Treatment of autoimmune disorders using chimeric antigen receptor therapy

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 375,776, filed September 15, 2022, and U.S. Provisional Application No. 63 / 507,141, filed June 9, 2023. The contents of the foregoing applications are hereby incorporated by reference in their entirety. Technical Field

[0003] The present invention generally relates to methods of making immune effector cells (e.g., T cells or NK cells) engineered to express chimeric antigen receptors (CARs), compositions comprising these immune effector cells, and their therapeutic uses for treating autoimmune diseases or disorders. Background Art

[0004] Current therapies for severe autoimmune diseases such as systemic lupus erythematosus (SLE) include conventional immunomodulatory and anti-inflammatory agents, such as antimalarials, glucocorticoids, and immunosuppressants (e.g., methotrexate, azathioprine, mycophenolate mofetil, and cyclophosphamide), and biologics (e.g., belimumab and, more recently, anifrolumab, as well as rituximab (often used in severe stages of the disease)). For patients with severe, refractory SLE (srSLE), with or without renal involvement, who have failed immunosuppressive and biologic therapies, treatment options are very limited. Autologous stem cell transplantation (ASCT) is an option; however, it remains experimental and is associated with significant toxicity, including death.

[0005] Therefore, there is an unmet need for new treatments for severe autoimmune diseases, including srSLE. Summary of the Invention

[0006] This disclosure is about preparing the method for the immune effector cells (such as T cells or NK cells) engineered to express CAR, and the compositions produced using such methods. It is also disclosed that such compositions are used to treat the method for the disease of a subject, the disease is, for example, autoimmune disease or disorder, for example, lupus (for example, systemic lupus erythematosus (SLE), for example, severe refractory systemic lupus erythematosus (srSLE), or lupus nephritis), systemic sclerosis (for example, rapidly progressing systemic sclerosis (SSc) with significant lung involvement (for example, for autologous HSCT)), idiopathic inflammatory myopathy (for example, polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-related myositis, such as antisynthetase syndrome with ILD), vasculitis (for example, ANCA-related blood Severe refractory Sjögren's syndrome, severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-related diseases (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-related diseases), NMDAR+ encephalitis, or antibody-related neurological paraneoplastic diseases), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis.

[0007] In one aspect, the disclosure provides methods of treating a subject having an autoimmune disease or disorder, 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 significant pulmonary involvement (e.g., for autologous HSCT)), 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 Sjögren's syndrome, severe refractory A method of treating a subject with a neuroimmune disease (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-related 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-related disease), NMDAR+ encephalitis, or antibody-related neurological paraneoplastic disease), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis, the method comprising administering to the subject a population of cells (e.g., T cells) expressing a CD19 chimeric antigen receptor (CAR) or comprising a nucleic acid configured to express a CD19 CAR, wherein the cell population is prepared by a method comprising:

[0008] (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 co-stimulatory molecule on the surface of the cells;

[0009] (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 ("CD19 CAR"); and

[0010] (iii) harvesting the cell (e.g., T cell) population for storage (e.g., reconstituting the cell population in a cryopreservation medium) or administration, wherein:

[0011] (a) step (ii) is performed together with step (i) or not later than 20 hours after the start of step (i), for example not later than 12, 13, 14, 15, 16, 17, or 18 hours after the start of step (i), for example not later than 18 hours after the start of step (i), and

[0012] step (iii) is performed no later than 30 (e.g., 26) hours after the start of step (i), for example no later than 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the start of step (i), for example no later than 24 hours after the start of step (i),

[0013] (b) step (ii) is performed together with step (i) or not later than 20 hours after the start of step (i), for example not later than 12, 13, 14, 15, 16, 17, or 18 hours after the start of step (i), for example not later than 18 hours after the start of step (i), and

[0014] step (iii) is performed no later than 30 hours after the start of step (ii), for example no later than 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the start of step (ii), or

[0015] (c) the cell population from step (iii) does not expand, or expands by no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40%, e.g., by no more than 10%, compared to the cell population at the start of step (i), e.g., as assessed by viable cell number,

[0016] Optionally, the nucleic acid molecule in step (ii) is on a viral vector, optionally, the nucleic acid molecule in step (ii) is an RNA molecule on a viral vector, optionally, step (ii) comprises transducing the cell (e.g., T cell) population with a viral vector comprising a nucleic acid molecule encoding the CAR.

[0017] In some embodiments, the agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3 (e.g., an anti-CD3 antibody), and wherein 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 wherein the agent that stimulates the CD3 / TCR complex or the agent that stimulates a costimulatory molecule 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 a scFv), a small molecule, or a ligand (e.g., a naturally occurring ligand). wherein the agent that stimulates the CD3 / TCR complex or the agent that stimulates the co-stimulatory 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 the co-stimulatory molecule comprises an anti-CD28 antibody; optionally wherein the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody covalently attached to a colloidal polymer nanomatrix and the agent that stimulates the co-stimulatory molecule comprises an anti-CD28 antibody covalently attached to a colloidal polymer nanomatrix; optionally wherein the agent that stimulates the CD3 / TCR complex and the agent that stimulates the co-stimulatory molecule comprise T cell TransAct TM .

[0018] In some embodiments, step (i) increases the percentage of CAR-expressing cells in the cell population from step (iii), compared to cells prepared by an otherwise similar method except that step (i) is not included, for example, the cell population from step (iii) shows a higher percentage of CAR-expressing cells (e.g., at least 10%, 20%, 30%, 40%, 50%, or 60% higher).

[0019] In some embodiments:

[0020] (a) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the cell population 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 cell population at the start of step (i);

[0021] (b) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the cell population 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 as compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells, in the cell population at the start of step (i);

[0022] (c) the percentage of naive T cells expressing CAR, e.g., CD45RA+CD45RO-CCR7+ T cells expressing CAR, in the cell population increases during the duration of step (ii), e.g., by at least 30%, 35%, 40%, 45%, 50%, 55%, or 60% between 18-24 hours after the start of step (ii); or

[0023] (d) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the cell population from step (iii) is not decreased, or is decreased by no more than 5% or 10%, as compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells, in the cell population at the start of step (i).

[0024] In some embodiments:

[0025] (a) 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 prepared by an otherwise similar method except that step (iii) is performed more than 26 hours after initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after initiation of step (i);

[0026] (b) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the cell population 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 prepared by an otherwise similar method except that step (iii) is performed more than 26 hours after initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after initiation of step (i);

[0027] (c) the percentage of naive T cells expressing CAR, e.g., CD45RA+CD45RO-CCR7+ T cells expressing CAR, in the cell population from step (iii) is higher (e.g., at least 4, 6, 8, 10, or 12 times higher) than the percentage of naive T cells expressing CAR, e.g., CD45RA+CD45RO-CCR7+ T cells expressing CAR, in cells prepared by an otherwise similar method except that step (iii) is performed more than 26 hours after initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after initiation of step (i);

[0028] (d) the cell population 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 prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii);

[0029] (e) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the cell population 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 prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii); or

[0030] (f) the percentage of naive T cells expressing CAR, e.g., CD45RA+CD45RO-CCR7+ T cells expressing CAR, in the cell population from step (iii) is higher (e.g., at least 4, 6, 8, 10, or 12 times higher) than the percentage of naive T cells expressing CAR, e.g., CD45RA+CD45RO-CCR7+ T cells expressing CAR, in cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii).

[0031] In some embodiments:

[0032] (a) the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population 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 cell population at the beginning of step (i);

[0033] (b) the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the cell population from step (iii) is reduced by at least 20%, 25%, 30%, 35%, 40%, 45%, or 50% as compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells in the cell population at the start of step (i);

[0034] (c) the percentage of central memory T cells expressing the CAR, e.g., CCR7+CD45RO+ cells expressing the CAR, decreases during the duration of step (ii), e.g., by at least 8%, 10%, 12%, 14%, 16%, 18%, or 20% between 18 and 24 hours after the start of step (ii); or

[0035] (d) the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the cell population from step (iii) does not increase or increases by no more than 5% or 10% as compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells in the cell population at the beginning of step (i).

