the population of CAR-expressing cells or rapcaptagene autoleucel.

70. 70. The method of any one of claims 1 to 69, wherein the subject is monitored for signs of cytokine release syndrome, for example over at least 2, 2.5, 3, 3.5 or 4 days, for example about 3 days.

71. Leukapheresis was performed (i) before administration of corticosteroids and / or (ii) after an absolute T-cell count of 300 cells / mm ３ A method according to any one of claims 1 to 70, carried out when:

72. 1. A method of generating a population of cells (e.g., T cells) expressing a chimeric antigen receptor (CAR), comprising: (i) contacting (e.g., binding) a population of cells (e.g., T cells, e.g., T cells isolated from a frozen or fresh leukapheresis product) with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, wherein the population of cells is a patient suffering from an autoimmune disease or disorder, such as lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with severe pulmonary involvement (e.g., as seen in autoHSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myopathy), or the like; the subject has: myasthenia gravis (MG), overlap myositis, cancer-associated myositis, e.g., antisynthetase syndrome with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjogren's, severe refractory neuroimmune disease (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOGAD (anti-MOG-associated disease), multiple sclerosis (MS)), severe refractory rheumatoid arthritis, antibody-mediated neuroimmune disease (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG-associated disease), NMDAR+ encephalitis or antibody-related paraneoplastic neurological disease), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis; (ii) contacting the population of cells (e.g., T cells) with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding the CAR, thereby providing a population of cells (e.g., T cells) comprising the nucleic acid molecule, wherein optionally the CAR comprises a CD19 antigen-binding domain; and (iii) recovering the population of cells (e.g., T cells) for storage (e.g., reformulating the population of cells in cryopreservation medium) or administration. Including, (a) step (ii) is carried out together with step (i) or within 20 hours after the start of step (i), such as within 12, 13, 14, 15, 16, 17 or 18 hours after the start of step (i), for example within 18 hours after the start of step (i); and step (iii) is carried out within 30 (e.g. 26) hours after the start of step (i), such as within 22, 23, 24, 25, 26, 27, 28, 29 or 30 hours after the start of step (i), for example within 24 hours after the start of step (i); (b) step (ii) is carried out together with step (i) or within 20 hours after the start of step (i), such as within 12, 13, 14, 15, 16, 17 or 18 hours after the start of step (i), for example within 18 hours after the start of step (i); and Step (iii) is carried out within 30 hours after the start of step (ii), for example within 22, 23, 24, 25, 26, 27, 28, 29 or 30 hours after the start of step (ii); or (c) the population of cells from step (iii) is not expanded, or is expanded by no more than 5, 10, 15, 20, 25, 30, 35 or 40%, e.g., no more than 10%, compared to the population of cells at the start of step (i), e.g., as assessed by viable cell count; Optionally, the nucleic acid molecule of step (ii) is on a viral vector; optionally, the nucleic acid molecule of step (ii) is an RNA molecule on a viral vector; and optionally, step (ii) comprises transducing the population of cells (e.g., T cells) with a viral vector comprising a nucleic acid molecule encoding the CAR.

73. The agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3 (e.g., an anti-CD3 antibody), and the agent that stimulates a costimulatory molecule is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof; optionally, the agent that stimulates the CD3 / TCR complex or the agent that stimulates a costimulatory molecule is an antibody (e.g., a single domain antibody (e.g., a heavy chain variable domain antibody), a peptibody, a Fab fragment, or an scFv), a small molecule, or a ligand (e.g., a naturally occurring ligand, a recombinant ligand, or a 73. The method of claim 72, wherein the agent that stimulates the CD3 / TCR complex or the agent that stimulates a costimulatory molecule is selected from a chimeric ligand (or a chimeric ligand), optionally wherein the agent that stimulates the CD3 / TCR complex or the agent that stimulates a costimulatory molecule does not comprise a bead, optionally wherein the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody, and the agent that stimulates a costimulatory molecule comprises an anti-CD28 antibody, optionally wherein the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody covalently bound to a colloidal polymer nanomatrix, and the agent that stimulates a costimulatory molecule comprises an anti-CD28 antibody covalently bound to a colloidal polymer nanomatrix, optionally wherein the agent that stimulates the CD3 / TCR complex and the agent that stimulates a costimulatory molecule comprise T Cell TransAct™.

74. 74. The method of claim 72 or 73, wherein step (i) increases the percentage of CAR-expressing cells in the population of cells from step (iii), e.g., the population of cells from step (iii) exhibits a higher percentage of CAR-expressing cells (e.g., at least 10, 20, 30, 40, 50, or 60% higher) compared to cells made by an otherwise similar method without step (i).

75. (a) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ T cells, in said 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, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ cells, in said population of cells at the start of step (i); (b) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ T cells, in the population of cells from step (iii) is increased, e.g., by at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3-fold compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ cells, in the population of cells at the start of step (i); (c) the percentage of CAR-expressing naive T cells, e.g., CAR-expressing CD45RA+ CD45RO- CCR7+ T cells, in said population of cells increases during step (ii), e.g., increases by at least 30, 35, 40, 45, 50, 55, or 60% 18 to 24 hours after initiation of step (ii); or 75. The method of any one of claims 72 to 74, wherein (d) the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO-CCR7+ T cells, in the population of cells from step (iii) is not reduced, or is reduced by no more than 5 or 10%, compared to the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO-CCR7+ cells, in the population of cells at the start of step (i).

76. (a) the population of cells from step (iii) exhibits a higher percentage (e.g., at least 10, 20, 30, or 40% higher) of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ T cells, compared to cells produced by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (b) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ T cells, in said population of cells from step (iii) is higher (e.g., at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3 times higher) than the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ T cells, in cells made by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (c) the percentage of CAR-expressing naive T cells, e.g., CAR-expressing CD45RA+ CD45RO-CCR7+ T cells, in the population of cells from step (iii) is higher (e.g., at least 4, 6, 8, 10, or 12 fold) than the percentage of CAR-expressing naive T cells, e.g., CAR-expressing CD45RA+ CD45RO-CCR7+ T cells, in cells made by a similar method except that step (iii) is performed more than 26 hours after the initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the initiation of step (i); (d) the population of cells from step (iii) exhibits a higher percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ T cells (e.g., at least 10, 20, 30, or 40% higher), compared to cells produced by a similar method but further comprising, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days; (e) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ T cells, in the population of cells from step (iii) is higher (e.g., at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3 times higher) than the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO-CCR7+ T cells, in cells made by a similar method but further comprising, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days; or (f) the percentage of CAR-expressing naive T cells, e.g., CAR-expressing CD45RA+ CD45RO-CCR7+ T cells, in the population of cells from step (iii) is higher (e.g., at least 4, 6, 8, 10, or 12 fold) than the percentage of CAR-expressing naive T cells, e.g., CAR-expressing CD45RA+ CD45RO-CCR7+ T cells, in cells made by a similar method but further comprising the step of expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii).

77. (a) the percentage of central memory cells, e.g., central memory T cells, e.g., 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, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells at the start of step (i); (b) the percentage of central memory cells, e.g., central memory T cells, e.g., 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% compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in the population of cells at the start of step (i); (c) the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+ CD45RO+ cells, is decreased during step (ii), e.g., is decreased by at least 8, 10, 12, 14, 16, or 20% 18 to 24 hours after initiation of step (ii); or 77. The method of any one of claims 72 to 76, wherein (d) the percentage of central memory cells, such as central memory T cells, such as CCR7+ CD45RO+ T cells, in the population of cells from step (iii) is not increased, or is increased by no more than 5 or 10%, compared to the percentage of central memory cells, such as central memory T cells, such as CCR7+ CD45RO+ T cells, in the population of cells at the start of step (i).