[0036] In some embodiments:

[0037] (a) the cell population 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 prepared by an otherwise similar method except that step (iii) is performed more than 26 hours after initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after initiation of step (i);

[0038] (b) the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the cell population 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 prepared by an otherwise similar method except that step (iii) is performed more than 26 hours after initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after initiation of step (i);

[0039] (c) the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+CD45RO+ T cells, in the cell population 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 prepared by an otherwise 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);

[0040] (d) the cell population 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 prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii);

[0041] (e) the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the cell population 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 prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii); or

[0042] (f) the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+CD45RO+ T cells, in the cell population from step (iii) is lower (e.g., at least 10%, 20%, 30%, or 40%) than the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+CD45RO+ T cells, in cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii).

[0043] In some embodiments:

[0044] (a) the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the cell population from step (iii) is increased as compared to the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the cell population at the start of step (i);

[0045] (b) the percentage of CAR-expressing stem cell memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the cell population from step (iii) is increased as compared to the percentage of CAR-expressing stem cell memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the cell population at the beginning of step (i);

[0046] (c) the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the cell population from step (iii) is higher than the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in cells prepared by an otherwise 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

[0047] (d) the percentage of CAR-expressing stem cell memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the cell population from step (iii) is higher than the percentage of CAR-expressing stem cell memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in cells prepared by an otherwise 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);

[0048] (e) the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the cell population from step (iii) is higher than the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii); or

[0049] (f) the percentage of CAR-expressing stem cell memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the cell population from step (iii) is higher than the percentage of CAR-expressing stem cell memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii).

[0050] In some embodiments:

[0051] (a) the median gene set score (TEM up vs. TSCM down) of the cell population from step (iii) is about the same as or differs by no more than (e.g., increases by no more than) about 25%, 50%, 75%, 100%, or 125% from the median gene set score (TEM up vs. TSCM down) of the cell population from the start of step (i);

[0052] (b) the median gene set score (TEM up vs. TSCM down) of the population of cells from step (iii) is lower (e.g., at least about 100%, 150%, 200%, 250%, or 300% lower) than the median gene set score (TEM up vs. TSCM down) of:

[0053] cells prepared by an otherwise similar method except that step (iii) is performed 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

[0054] Cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii);

[0055] (c) the median gene set score (Treg up vs. Teff down) of the cell population from step (iii) is about the same as or differs by no more than (e.g., increases by no more than) about 25%, 50%, 100%, 150%, or 200% from the median gene set score (Treg up vs. Teff down) of the cell population from the start of step (i);

[0056] (d) the median gene set score (Treg up vs. Teff down) of the cell population from step (iii) is lower (e.g., at least about 50%, 100%, 125%, 150%, or 175% lower) than the median gene set score (Treg up vs. Teff down) of:

[0057] cells prepared by an otherwise similar method except that step (iii) is performed 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

[0058] Cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii);

[0059] (e) the median gene set score (downward stemness) of the cell population from step (iii) is about the same as or differs by no more than (e.g., increases by no more than) about 25%, 50%, 100%, 150%, 200%, or 250% from the median gene set score (downward stemness) of the cell population from the start of step (i);

[0060] (f) the median gene set score (downward stemness) of the population of cells from step (iii) is lower (e.g., at least about 50%, 100%, or 125% lower) than the median gene set score (downward stemness) of:

[0061] cells prepared by an otherwise similar method except that step (iii) is performed 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

[0062] Cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii);

[0063] (g) the median gene set score (upward hypoxia) of the cell population from step (iii) is about the same as or differs by no more than (e.g., increases by no more than) about 125%, 150%, 175%, or 200% from the median gene set score (upward hypoxia) of the cell population from the start of step (i);

[0064] (h) the median gene set score (hypoxia up) of the population of cells from step (iii) is lower (e.g., at least about 40%, 50%, 60%, 70%, or 80% lower) than the median gene set score (hypoxia up) of:

[0065] cells prepared by an otherwise similar method except that step (iii) is performed 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

[0066] Cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii);

[0067] (j) the median gene set score (autophagy up) of the cell population from step (iii) is about the same as or differs by no more than (e.g., increases by no more than) about 180%, 190%, 200%, or 210% from the median gene set score (autophagy up) of the cell population from the start of step (i); or

[0068] (k) the median gene set score (autophagy up) of the population of cells from step (iii) is lower (e.g., at least about 20%, 30%, or 40% lower) than the median gene set score (autophagy up) of:

[0069] cells prepared by an otherwise similar method except that step (iii) is performed 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

[0070] Cells prepared by an otherwise similar method except further comprising expanding the cell (eg, T cell) population in vitro for more than 3 days, eg, 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii).

[0071] In some embodiments, the cell population from step (iii) secretes IL-2 at a higher level (e.g., at least 2, 4, 6, 8, 10, 12, or 14 times higher) after incubation with cells expressing the antigen recognized by the CAR, compared to cells prepared by an otherwise 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 compared to cells prepared by an otherwise similar method except that the cell (e.g., T cell) population is expanded in vitro for more than 3 days (e.g., 5, 6, 7, 8, or 9 days) after step (ii) and before step (iii).

[0072] In some embodiments, the cell population from step (iii) persists for a longer period of time or expands at a higher level after in vivo administration to a subject compared to cells prepared by an otherwise similar method except that step (iii) is performed more than 26 hours after initiation of step (i) (e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after initiation of step (i)), or compared to cells prepared by an otherwise similar method except that step (iii) is performed more than 26 hours after initiation of step (i) (e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after initiation of step (i)), or compared to cells prepared by an otherwise similar method except that step (iii) is performed more than 3 days (e.g., 5, 6, 7, 8, or 9 days) after step (ii) and before step (iii).

[0073] In some embodiments, the cell population from step (iii) exhibits greater activity (e.g., greater activity at a low dose, e.g., no more than 0.15 x 10 T cells) after in vivo administration to a subject compared to cells prepared by an otherwise similar method except that step (iii) is performed more than 26 hours after initiation of step (i) (e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after initiation of step (i)), or compared to cells prepared by an otherwise similar method except that step (iii) is performed more than 26 hours after initiation of step (i) (e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after initiation of step (i)). 6 , 0.2 x 10 6 , 0.25 x 106 , or 0.3 x 10 6 The dose of viable cells expressing CAR is used.

[0074] In some embodiments, the cell population 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 cell population at the start of step (i), e.g., as assessed by viable cell number, optionally wherein the number of viable cells in the cell population from step (iii) is reduced compared to the number of viable cells in the cell population at the start of step (i).

[0075] In some embodiments, the cell population from step (iii) does not expand or expands for less than 2 hours, eg, less than 1 or 1.5 hours, compared to the cell population at the beginning of step (i).

[0076] In some embodiments, steps (i) and / or (ii) are performed 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), a LSD1 inhibitor, a MALT1 inhibitor, or a combination thereof.

[0077] In some embodiments, steps (i) and / or (ii) are performed in a serum-free cell culture medium containing a serum replacement. In some embodiments, the serum replacement is CTS TM Immune cell serum replacement (ICSR).

[0078] In some embodiments, the method further comprises, before step (i):

[0079] (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 removal (e.g., a fresh product from a thymectomy)) from an entity, such as a laboratory, hospital, or healthcare provider, and

[0080] (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 ablation (e.g., a fresh product from a thymectomy)), optionally wherein:

[0081] step (iii) is performed no later than 35 hours after the start of step (v), for example no later than 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the start of step (v), for example no later than 30 hours after the start of step (v), or

[0082] For example, the cell population 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 cell population at the end of step (v), as assessed by viable cell number.

[0083] 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 cryopreserved T cells isolated from whole blood, bone marrow, or an organ biopsy or removal (e.g., thymectomy)) from an entity, such as a laboratory, hospital, or healthcare provider.

[0084] In some embodiments, the method further comprises, before step (i):

[0085] (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 removal (e.g., a cryopreserved product from a thymectomy)) from an entity, such as a laboratory, hospital, or healthcare provider, and

[0086] (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, e.g., a cryopreserved whole blood product, a cryopreserved bone marrow product, or a cryopreserved organ biopsy or ablation (e.g., a cryopreserved product from a thymectomy)), optionally wherein:

[0087] step (iii) is performed no later than 35 hours after the start of step (v), for example no later than 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the start of step (v), for example no later than 30 hours after the start of step (v), or

[0088] For example, the cell population 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 cell population at the end of step (v), as assessed by viable cell number.