78. (a) the population of cells from step (iii) exhibits a lower percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells (e.g., at least 10, 20, 30, or 40% lower), compared to cells produced by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (b) the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in the population of cells from step (iii) is lower (e.g., at least 20, 30, 40, or 50% lower) than the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in cells made by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (c) the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+ CD45RO+ T cells, in the population of cells from step (iii) is lower (e.g., at least 10, 20, 30, or 40% lower) than the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+ CD45RO+ T cells, in cells generated by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); (d) the population of cells from step (iii) exhibits a lower percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells (e.g., at least 10, 20, 30, or 40% lower), compared to cells produced by a similar method but further comprising, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days; (e) the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in the population of cells from step (iii) is lower (e.g., at least 20, 30, 40, or 50% lower) than the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+ CD45RO+ T cells, in cells made by a similar method, but further comprising, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days; or (f) the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+ CD45RO+ T cells, in the population of cells from step (iii) is lower (e.g., at least 10, 20, 30, or 40% lower) than the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+ CD45RO+ T cells, in cells made by a similar method but further comprising the step of expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii).

79. (a) is the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) increased compared to the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells at the start of step (i); (b) is the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) increased compared to the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells at the start of step (i); (c) the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) is higher than the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in cells made by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i); or (d) whether the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) is higher than the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in cells generated by a similar method except that step (iii) is performed more than 26 hours after the initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the initiation of step (i); (e) the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) is higher than the percentage of stem memory T cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in cells made by a similar method, but further comprising, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days; or (f) the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) is higher than the percentage of CAR-expressing stem memory T cells, e.g., CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in cells made by a similar method but further comprising the step of expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii).

80. (a) the median GeneSetScore (Up TEM vs. Down TSCM) of the population of cells from step (iii) is about the same as, or differs by (e.g., increases by) no more than about 25, 50, 75, 100, or 125% from, the median GeneSetScore (Up TEM vs. Down TSCM) of the population of cells at the start of step (i); (b) the median GeneSetScore (Up TEM vs. Down TSCM) of the population of cells from step (iii) is Cells produced by a similar method, except that step (iii) is carried out more than 26 hours after the start of step (i), for example more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i); or Cells made by a similar method, but further comprising, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days. lower (e.g., at least about 100, 150, 200, 250, or 300% lower) than the median GeneSetScore (Up TEM vs. Down TSCM); (c) the median GeneSetScore (Up Treg vs. Down Teff) of the population of cells from step (iii) is about the same as, or differs by (e.g., increases 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 at the start of step (i); (d) the median GeneSetScore (Up Treg vs. Down Teff) of the population of cells from step (iii) is Cells produced by a similar method, except that step (iii) is carried out more than 26 hours after the start of step (i), for example more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i); or Cells made by a similar method, but further comprising, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days. lower (e.g., at least about 50, 100, 125, 150, or 175% lower) than the median GeneSetScore (Up Treg vs. Down Teff) of (e) the median GeneSetScore (Down stemness) of the population of cells from step (iii) is about the same as or differs from (e.g., increases by) no more than about 25, 50, 100, 150, 200, or 250% of the median GeneSetScore (Down stemness) of the population of cells at the start of step (i); (f) the median GeneSetScore (Down stemness) of the population of cells from step (iii) is Cells produced by a similar method, except that step (iii) is carried out more than 26 hours after the start of step (i), for example more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i); or Cells made by a similar method, but further comprising, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days. lower (e.g., at least about 50, 100, or 125% lower) than the median GeneSetScore (Down stemness); (g) the median GeneSetScore (Up hypothesis) of the population of cells from step (iii) is about the same as or differs by (e.g., increases by) no more than about 125, 150, 175, or 200% from the median GeneSetScore (Up hypothesis) of the population of cells at the start of step (i); (h) the median GeneSetScore (Up hypoxia) of the population of cells from step (iii) is Cells produced by a similar method, except that step (iii) is carried out more than 26 hours after the start of step (i), for example more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i); or Cells made by a similar method, but further comprising, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days. lower (e.g., at least about 40, 50, 60, 70, or 80% lower) than the median GeneSetScore (Up hypoxia); (j) the median GeneSetScore (Up autophagy) of the population of cells from step (iii) is about the same as or differs from (e.g., increases by) no more than about 180, 190, 200, or 210% of the median GeneSetScore (Up autophagy) of the population of cells at the start of step (i); or (k) the median GeneSetScore (Up autophagey) of the population of cells from step (iii) is Cells produced by a similar method, except that step (iii) is carried out more than 26 hours after the start of step (i), for example more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i); or Cells made by a similar method, but further comprising, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days.

80. The method of any one of claims 72 to 79, wherein the median GeneSetScore (Up autophagy) is lower (e.g., at least about 20, 30, or 40% lower).

81. 81. The method of any one of claims 72-80, wherein the population of cells from step (iii), after incubation with cells expressing the antigen recognized by the CAR, secrete IL-2 at a higher (e.g., at least 2, 4, 6, 8, 10, 12 or 14 fold) level than cells produced by a similar method except that step (iii) is carried out more than 26 hours after the start of step (i), such as more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i), or than cells produced by a similar method except that the method further comprises expanding the population of cells (e.g., T cells) in vitro for more than 3 days, such as 5, 6, 7, 8 or 9 days, after step (ii) and before step (iii).

82. 82. The method of any one of claims 72-81, wherein the population of cells from step (iii), after administration in vivo to the subject, persists longer or proliferates to a higher level than cells made by a similar method except that step (iii) is carried out more than 26 hours after the start of step (i), such as more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i), or compared to cells made by a similar method except that the method further comprises expanding the population of cells (e.g., T cells) in vitro for more than 3 days, such as 5, 6, 7, 8 or 9 days after step (ii) and before step (iii).

83. The population of cells from step (iii), after administration in vivo to the subject, exhibits a stronger activity (e.g., a lower dose, e.g., 0.15×10) than cells produced by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i), or than cells produced by a similar method except that step (iii) further comprises expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). ６ , 0.2 × 10 ６ , 0.25×10 ６ or 0.3 x 10 ６ The method of any one of claims 72 to 82, wherein the antibody exhibits a potent activity at a dose of 1000 to 1000 viable CAR-expressing cells.

84. 84. The method of any one of claims 72 to 83, wherein the population of cells from step (iii) is not expanded, or is expanded by no more than 5, 10, 15, 20, 25, 30, 35 or 40% compared to the population of cells at the start of step (i), e.g. as assessed by viable cell count, and optionally the number of viable cells in the population of cells from step (iii) is reduced from the number of viable cells in the population of cells at the start of step (i).

85. 85. The method of any one of claims 72 to 84, wherein the population of cells from step (iii) is not expanded or is expanded by less than 2 hours, such as less than 1 or 1.5 hours, compared to the population of cells at the start of step (i).

86. 86. The method of any one of claims 72 to 85, wherein steps (i) and / or (ii) are carried out in a cell culture medium (e.g., serum-free medium) comprising IL-2, IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)), IL-7, IL-21, IL-6 (e.g., IL-6 / sIL-6Ra), an LSD1 inhibitor, a MALT1 inhibitor, or a combination thereof.

87. 87. The method of any one of claims 72 to 86, wherein steps (i) and / or (ii) are carried out in a serum-free cell culture medium comprising a serum replacement.

88. 88. The method of claim 87, wherein the serum replacement is CTS™ Immune Cell Serum Replacement (ICSR).

89. Before step (i), (iv) (optionally) receiving a fresh leukapheresis product (or an alternative source of hematopoietic tissue, such as a fresh whole blood product, a fresh bone marrow product, or a fresh organ biopsy or resection (e.g., a fresh product from a thymus removal)) from an entity, e.g., a laboratory, hospital, or healthcare provider; and (v) isolating the population of cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells) contacted in step (i) from a fresh leukapheresis product (or an alternative source of hematopoietic tissue, such as a fresh whole blood product, a fresh bone marrow product, or a fresh organ biopsy or resection (e.g., a fresh product from a thymus removal)). and optionally further comprising: Step (iii) is carried out within 35 hours after the start of step (v), such as within 27, 28, 29, 30, 31, 32, 33, 34 or 35 hours after the start of step (v), for example within 30 hours after the start of step (v); or 89. The method of any one of claims 72 to 88, wherein the population of cells from step (iii) is not expanded, or is expanded by no more than 5, 10, 15, 20, 25, 30, 35 or 40%, such as no more than 10%, compared to the population of cells at the end of step (v), as assessed by viable cell count.