[0089] In some embodiments, the method further comprises step (vi):

[0090] A portion of the cell population from step (iii) is cultured 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 the level of CAR expression in the portion is measured (e.g., the percentage of viable cells in the portion that express CAR is measured), optionally wherein:

[0091] Step (iii) comprises harvesting and freezing the cell (e.g., T cell) population, and step (vi) comprises thawing a portion of the cell population 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, 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 cells expressing CAR in the portion).

[0092] In some embodiments, the cell population at the start of step (i) or step (1) is enriched for cells expressing IL6R (e.g., cells positive for IL6Rα and / or IL6Rβ). In some embodiments, the cell population at the start of step (i) or step (1) comprises no less than 50%, 60%, or 70% of cells expressing IL6R (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 comprising IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)). In some embodiments, IL-15 increases the ability of the cell population to expand, e.g., after 10, 15, 20, or 25 days. In some embodiments, IL-15 increases the percentage of cells expressing IL6Rβ in the cell population.

[0094] In one aspect, the disclosure provides methods of treating a subject having an autoimmune disease or disorder, 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 significant lung involvement (e.g., for autologous HSCT)), 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 Sjögren's disease, syndrome, severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-related diseases (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-related diseases), NMDAR+ encephalitis, or antibody-related neurological paraneoplastic diseases), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis, the method comprising administering to the subject a cell population engineered to express a CD19 CAR ("CAR-expressing cell population"), the population comprising:

[0095] (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 cell population before being engineered to express a CAR;

[0096] (b) naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ T cells, that vary by about 5% to about 10%, e.g., as compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ cells in the same cell population before being engineered to express a CAR;

[0097] (c) an increase in the 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 cell population before being engineered to express the CAR, e.g., an increase of at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3 fold;

[0098] (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 cell population before being engineered to express a CAR;

[0099] (e) central memory cells (e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells) that vary within about 5% to about 10% as compared to the percentage of central memory cells (e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells) in the same cell population before being engineered to express a CAR;

[0100] (f) a decrease in the 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 cell population before being engineered to express a CAR, e.g., a decrease of at least 20%, 25%, 30%, 35%, 40%, 45%, or 50%;

[0101] (g) about the same percentage of stem cell memory T cells (e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells) as compared to the percentage of stem cell memory T cells (e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells) in the same cell population before being engineered to express a CAR;

[0102] (h) a change of about 5% to about 10% in the percentage of stem cell memory T cells (e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells) as compared to the percentage of stem cell memory T cells (e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells) in the same cell population before being engineered to express a CAR; or

[0103] (i) increasing the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor beta+CCR7+CD62L+ T cells, as compared to the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor beta+CCR7+CD62L+ T cells, in the same cell population before being engineered to express the CAR.

[0104] In one aspect, the disclosure provides methods of treating a subject having an autoimmune disease or disorder, 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 significant lung involvement (e.g., for autologous HSCT)), 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 Sjögren's disease, syndrome, severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-related diseases (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-related diseases), NMDAR+ encephalitis, or antibody-related neurological paraneoplastic diseases), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis, the method comprising administering to the subject a cell population engineered to express a CD19 CAR ("CAR-expressing cell population"), wherein:

[0105] (a) the median gene set score (TEM up vs. TSCM down) of the cell population is about the same as or differs by no more than (e.g., increases by no more than) about 25%, 50%, 75%, 100%, or 125% from the median gene set score (TEM up vs. TSCM down) of the same cell population before being engineered to express a CAR;

[0106] (b) the median gene set score (Treg up vs. Teff down) of the cell population is about the same as or differs by no more than (e.g., increases by no more than) about 25%, 50%, 100%, 150%, or 200% from the median gene set score (Treg up vs. Teff down) of the cell population before being engineered to express the CAR;

[0107] (c) the median gene set score (downward stemness) of the cell population is about the same as or differs by no more than (e.g., increases by no more than) about 25%, 50%, 100%, 150%, 200%, or 250% from the median gene set score (downward stemness) of the cell population before being engineered to express the CAR;

[0108] (d) the median gene set score (upward hypoxia) of the cell population is about the same as or differs by no more than (e.g., increases by no more than) about 125%, 150%, 175%, or 200% from the median gene set score (upward hypoxia) of the cell population before being engineered to express the CAR; or

[0109] (e) the median gene set score (autophagy up) of the cell population is approximately the same as or differs by no more than (e.g., increases by no more than) about 180%, 190%, 200%, or 210% from the median gene set score (autophagy up) of the cell population before being engineered to express the CAR.

[0110] In one aspect, the disclosure provides methods of treating a subject having an autoimmune disease or disorder, 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 significant pulmonary involvement (e.g., for autologous HSCT)), 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 Sjögren's syndrome, Severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-related diseases (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-related diseases), NMDAR+ encephalitis, or antibody-related neurological paraneoplastic diseases), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis, the method comprising administering rapcabtagene autoleucel to the subject.

[0111] In one aspect, the present disclosure provides a method of treating a subject having a severe refractory autoimmune disease, the method comprising administering radakinumab to the subject.

[0112] In some embodiments, the severe refractory autoimmune disease is selected from systemic lupus erythematosus, lupus nephritis, idiopathic inflammatory myopathy, systemic sclerosis, and ANCA-associated vasculitis.

[0113] In some embodiments, the lupus is systemic lupus erythematosus. In some embodiments, the SLE is severe refractory SLE (srSLE).

[0114] In some embodiments, the CD19 CAR comprises a CD19 binding domain, a transmembrane domain, and an intracellular signaling domain.

[0115] In some embodiments:

[0116] (a) the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of 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,

[0117] (b) the transmembrane domain comprises the transmembrane domain of CD8,

[0118] (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

[0119] (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 thereto.

[0120] In one aspect, the present disclosure provides a method of treating a subject with severe refractory systemic lupus erythematosus (srSLE), the method comprising administering to the subject a cell population comprising a CD19 chimeric antigen receptor (CD19 CAR), or a cell population comprising a nucleic acid encoding the CD19 CAR, in an amount sufficient to treat the srSLE.

[0121] wherein the CAR comprises a CD19 binding domain, a transmembrane domain, and an intracellular signaling domain, and wherein the transmembrane domain comprises the transmembrane domain of a CD8 protein;

[0122] thereby treating the srSLE.

[0123] In some embodiments:

[0124] (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

[0125] (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 thereto.

[0126] In some embodiments, the population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is approximately 0.5 x 10 6 Up to 50x 10 6 viable cells expressing CAR, for example, about 5x10 6 The method further comprises administering to a patient a dose of 5 x 10 viable CAR-expressing cells, optionally wherein the CAR-expressing cells (e.g., CD19 CAR-expressing cells) are administered at a population of 5 x 10 6 A dose of viable cells expressing CAR is administered.

[0127] In some embodiments, the population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is approximately 2.5 x 10 6 to 2.5x 10 8 viable cells expressing CAR, for example, about 1.25 x 10 7 The method further comprises administering to a patient a dose of 1.25 x 10 viable CAR-expressing cells, optionally wherein the CAR-expressing cells (e.g., CD19 CAR-expressing cells) are administered at a dose of 1.25 x 10 7 A dose of viable cells expressing CAR is administered.

[0128] In some embodiments, the population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is approximately 1.25 x 10 7 to 1.25x 10 9 viable cells expressing CAR, for example, about 1.25 x 10 8 The method further comprises administering to a patient a dose of 1.25 x 10 viable CAR-expressing cells, optionally wherein the CAR-expressing cells (e.g., CD19 CAR-expressing cells) are administered at a dose of 1.25 x 10 8 A dose of viable cells expressing CAR is administered.

[0129] In some embodiments, the population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is approximately 2.5 x 10 6 to 2.5x 10 8 viable cells expressing CAR, for example, about 1x10 7 or 5x 10 7 A dose of viable cells expressing CAR is administered.

[0130] In one aspect, the present disclosure provides a method of treating a subject having severe refractory systemic lupus erythematosus (srSLE), the method comprising administering to the subject radakinumab in an amount sufficient to treat the srSLE.

[0131] thereby treating the srSLE.

[0132] In some embodiments, the amount of Reda Kiolan is about 0.5 x 10 6 Up to 50x 10 6 CAR-positive living cells, for example, about 5 x 10 6 CAR-positive living cells, optionally wherein radaxylamine is administered at a dose of 5 x 10 6 The dose of CAR-positive live cells was administered.

[0133] In some embodiments, the amount of Reda Kiolan is about 2.5 x 10 6 to 2.5x 10 8 CAR-positive live cells, for example, about 1.25 x 10 7 CAR-positive living cells, optionally wherein radaxylamine is administered at a dose of 1.25 x 10 7 The dose of CAR-positive viable cells was administered.

[0134] In some embodiments, the amount of Reda Kiolan is about 1.25 x 10 7 to 1.25x 10 9 CAR-positive live cells, for example, about 1.25 x 10 8 CAR-positive living cells, optionally wherein radaxylamine is administered at a dose of 1.25 x 10 8 The dose of CAR-positive viable cells was administered.

[0135] In some embodiments, the amount of Reda Kiolan is about 2.5 x 10 6 to 2.5x 10 8 CAR-positive living cells, for example, about 1 x 10 7 or 5x 10 7 The dose of CAR-positive live cells was administered.

[0136] In one aspect, the present 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), the method comprising administering to the subject a population of cells expressing a CD19 chimeric antigen receptor (CD19 CAR) or comprising a nucleic acid configured to express a CD19 CAR, wherein the cells are administered at a rate of 0.5–50 x 10 6viable CAR+ T cells (e.g., 5-12.5 x 10 6 The dose of CAR+ T cells (viable) was administered.

[0137] In one aspect, the present 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 radaxylamine, wherein radaxylamine is administered at a dose of 0.5-50 x 10 6 CAR+ T cells (e.g., 5-12.5x10 6 The dose of CAR+ T cells (viable) was administered.

[0138] In some embodiments, the lupus is systemic lupus erythematosus. In some embodiments, the SLE is severe refractory SLE (srSLE), wherein optionally the subject has renal involvement.

[0139] In some embodiments, the CAR comprises a CD19 binding domain, a transmembrane domain, and an intracellular signaling domain.

[0140] In some embodiments:

[0141] (a) the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of 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,

[0142] (b) the transmembrane domain comprises the transmembrane domain of CD8,

[0143] (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

[0144] (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 thereto.

[0145] In some embodiments, the CD19 binding domain comprises a heavy chain complementarity determining region 1 (HC CDR1), HC CDR2, HC CDR3, a light chain complementarity determining region 1 (LC CDR1), LC CDR2, and LC CDR3, wherein:

[0146] (a) the HC CDR1 comprises the amino acid sequence of SEQ ID NO: 295;

[0147] (b) the HC CDR2 comprises the amino acid sequence of SEQ ID NO: 296;

[0148] (c) the HC CDR3 comprises the amino acid sequence of SEQ ID NO: 297;

[0149] (d) the LC CDR1 comprises the amino acid sequence of SEQ ID NO: 298;

[0150] (e) the LC CDR2 comprises the amino acid sequence of SEQ ID NO: 299; and

[0151] (f) The LC CDR3 comprises the amino acid sequence of SEQ ID NO: 300.

[0152] In some embodiments, the CD19 binding domain comprises a VH and a VL, wherein the VH and VL are connected by a linker, optionally wherein the linker comprises the amino acid sequence of SEQ ID NO: 63 or 104.

[0153] In some embodiments, the CD 19 binding domain is connected to the transmembrane domain by a hinge region, optionally wherein:

[0154] (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

[0155] (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 thereto.

[0156] In some embodiments, the intracellular signaling domain comprises a primary signaling domain, optionally wherein the primary signaling domain comprises a functional signaling domain derived from CD3 zeta, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (ICOS), Fc epsilon RI, DAP10, DAP12, or CD66d, optionally wherein:

[0157] (a) the primary signaling domain comprises a functional signaling domain derived from CD3ζ,

[0158] (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 thereto, or

[0159] (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 thereto.

[0160] In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain, optionally wherein the costimulatory signaling domain comprises a protein derived from 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, 4-1BB (CD137), B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LI GHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, I TGA4, 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, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM 1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, C D100 (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 functional signaling domain of a ligand that specifically binds to CD83, optionally wherein:

[0161] (a) the costimulatory signaling domain comprises a functional signaling domain derived from 4-1BB,

[0162] (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

[0163] (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 thereto.

[0164] In some embodiments, the intracellular signaling domain comprises a functional signaling domain derived from 4-1BB and a functional signaling domain derived from CD3 zeta, optionally wherein 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), optionally wherein 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.

[0165] In some embodiments, the CAR further comprises a leader sequence comprising the amino acid sequence of SEQ ID NO: 1.

[0166] In some embodiments, the CD19 CAR comprises the amino acid sequence of SEQ ID NO: 301, or a sequence at least 80%, 85%, 90%, 95%, or 99% identical thereto.

[0167] 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.

[0168] In some embodiments, the subject has been previously treated with one or more of the following, or is concurrently treated with one or more of the following: an antimalarial (e.g., hydroxychloroquine or quinacrine), a glucocorticoid (e.g., prednisone), a calcineurin inhibitor, an immunomodulator (e.g., methotrexate, azathioprine, mycophenolate mofetil, cyclophosphamide, or tacrolimus), a biologic (e.g., belimumab, rituximab, a disease-modifying antirheumatic drug (DMARD) (e.g., leflunomide).

[0169] In some embodiments, the subject has been identified as non-responsive to treatment including two or more immunosuppressive therapies (eg, mycophenolate mofetil or cyclophosphamide in combination with a glucocorticoid) and a biologic agent.

[0170] In some embodiments, the subject has not previously received therapy comprising a CD19 CAR (e.g., radaxylex), adoptive T cell therapy, or a gene therapy product.

[0171] In some embodiments, the subject receives lymphocyte depletion therapy prior to administration of the CD19 CAR (e.g., radaxylamine).

[0172] In some embodiments, the subject receives lymphocyte depletion therapy about two weeks prior to administration of the CD19 CAR (e.g., radaxylamine).

[0173] In some embodiments, the lymphocyte depletion therapy comprises fludarabine (e.g., 25 mg / m 2 IV, three times a day) and cyclophosphamide (eg, 250 mg / m 2 IV, three doses per day).

[0174] In some embodiments, the method further comprises administering to the subject a second therapeutic agent.

[0175] In some embodiments, the second therapeutic agent is administered before, simultaneously with, or after administration of the CAR-expressing cell population or radaxapolone.

[0176] 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, for about 3 days.

[0177] In some embodiments, (i) before administration of corticosteroids and / or (ii) when the absolute T cell count is ≥300 / mm 3 Leukocyte apheresis was performed.

[0178] In one aspect, the present disclosure provides a method for preparing a population of cells (e.g., T cells) expressing a chimeric antigen receptor (CAR), the method comprising:

[0179] (i) contacting (e.g., binding) a cell population (e.g., T cells, e.g., T cells isolated from frozen or fresh leukapheresis product) with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a co-stimulatory molecule on the surface of the cell, wherein the cell population is from a subject having an autoimmune disease or disorder, 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 significant lung involvement (e.g., for autologous HSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis), or a combination thereof. , cancer-related myositis (e.g., antisynthetase syndrome with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjögren's syndrome, severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-related 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-related disease), NMDAR+ encephalitis, or antibody-related neurological paraneoplastic diseases), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis;

[0180] (ii) contacting the cell (e.g., T cell) population with a nucleic acid molecule (e.g., DNA or RNA molecule) encoding the CAR, thereby providing a cell (e.g., T cell) population comprising the nucleic acid molecule, wherein optionally the CAR comprises a CD19 antigen binding domain; and

[0181] (iii) harvesting the cell (e.g., T cell) population for storage (e.g., reconstituting the cell population in a cryopreservation medium) or administration, wherein:

[0182] (a) step (ii) is performed together with step (i) or not later than 20 hours after the start of step (i), for example not later than 12, 13, 14, 15, 16, 17, or 18 hours after the start of step (i), for example not later than 18 hours after the start of step (i), and

[0183] step (iii) is performed no later than 30 (e.g., 26) hours after the start of step (i), for example no later than 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the start of step (i), for example no later than 24 hours after the start of step (i),

[0184] (b) step (ii) is performed together with step (i) or not later than 20 hours after the start of step (i), for example not later than 12, 13, 14, 15, 16, 17, or 18 hours after the start of step (i), for example not later than 18 hours after the start of step (i), and

[0185] step (iii) is performed no later than 30 hours after the start of step (ii), for example no later than 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the start of step (ii), or

[0186] (c) the cell population from step (iii) does not expand, or expands by no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40%, e.g., by no more than 10%, compared to the cell population at the start of step (i), e.g., as assessed by viable cell number,

[0187] Optionally, the nucleic acid molecule in step (ii) is on a viral vector, optionally, the nucleic acid molecule in step (ii) is an RNA molecule on a viral vector, optionally, step (ii) comprises transducing the cell (e.g., T cell) population with a viral vector comprising a nucleic acid molecule encoding the CAR.