90. 89. The method of any one of claims 72-88, further comprising receiving cryopreserved T cells isolated from a leukapheresis product (or an alternative source of hematopoietic tissue such as whole blood, bone marrow, or cryopreserved T cells isolated from an organ biopsy or resection (e.g., thymus removal)) from an entity, e.g., a laboratory, hospital, or healthcare provider, prior to step (i).

91. Before step (i), (iv) (optionally) receiving a cryopreserved leukapheresis product (or an alternative source of hematopoietic tissue, such as a cryopreserved whole blood product, a cryopreserved bone marrow product, or a cryopreserved organ biopsy or resection (e.g., a cryopreserved product from a thymus removal)) from an entity, e.g., a laboratory, hospital, or healthcare provider; and (v) isolating the population of cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells) contacted in step (i) from a cryopreserved leukapheresis product (or an alternative source of hematopoietic tissue, such as a cryopreserved whole blood product, a cryopreserved bone marrow product, or a cryopreserved organ biopsy or resection (e.g., a cryopreserved product from a thymus removal)). and optionally further comprising: Step (iii) is carried out within 35 hours after the start of step (v), such as within 27, 28, 29, 30, 31, 32, 33, 34 or 35 hours after the start of step (v), for example within 30 hours after the start of step (v); or 89. The method of any one of claims 72 to 88, wherein the population of cells from step (iii) is not expanded, or is expanded by no more than 5, 10, 15, 20, 25, 30, 35 or 40%, such as no more than 10%, compared to the population of cells at the end of step (v), as assessed by viable cell count.

92. Step (vi): culturing a portion of the population of cells from step (iii) for at least 2, 2.5, 3, 3.5, 4, 4.5, 5, 5, 6, 6.5, or 7 days, e.g., at least 2 days and no more than 7 days, and measuring the level of CAR expression in said portion (e.g., measuring the percentage of surviving CAR-expressing cells in said portion). and optionally further comprising:

92. The method of any one of claims 72 to 91, wherein step (iii) comprises harvesting and freezing the population of cells (e.g., T cells), and step (vi) comprises thawing a portion of the population of cells from step (iii), culturing the portion for at least 2, 2.5, 3, 3.5, 4, 4.5, 5.5, 6, 6.5 or 7 days, such as at least 2 days and not more than 7 days, and measuring the level of CAR expression in the portion (e.g., measuring the percentage of surviving CAR-expressing cells in the portion).

93. 93. The method of any one of claims 72 to 92, wherein the population of cells at the start of step (i) or step (1) is enriched for IL6R-expressing cells (e.g., cells positive for IL6Rα and / or IL6Rβ).

94. 94. The method of any one of claims 72 to 93, wherein the population of cells at the start of step (i) or step (1) comprises 50, 60, or 70% or more IL6R-expressing cells (e.g., cells positive for IL6Rα and / or IL6Rβ).

95. 95. The method of any one of claims 72 to 94, wherein steps (i) and (ii) or steps (1) and (2) are carried out in a cell culture medium containing IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)).

96. 96. The method of claim 95, wherein IL-15 increases the ability of the population of cells to proliferate, for example after 10, 15, 20 or 25 days.

97. 96. The method of claim 95, wherein IL-15 increases the percentage of IL6Rβ-expressing cells in the population of cells.

98. 98. The method of any one of claims 72 to 97, wherein the lupus is systemic lupus erythematosus.

99. 99. The method of claim 98, wherein the SLE is severe refractory SLE (srSLE).

100. The method of any one of claims 72 to 99, wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

101. 101. The method of claim 100, wherein the antigen binding domain binds to a lupus-associated B cell antigen (e.g., CD19).

102. 102. The method of claim 100 or 101, wherein the antigen binding domain comprises a CDR, VH, VL, scFv or CAR sequence disclosed herein.