[0188] In some embodiments, the agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3 (e.g., an anti-CD3 antibody), and wherein 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 wherein the agent that stimulates the CD3 / TCR complex or the agent that stimulates a costimulatory molecule 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 a scFv), a small molecule, or a ligand (e.g., a naturally occurring ligand). wherein the agent that stimulates the CD3 / TCR complex or the agent that stimulates the co-stimulatory 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 the co-stimulatory molecule comprises an anti-CD28 antibody; optionally wherein the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody covalently attached to a colloidal polymer nanomatrix and the agent that stimulates the co-stimulatory molecule comprises an anti-CD28 antibody covalently attached to a colloidal polymer nanomatrix; optionally wherein the agent that stimulates the CD3 / TCR complex and the agent that stimulates the co-stimulatory molecule comprise T cell TransAct TM .

[0189] In some embodiments, step (i) increases the percentage of CAR-expressing cells in the cell population from step (iii), compared to cells prepared by an otherwise similar method except that step (i) is not included, for example, the cell population from step (iii) shows a higher percentage of CAR-expressing cells (e.g., at least 10%, 20%, 30%, 40%, 50%, or 60% higher).

[0190] In some embodiments:

[0191] (a) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the cell population 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 cell population at the start of step (i);

[0192] (b) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the cell population 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 as compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells, in the cell population at the start of step (i);

[0193] (c) the percentage of naive T cells expressing CAR, e.g., CD45RA+CD45RO-CCR7+ T cells expressing CAR, in the cell population increases during the duration of step (ii), e.g., by at least 30%, 35%, 40%, 45%, 50%, 55%, or 60% between 18-24 hours after the start of step (ii); or

[0194] (d) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the cell population from step (iii) is not decreased, or is decreased by no more than 5% or 10%, as compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells, in the cell population at the start of step (i).

[0195] In some embodiments:

[0196] (a) 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 prepared by an otherwise similar method except that step (iii) is performed more than 26 hours after initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after initiation of step (i);

[0197] (b) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the cell population 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 prepared by an otherwise similar method except that step (iii) is performed more than 26 hours after initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after initiation of step (i);

[0198] (c) the percentage of naive T cells expressing CAR, e.g., CD45RA+CD45RO-CCR7+ T cells expressing CAR, in the cell population from step (iii) is higher (e.g., at least 4, 6, 8, 10, or 12 times higher) than the percentage of naive T cells expressing CAR, e.g., CD45RA+CD45RO-CCR7+ T cells expressing CAR, in cells prepared by an otherwise similar method except that step (iii) is performed more than 26 hours after initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after initiation of step (i);

[0199] (d) the cell population 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 prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii);

[0200] (e) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the cell population 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 prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii); or

[0201] (f) the percentage of naive T cells expressing CAR, e.g., CD45RA+CD45RO-CCR7+ T cells expressing CAR, in the cell population from step (iii) is higher (e.g., at least 4, 6, 8, 10, or 12 times higher) than the percentage of naive T cells expressing CAR, e.g., CD45RA+CD45RO-CCR7+ T cells expressing CAR, in cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii).

[0202] In some embodiments:

[0203] (a) the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the cell population 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 cell population at the beginning of step (i);

[0204] (b) the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the cell population from step (iii) is reduced by at least 20%, 25%, 30%, 35%, 40%, 45%, or 50% as compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells in the cell population at the start of step (i);

[0205] (c) the percentage of central memory T cells expressing the CAR, e.g., CCR7+CD45RO+ cells expressing the CAR, decreases during the duration of step (ii), e.g., by at least 8%, 10%, 12%, 14%, 16%, 18%, or 20% between 18 and 24 hours after the start of step (ii); or

[0206] (d) the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the cell population from step (iii) does not increase or increases by no more than 5% or 10% as compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells in the cell population at the beginning of step (i).

[0207] In some embodiments:

[0208] (a) the cell population 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 prepared by an otherwise similar method except that step (iii) is performed more than 26 hours after initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after initiation of step (i);

[0209] (b) the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the cell population 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 prepared by an otherwise similar method except that step (iii) is performed more than 26 hours after initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after initiation of step (i);

[0210] (c) the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+CD45RO+ T cells, in the cell population 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 prepared by an otherwise 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);

[0211] (d) the cell population 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 prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii);

[0212] (e) the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the cell population 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 prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii); or

[0213] (f) the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+CD45RO+ T cells, in the cell population from step (iii) is lower (e.g., at least 10%, 20%, 30%, or 40%) than the percentage of CAR-expressing central memory T cells, e.g., CAR-expressing CCR7+CD45RO+ T cells, in cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii).

[0214] In some embodiments:

[0215] (a) the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the cell population from step (iii) is increased as compared to the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the cell population at the start of step (i);

[0216] (b) the percentage of CAR-expressing stem cell memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the cell population from step (iii) is increased as compared to the percentage of CAR-expressing stem cell memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the cell population at the beginning of step (i);

[0217] (c) the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the cell population from step (iii) is higher than the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in cells prepared by an otherwise 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

[0218] (d) the percentage of CAR-expressing stem cell memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the cell population from step (iii) is higher than the percentage of CAR-expressing stem cell memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in cells prepared by an otherwise 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);

[0219] (e) the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the cell population from step (iii) is higher than the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii); or

[0220] (f) the percentage of CAR-expressing stem cell memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the cell population from step (iii) is higher than the percentage of CAR-expressing stem cell memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii).

[0221] In some embodiments:

[0222] (a) the median gene set score (TEM up vs. TSCM down) of the cell population from step (iii) is about the same as or differs by no more than (e.g., increases by no more than) about 25%, 50%, 75%, 100%, or 125% from the median gene set score (TEM up vs. TSCM down) of the cell population from the start of step (i);

[0223] (b) the median gene set score (TEM up vs. TSCM down) of the population of cells from step (iii) is lower (e.g., at least about 100%, 150%, 200%, 250%, or 300% lower) than the median gene set score (TEM up vs. TSCM down) of:

[0224] cells prepared by an otherwise similar method except that step (iii) is performed 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

[0225] Cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii);

[0226] (c) the median gene set score (Treg up vs. Teff down) of the cell population from step (iii) is about the same as or differs by no more than (e.g., increases by no more than) about 25%, 50%, 100%, 150%, or 200% from the median gene set score (Treg up vs. Teff down) of the cell population from the start of step (i);

[0227] (d) the median gene set score (Treg up vs. Teff down) of the cell population from step (iii) is lower (e.g., at least about 50%, 100%, 125%, 150%, or 175% lower) than the median gene set score (Treg up vs. Teff down) of:

[0228] cells prepared by an otherwise similar method except that step (iii) is performed 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

[0229] Cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii);

[0230] (e) the median gene set score (downward stemness) of the cell population from step (iii) is about the same as or differs by no more than (e.g., increases by no more than) about 25%, 50%, 100%, 150%, 200%, or 250% from the median gene set score (downward stemness) of the cell population from the start of step (i);

[0231] (f) the median gene set score (downward stemness) of the population of cells from step (iii) is lower (e.g., at least about 50%, 100%, or 125% lower) than the median gene set score (downward stemness) of:

[0232] cells prepared by an otherwise similar method except that step (iii) is performed 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

[0233] Cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii);

[0234] (g) the median gene set score (upward hypoxia) of the cell population from step (iii) is about the same as or differs by no more than (e.g., increases by no more than) about 125%, 150%, 175%, or 200% from the median gene set score (upward hypoxia) of the cell population from the start of step (i);

[0235] (h) the median gene set score (hypoxia up) of the population of cells from step (iii) is lower (e.g., at least about 40%, 50%, 60%, 70%, or 80% lower) than the median gene set score (hypoxia up) of:

[0236] cells prepared by an otherwise similar method except that step (iii) is performed 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

[0237] Cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii);

[0238] (j) the median gene set score (autophagy up) of the cell population from step (iii) is about the same as or differs by no more than (e.g., increases by no more than) about 180%, 190%, 200%, or 210% from the median gene set score (autophagy up) of the cell population from the start of step (i); or

[0239] (k) the median gene set score (autophagy up) of the population of cells from step (iii) is lower (e.g., at least about 20%, 30%, or 40% lower) than the median gene set score (autophagy up) of:

[0240] cells prepared by an otherwise similar method except that step (iii) is performed 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

[0241] Cells prepared by an otherwise similar method except further comprising expanding the cell (eg, T cell) population in vitro for more than 3 days, eg, 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii).

[0242] In some embodiments, the cell population from step (iii) secretes IL-2 at a higher level (e.g., at least 2, 4, 6, 8, 10, 12, or 14 times higher) after incubation with cells expressing the antigen recognized by the CAR, compared to cells prepared by an otherwise 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 compared to cells prepared by an otherwise similar method except that the cell (e.g., T cell) population is expanded in vitro for more than 3 days (e.g., 5, 6, 7, 8, or 9 days) after step (ii) and before step (iii).

[0243] In some embodiments, the cell population from step (iii) persists for a longer period of time or expands at a higher level after in vivo administration to a subject compared to cells prepared by an otherwise similar method except that step (iii) is performed more than 26 hours after initiation of step (i) (e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after initiation of step (i)), or compared to cells prepared by an otherwise similar method except that step (iii) is performed more than 26 hours after initiation of step (i) (e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after initiation of step (i)), or compared to cells prepared by an otherwise similar method except that step (iii) is performed more than 3 days (e.g., 5, 6, 7, 8, or 9 days) after step (ii) and before step (iii).

[0244] In some embodiments, the cell population from step (iii) exhibits greater activity (e.g., greater activity at a low dose, e.g., no more than 0.15 x 10 T cells) after in vivo administration to a subject compared to cells prepared by an otherwise similar method except that step (iii) is performed more than 26 hours after initiation of step (i) (e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after initiation of step (i)), or compared to cells prepared by an otherwise similar method except that step (iii) is performed more than 26 hours after initiation of step (i) (e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after initiation of step (i)). 6 , 0.2x 10 6 , 0.25x 106 , or 0.3x 10 6 The dose of viable cells expressing CAR is used.

[0245] In some embodiments, the cell population 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 cell population at the start of step (i), e.g., as assessed by viable cell number, optionally wherein the number of viable cells in the cell population from step (iii) is reduced compared to the number of viable cells in the cell population at the start of step (i).

[0246] In some embodiments, the cell population from step (iii) does not expand or expands for less than 2 hours, eg, less than 1 or 1.5 hours, compared to the cell population at the beginning of step (i).

[0247] In some embodiments, steps (i) and / or (ii) are performed 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), a LSD1 inhibitor, a MALT1 inhibitor, or a combination thereof.

[0248] In some embodiments, steps (i) and / or (ii) are performed in a serum-free cell culture medium containing a serum replacement. In some embodiments, the serum replacement is CTS TM Immune cell serum replacement (ICSR).

[0249] In some embodiments, the method further comprises, before step (i):

[0250] (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 removal (e.g., a fresh product from a thymectomy)) from an entity, such as a laboratory, hospital, or healthcare provider, and

[0251] (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 ablation (e.g., a fresh product from a thymectomy)), optionally wherein:

[0252] step (iii) is performed no later than 35 hours after the start of step (v), for example no later than 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the start of step (v), for example no later than 30 hours after the start of step (v), or

[0253] For example, the cell population 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 cell population at the end of step (v), as assessed by viable cell number.

[0254] 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 cryopreserved T cells isolated from whole blood, bone marrow, or an organ biopsy or removal (e.g., thymectomy)) from an entity, such as a laboratory, hospital, or healthcare provider.

[0255] In some embodiments, the method further comprises, before step (i):

[0256] (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 removal (e.g., a cryopreserved product from a thymectomy)) from an entity, such as a laboratory, hospital, or healthcare provider, and

[0257] (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, e.g., a cryopreserved whole blood product, a cryopreserved bone marrow product, or a cryopreserved organ biopsy or ablation (e.g., a cryopreserved product from a thymectomy)), optionally wherein:

[0258] step (iii) is performed no later than 35 hours after the start of step (v), for example no later than 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the start of step (v), for example no later than 30 hours after the start of step (v), or

[0259] For example, the cell population 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 cell population at the end of step (v), as assessed by viable cell number.

[0260] In some embodiments, the method further comprises step (vi):

[0261] A portion of the cell population from step (iii) is cultured 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 the level of CAR expression in the portion is measured (e.g., the percentage of viable cells in the portion that express CAR is measured), optionally wherein:

[0262] Step (iii) comprises harvesting and freezing the cell (e.g., T cell) population, and step (vi) comprises thawing a portion of the cell population 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, 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 cells expressing CAR in the portion).

[0263] In some embodiments, the cell population at the start of step (i) or step (1) is enriched for cells expressing IL6R (e.g., cells positive for IL6Rα and / or IL6Rβ). In some embodiments, the cell population at the start of step (i) or step (1) comprises no less than 50%, 60%, or 70% of cells expressing IL6R (e.g., cells positive for IL6Rα and / or IL6Rβ).

[0264] In some embodiments, steps (i) and (ii) or steps (1) and (2) are performed in a cell culture medium comprising IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)). In some embodiments, IL-15 increases the ability of the cell population to expand, e.g., after 10, 15, 20, or 25 days. In some embodiments, IL-15 increases the percentage of cells expressing IL6Rβ in the cell population.

[0265] In some embodiments, the lupus is systemic lupus erythematosus. In some embodiments, the SLE is severe refractory SLE (srSLE).

[0266] In some embodiments, the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

[0267] In some embodiments, the antigen binding domain binds to a lupus-associated B cell antigen (e.g., CD19).

[0268] In some embodiments, the antigen binding domain comprises a CDR, VH, VL, scFv, or CAR sequence disclosed herein.

[0269] In some embodiments, the antigen binding domain comprises a CD19 binding domain comprising a heavy chain complementarity determining region 1 (HC CDR1), HC CDR2, HC CDR3, a light chain complementarity determining region 1 (LC CDR1), LC CDR2, and LC CDR3, wherein:

[0270] (a) the HC CDR1 comprises the amino acid sequence of SEQ ID NO: 295;

[0271] (b) the HC CDR2 comprises the amino acid sequence of SEQ ID NO: 296;

[0272] (c) the HC CDR3 comprises the amino acid sequence of SEQ ID NO: 297;

[0273] (d) the LC CDR1 comprises the amino acid sequence of SEQ ID NO: 298;

[0274] (e) the LC CDR2 comprises the amino acid sequence of SEQ ID NO: 299; and

[0275] (f) The LC CDR3 comprises the amino acid sequence of SEQ ID NO: 300.

[0276] In some embodiments, the antigen binding domain comprises a VH and a VL, wherein the VH and VL are connected by a linker, optionally wherein the linker comprises the amino acid sequence of SEQ ID NO: 63 or 104.

[0277] In some embodiments:

[0278] (a) the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of 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,

[0279] (b) the transmembrane domain comprises the transmembrane domain of CD8,

[0280] (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

[0281] (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 thereto.

[0282] In some embodiments, the antigen binding domain is connected to the transmembrane domain by a hinge region, optionally wherein:

[0283] (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

[0284] (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 thereto.

[0285] In some embodiments, the intracellular signaling domain comprises a primary signaling domain, optionally wherein the primary signaling domain comprises a functional signaling domain derived from CD3 zeta, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (ICOS), Fc epsilon RI, DAP10, DAP12, or CD66d, optionally wherein:

[0286] (a) the primary signaling domain comprises a functional signaling domain derived from CD3ζ,

[0287] (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 thereto, or

[0288] (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 thereto.

[0289] In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain, optionally wherein the costimulatory signaling domain comprises a protein derived from 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, 4-1BB (CD137), B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LI GHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, I TGA4, 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, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM 1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, C D100 (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 functional signaling domain of a ligand that specifically binds to CD83, optionally wherein:

[0290] (a) the costimulatory signaling domain comprises a functional signaling domain derived from 4-1BB,

[0291] (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

[0292] (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 thereto.