103. the antigen-binding domain comprises a CD19-binding domain comprising heavy chain complementarity-determining region 1 (HC CDR1), HC CDR2, HC CDR3, light chain complementarity-determining region 1 (LC CDR1), LC CDR2, and LC CDR3; (a) the HC CDR1 comprises the amino acid sequence of SEQ ID NO:295; (b) the HC CDR2 comprises the amino acid sequence of SEQ ID NO: 296; (c) the HC CDR3 comprises the amino acid sequence of SEQ ID NO: 297; (d) the LC CDR1 comprises the amino acid sequence of SEQ ID NO: 298; (e) the LC CDR2 comprises the amino acid sequence of SEQ ID NO: 299; and (f) the LC CDR3 comprises the amino acid sequence of SEQ ID NO:

300.

104. 104. The method of any one of claims 100 to 103, wherein the antigen-binding domain comprises a VH and a VL, wherein the VH and VL are linked by a linker, and optionally the linker comprises the amino acid sequence of SEQ ID NO: 63 or 104.

105. (a) the transmembrane domain comprises a transmembrane domain of a protein selected from the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154; or (b) the transmembrane domain comprises the transmembrane domain of CD8; or (c) the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; or (d) the nucleic acid molecule comprises a nucleic acid sequence encoding the transmembrane domain, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 17 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereof.

106. the antigen binding domain is linked to the transmembrane domain by a hinge region, and optionally (a) the hinge region comprises the amino acid sequence of SEQ ID NO: 2, 3, or 4, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; or (b) the nucleic acid molecule comprises a nucleic acid sequence encoding the hinge region, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 13, 14, or 15, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereof.

107. the intracellular signaling domain comprises a primary signaling domain, and optionally the primary signaling domain comprises a functional signaling domain derived from CD3ζ, TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (ICOS), FcεRI, DAP10, DAP12, or CD66d, and optionally (a) the primary signaling domain comprises a functional signaling domain derived from CD3ζ; or (b) the primary signaling domain comprises the amino acid sequence of SEQ ID NO: 9 or 10, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereof; or (c) the nucleic acid molecule comprises a nucleic acid sequence encoding the primary signaling domain, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 20 or 21, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereof.

108. The intracellular signaling domain comprises a costimulatory signaling domain, and optionally the costimulatory signaling domain is selected from the group consisting of an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), an activating NK cell receptor, BTLA, a Toll Ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, C DS, ICAM-1, 4-1BB (CD137), B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), N Kp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, C D49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (C D229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD28-OX40, CD28-4-1BB, or CD83; and optionally, a ligand that specifically binds to (a) the costimulatory signaling domain comprises a functional signaling domain derived from 4-1BB; (b) the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereof; or (c) the nucleic acid molecule comprises a nucleic acid sequence encoding the costimulatory signaling domain, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 18, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereof.

109. 109. The method of any one of claims 100-108, wherein the intracellular signaling domain comprises a functional signaling domain derived from 4-1BB and a functional signaling domain derived from CD3ζ, and optionally the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7 (or an amino acid sequence with at least about 85%, 90%, 95%, or 99% sequence identity thereof) and the amino acid sequence of SEQ ID NO: 9 or 10 (or an amino acid sequence with at least about 85%, 90%, 95%, or 99% sequence identity thereof), and optionally the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 9 or 10.

110. The method of any one of claims 100 to 109, wherein the CAR further comprises a leader sequence comprising the amino acid sequence of SEQ ID NO:

1.

111. 101. The method of claim 100, wherein the CAR comprises a CD19 CAR comprising the amino acid sequence of SEQ ID NO: 301, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereof.

112. 101. The method of claim 100, wherein the nucleic acid molecule encoding the CD19 CAR comprises the nucleotide sequence of SEQ ID NO: 302, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereof.

113. 113. The method of any one of claims 72-112, wherein the subject has been previously treated with one or more of an antimalarial agent (e.g., hydroxychloroquine or quinacrine), a glucocorticoid (e.g., prednisone), a calcineurin inhibitor, an immunomodulatory agent (e.g., methotrexate, azathioprine, mycophenolate mofetil, cyclophosphamide, or tacrolimus), a biologic agent (e.g., belimumab, rituximab, a disease-modifying antirheumatic drug (DMARD) (e.g., leflunomide).