[0293] In some embodiments, the intracellular signaling domain comprises a functional signaling domain derived from 4-1BB and a functional signaling domain derived from CD3 zeta, optionally wherein 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), optionally wherein 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.

[0294] In some embodiments, the CAR further comprises a leader sequence comprising the amino acid sequence of SEQ ID NO: 1.

[0295] In some embodiments, the CAR comprises a CD19 CAR comprising the amino acid sequence of SEQ ID NO: 301, or a sequence at least 80%, 85%, 90%, 95%, or 99% identical thereto.

[0296] 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.

[0297] In some embodiments, the subject has been previously treated with one or more of the following: an antimalarial (e.g., hydroxychloroquine or quinacrine), a glucocorticoid (e.g., prednisone), a calcineurin inhibitor, an immunomodulator (e.g., methotrexate, azathioprine, mycophenolate mofetil, cyclophosphamide, or tacrolimus), a biologic (e.g., belimumab, rituximab, a disease-modifying antirheumatic drug (DMARD) (e.g., leflunomide).

[0298] In some embodiments, the subject has been identified as non-responsive to treatment including two or more immunosuppressive therapies (eg, mycophenolate mofetil or cyclophosphamide in combination with a glucocorticoid) and a biologic agent.

[0299] In some embodiments, the subject has not previously received therapy comprising a CD19 CAR, adoptive T cell therapy, or a gene therapy product.

[0300] In some embodiments, (i) before administration of corticosteroids and / or (ii) when the absolute T cell count is ≥300 / mm 3 Leukocyte apheresis was performed.

[0301] 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) prepared by the methods described herein.

[0302] In some embodiments, the population comprises autoreactive B cells (e.g., autoreactive B cells that do not express CAR).

[0303] In one aspect, the present disclosure provides a pharmaceutical composition comprising a CAR-expressing cell population described herein and a pharmaceutically acceptable carrier.

[0304] In one aspect, the present disclosure provides a CAR-expressing cell population or a pharmaceutical composition comprising the CAR-expressing cell population 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 CAR-expressing cell population or an effective amount of the pharmaceutical composition.

[0305] In one aspect, the present 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:

[0306] A cell population expressing a CD19 chimeric antigen receptor (CD19 CAR) or comprising a nucleic acid configured to express a CD19 CAR, and

[0307] a second therapy selected from an antimalarial agent or a stable immunosuppressant,

[0308] wherein the second therapy and the CD19 CAR cells are present in the subject concurrently, e.g., wherein the second therapy is administered while the CD19 CAR cells are present in the subject.

[0309] In one aspect, the present 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:

[0310] Reda Kiolon Race, and

[0311] a second therapy selected from an antimalarial agent or a stable immunosuppressant,

[0312] wherein the second therapy and radaxolamine are present in the subject concurrently, e.g., wherein the second therapy is administered while radaxolamine is present in the subject.

[0313] In some aspects, the present disclosure provides radakinumab prepared from autologous cells of a subject having 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 significant lung involvement (e.g., for autologous HSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-related myositis, e.g., antisynthetase syndrome with ILD), vasculitis (e.g., AN CA-associated vasculitis), severe refractory Sjögren's syndrome, severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-related 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-related disease), NMDAR+ encephalitis, or antibody-related neurological paraneoplastic diseases), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis.

[0314] In one aspect, the present disclosure provides a pharmaceutical composition comprising radaxylex and a pharmaceutically acceptable carrier.

[0315] In one aspect, the present disclosure provides radaxolamine or a pharmaceutical composition comprising radaxolamine for use in a method of modulating an immune response in a subject having 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 significant lung involvement (e.g., for autologous HSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-related myositis, e.g., antisynthetase syndrome with ILD), vasculitis (e.g., ANCA-associated vasculitis), or a combination thereof. The method is for treating a patient with severe refractory Sjögren's syndrome, a severe refractory neuroimmune disease (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-related 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-related disease), NMDAR+ encephalitis, or antibody-related neurological paraneoplastic disease), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis, the method comprising administering to the subject an effective amount of a radaxylamine group or an effective amount of a pharmaceutical composition.

[0316] In one aspect, the present disclosure provides radaxacept or a pharmaceutical composition comprising radaxacept for use in a method of modulating an immune response in a subject having 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 radaxacept or an effective amount of the pharmaceutical composition.

[0317] Radakioren is for use in treating a subject 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 significant pulmonary involvement (e.g., for autologous HSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-related myositis, e.g., antisynthetase syndrome with ILD), vasculitis (e.g., ANCA-associated myositis), or inflammatory bowel disease. Severe refractory Sjögren's syndrome, severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-related diseases (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-related diseases), NMDAR+ encephalitis, or antibody-related neurological paraneoplastic diseases), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis.

[0318] Radix Axiopeptide for use in treating a subject having severe refractory systemic lupus erythematosus (srSLE), wherein Radix Axiopeptide is formulated for administration in an amount sufficient to treat srSLE.

[0319] 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), radaxolamine is formulated for use at a dose of 0.5–50 x 10 6 viable CAR+ T cells (e.g., 5-12.5 x 10 6 The dose of CAR+ T cells (viable) was administered.

[0320] Radakioren for use in 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), and a second therapy.

[0321] wherein the second therapy is selected from an antimalarial agent or a stable immunosuppressant, and

[0322] wherein the second therapy and radaxolamine are present in the subject concurrently, e.g., wherein the second therapy is administered while radaxolamine is present in the subject.

[0323] Although methods and materials similar or equivalent to those described herein can be used for practice of the present invention or test, suitable methods and materials are described below. All publications, patent applications, patents and other references (such as sequence database reference numbers) mentioned herein are incorporated by reference in their entirety. For example, all GenBank, Unigene and Entrez sequences (such as in any table herein) mentioned herein are incorporated by reference. When a gene or protein is with reference to multiple sequence accession numbers, all sequence variants are contained.

[0324] Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. Titles, subheadings, or numbered or lettered elements, such as (a), (b), (i), etc., are presented only for ease of reading. The use of titles, numbered, or lettered elements in this document does not require that the steps or elements be performed in alphabetical order, or that the steps or elements be discrete from one another. Other features, objects, and advantages of the present invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0325] Figure 1 Schematic diagram showing the clinical trial design for the open-label, multicenter, Phase 1 / 2 study designed to evaluate the safety, efficacy, and cell kinetics of ARM-CD19 CAR T cells in participants with severe, refractory autoimmune disorders. DETAILED DESCRIPTION

[0326] definition

[0327] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0328] The terms "a" and "an" refer to one or to more than one (ie, to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0329] When referring to a measurable value such as an amount, a time interval, and the like, the term "about" is intended to encompass 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 stated value, as such variations are appropriate for performing the disclosed methods.

[0330] The compositions and methods of the present invention encompass polypeptides and nucleic acids having a specified sequence, or a sequence substantially identical or similar thereto, such as a sequence having at least 85%, 90% or 95% identity or higher identity to a specified sequence. In the context of amino acid sequences, the term "substantially identical" is used herein to refer to a first amino acid sequence containing a sufficient or minimum number of amino acid residues that i) are identical to the aligned amino acid residues in the second amino acid sequence, or ii) are conservative substitutions of the aligned amino acid residues in the second amino acid sequence, so that the first amino acid sequence and the second amino acid sequence can have a common domain and / or common functional activity, such as an amino acid sequence containing a common domain with at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence (e.g., a sequence provided herein).

[0331] In the context of nucleotide sequences, the term "substantially identical" is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to the 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, such as a nucleotide sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence (e.g., a sequence provided herein).

[0332] The term "variant" refers to a polypeptide having an amino acid sequence substantially identical to a reference amino acid sequence, or encoded by a substantially identical nucleotide sequence. In some embodiments, the variant is a functional variant.

[0333] The term "functional variant" refers to a polypeptide having an amino acid sequence substantially identical to a reference amino acid sequence, or encoded by a nucleotide sequence substantially identical to a reference amino acid sequence, and capable of having one or more activities of the reference amino acid sequence.

[0334] The term cytokine (e.g., IL-2, IL-7, IL-15, IL-21, or IL-6) includes full-length, fragments, or variants, such as functional variants, of naturally occurring cytokines, including fragments and functional variants thereof having at least 10%, 30%, 50%, or 80% of the activity (e.g., naturally occurring cytokine immunomodulatory activity). In some embodiments, the cytokine has an amino acid sequence substantially identical to a naturally occurring cytokine (e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), or is encoded by a nucleotide sequence substantially identical to a naturally occurring nucleotide sequence encoding the cytokine (e.g., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity). In some embodiments, as described above and below, the cytokine further comprises a receptor domain, such as a cytokine receptor domain (eg, IL-15 / IL-15R).