114. 114. The method of any one of claims 72-113, wherein the subject has been identified as having failed treatment including two or more immunosuppressive therapies (e.g., a combination of mycophenolate or cyclophosphamide with a glucocorticoid) and one biologic agent.

115. 115. The method of any one of claims 72-114, wherein the subject has not previously received a therapy comprising a CD19 CAR, adoptive T cell therapy, or gene therapy product.

116. Leukapheresis was performed (i) before administration of corticosteroids and / or (ii) after an absolute T-cell count of 300 cells / mm ３ The method of any one of claims 72 to 115, wherein the method is carried out when

117. 117. A population of CAR-expressing cells (e.g., autologous or allogeneic CAR-expressing T cells or NK cells) produced by the method of any one of claims 72 to 116.

118. 118. The population of CAR-expressing cells of claim 117, comprising autoreactive B cells (e.g., autoreactive B cells that do not express a CAR).

119. 119. A pharmaceutical composition comprising a population of CAR-expressing cells of claim 117 or 118 and a pharmaceutically acceptable carrier.

120. 120. The population of CAR-expressing cells of claim 117 or 118 or the pharmaceutical composition of claim 119 for use in a method of modulating an immune response in a subject with lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), the method comprising administering to the subject an effective amount of the population of CAR-expressing cells or an effective amount of the pharmaceutical composition.

121. 1. A method of treating a subject having an autoimmune disease, e.g., lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), comprising administering to the subject: a population of cells that express, or contain a nucleic acid configured to express, a CD19 chimeric antigen receptor (CD19 CAR); and A second therapy selected from an antimalarial or stable immunosuppressant wherein the second therapy and CD19 CAR cells are present in the subject at the same time, e.g., the second therapy is administered at a time when the CD19 CAR cells are present in the subject.

122. 1. A method of treating a subject having an autoimmune disease, e.g., lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), comprising administering to the subject: Lapcaptagene Autoleucel, and A second therapy selected from an antimalarial or stable immunosuppressant wherein the second therapy and lapcaptagene autoleucel are present in the subject at the same time, for example, the second therapy is administered at a time when lapcaptagene autoleucel is present in the subject.

123. Autoimmune diseases or disorders, such as lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with severe pulmonary involvement (e.g., as seen in autoHSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-associated myositis, e.g., antisynthetase syndrome with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjogren's disease, severe refractory myopathy, and rapcavtagene autoleucel made from autologous cells for subjects with curative neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOGAD (anti-MOG associated disease), multiple sclerosis (MS)), severe refractory rheumatoid arthritis, antibody-mediated neuroimmune diseases (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG associated disease), NMDAR+ encephalitis or antibody-related paraneoplastic neurological diseases), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis.

124. A pharmaceutical composition comprising the lapcaptagene autoleucel of claim 123 and a pharmaceutically acceptable carrier.

125. autoimmune diseases or disorders, such as lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with severe pulmonary involvement (e.g., as seen in autoHSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-associated myositis, e.g., antisynthetase syndrome associated with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjogren's disease, severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-related disease (MOG) 125. The lapcaptagen autleucel of claim 123 or the pharmaceutical composition of claim 124 for use in a method of modulating the immune response of a subject with rheumatoid arthritis (AD), multiple sclerosis (MS), severe refractory rheumatoid arthritis, an antibody-mediated neuroimmune disease (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG associated disease), NMDAR+ encephalitis or antibody-related paraneoplastic neurological disease), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris or amyotrophic lateral sclerosis, the method comprising the step of administering to the subject an effective amount of lapcaptagen autleucel or an effective amount of the pharmaceutical composition.

126. The lapcaptagen autreucel of claim 123 or the pharmaceutical composition of claim 124 for use in a method for modulating the immune response of a subject with lupus (e.g., systemic lupus erythematosus (SLE), e.g., severe refractory systemic lupus erythematosus (srSLE) or lupus nephritis), the method comprising administering to the subject an effective amount of lapcaptagen autreucel or an effective amount of the pharmaceutical composition.

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