[0335] The term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct comprising at least one extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as "intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule as defined below. In some embodiments, the domains in the CAR polypeptide construct are in the same polypeptide chain, e.g., comprising a chimeric fusion protein. In some embodiments, e.g., as provided in an RCAR as described herein, the domains in the CAR polypeptide construct are discontinuous with each other, e.g., in different polypeptide chains.

[0336] In some embodiments, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., a primary signaling domain of CD3-ζ). In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived 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, wherein the intracellular signaling domain comprises a functional signaling domain derived 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, wherein the intracellular signaling domain comprises a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain, wherein the intracellular signaling domain comprises two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain, wherein the intracellular signaling domain comprises 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, CAR comprises an optional leader sequence at the amino-terminus (N-terminus) of the CAR fusion protein. In some embodiments, CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cut from the antigen recognition domain (e.g., scFv) during cell processing and CAR localization to the cell membrane.

[0337] A CAR comprising an antigen binding domain (e.g., scFv (single domain antibody) or TCR (e.g., TCRα binding domain or TCRβ binding domain)) targeting a specific antigen X is also referred to as an XCAR (wherein X can be an antigen as described herein). For example, a CAR comprising an antigen binding domain targeting CD19 is referred to as a CD19 CAR. CAR can be expressed in any cell, for example, an immune effector cell (e.g., T cell or NK cell) as described herein.

[0338] The term "signaling domain" refers to a functional portion of a protein that acts by transmitting information within the cell to act as an effector, either by generating second messengers or by responding to such messengers, to regulate cellular activity via a defined signaling pathway.

[0339] As used herein, the term "antibody" 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. Antibodies can be tetramers of immunoglobulin molecules.

[0340] 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., an antigen-determining variable region of an intact antibody) sufficient to confer recognition and specific binding to a target (e.g., an antigen) on the antibody fragment. 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 (e.g., sdAb (VL or VH)), Camelid VHH domains, and multispecific molecules formed from antibody fragments such as bivalent fragments comprising two or more (e.g., two) Fab fragments linked by a disulfide bridge at the hinge region, or two or more (e.g., two) isolated CDRs or other epitope-binding fragments of an antibody linked. Antibody fragments can also be incorporated into single-domain antibodies, multibodies, 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 into scaffolds based on polypeptides such as fibronectin type III (Fn3) (see US Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0341] 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 chain and heavy chain variable regions are continuously connected by a short flexible polypeptide linker and can be expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise indicated, as used herein, scFv can have VL and VH variable regions in any order, for example, relative to the N-terminus and C-terminus of the polypeptide, and the scFv can comprise VL-linker-VH or can comprise VH-linker-VL. In some embodiments, scFv can comprise NH2-V L -Connector-V H -COOH or NH2-V H -Connector-V L The structure of -COOH.

[0342] As used herein, the term "complementarity determining region" or "CDR" refers to the sequence of amino acids that confer antigen specificity and binding affinity within the antibody variable region. For example, in general, there are three CDRs (such as HCDR1, HCDR2, and HCDR3) in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, and LCDR3) in each light chain variable region. The precise amino acid sequence boundaries of a given CDR can be determined using any of many well-known schemes, including those described by the following documents: Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Josiah" numbering scheme), or a combination thereof. In the combined Kabat and Joshua numbering scheme, in some embodiments, a CDR corresponds to an amino acid residue that is part of a Kabat CDR, a Joshua CDR, or both.

[0343] The portion of the CAR composition of the invention comprising an antibody or antibody fragment thereof can exist in a variety of forms, for example, wherein 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, for example, a human or humanized antibody (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 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 the CAR composition of the invention comprises an antibody fragment. In some embodiments, the CAR comprises an antibody fragment comprising a scFv.

[0344] As used herein, the term "binding domain" or "antibody molecule" (also referred to herein as "anti-target binding domain") refers to a protein comprising at least one immunoglobulin variable domain sequence, such as an immunoglobulin chain or a fragment thereof. The term "binding domain" or "antibody molecule" encompasses antibodies and antibody fragments. In some embodiments, the antibody molecule is a multispecific antibody molecule, for example, it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence in a plurality has binding specificity to a first epitope and a second immunoglobulin variable domain sequence in a plurality has binding specificity to a second epitope. In some embodiments, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibodies are specific for no more than two antigens. The bispecific antibody molecule is characterized in that a first immunoglobulin variable domain sequence having binding specificity to a first epitope and a second immunoglobulin variable domain sequence having binding specificity to a second epitope are provided.

[0345] The term "bispecific antibody" refers to a molecule that combines the antigen-binding sites of two antibodies in a single molecule. Therefore, a bispecific antibody can bind to two different antigens 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. As known to those skilled in the art, the forms of the bispecific antibodies of the present invention include, but are not limited to, diabodies, single-chain diabodies, Fab dimerization (Fab-Fab), Fab-scFv, and tandem antibodies.

[0346] The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in the naturally occurring conformation of an antibody molecule and generally determines the class to which the antibody belongs.

[0347] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in the naturally occurring conformation of antibody molecules. Kappa (kappa) and lambda (lambda) light chains refer to the two major antibody light chain isotypes.

[0348] The term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as, for example, antibodies expressed by phage or yeast expression systems. The term should also be interpreted as meaning an antibody produced by synthesizing DNA molecules encoding the antibody and DNA molecules expressing the antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology available and well known in the art.

[0349] The term "antigen" or "Ag" refers to a molecule that causes an immune response. An immune response can involve the production of antibodies or the activation of specific immunocompetent cells or both. Technicians will understand that virtually any macromolecule, including all proteins or peptides, can serve as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. Technicians will understand that any DNA comprising a nucleotide sequence or partial nucleotide sequence encoding a protein that causes an immune response will therefore encode an "antigen" (when the term is used herein). In addition, those skilled in the art will understand that antigens do not need to be encoded only by the full-length nucleotide sequence of a gene. It is apparent that the present invention includes but is not limited to using partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to encode polypeptides that cause a desired immune response. In addition, it will be understood that antigens do not need to be encoded by a "gene" at all. It is apparent that antigens can be synthesized or can be derived from a biological sample, or can be a macromolecule other than a polypeptide. Such biological samples can include but are not limited to tissue samples, tumor samples, cells, or fluids with other biological components.

[0350] The term "autologous" refers to any material originating from the same individual that is subsequently reintroduced into the individual.

[0351] 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 distinct to interact antigenically.

[0352] The term "xenogeneic" refers to a transplant that originates from an animal of a different species.

[0353] As used herein, the term "apheresis" refers to an art-recognized extracorporeal process by which blood is removed from a donor or patient and passed through a device that separates one or more selected specific components and returns the remainder to the donor's or patient's circulation (e.g., by transfusion). Thus, in the context of "apheresis sample," it refers to a sample obtained using apheresis.

[0354] As used herein, "lupus" refers to all types and manifestations of lupus. Manifestations of lupus include, but are not limited to, systemic lupus erythematosus (including severe, refractory SLE (srSLE); lupus nephritis; cutaneous manifestations (e.g., visible manifestations of cutaneous lupus erythematosus, e.g., skin lesions or rashes); CNS lupus; cardiovascular, pulmonary, hepatic, hematologic, gastrointestinal, and musculoskeletal manifestations; neonatal lupus erythematosus; juvenile systemic lupus erythematosus; drug-induced lupus erythematosus; antiphospholipid syndrome; and complement deficiency syndromes that lead to lupus manifestations.

[0355] "Derived from" (as the term is used herein) refers to a relationship between a first molecule and a second molecule. It generally refers to the structural similarity between the first molecule and the second molecule and does not imply or include a limitation on the process or source of the first molecule derived from the second molecule. For example, in the case of an intracellular signaling domain derived from a CD3 zeta molecule, the intracellular signaling domain retains sufficient CD3 zeta structure such that it has the desired function, i.e., the ability to generate a signal under appropriate conditions. It does not imply or include a limitation on a particular process for generating the intracellular signaling domain, e.g., it does not mean that in order to provide the intracellular signaling domain, one must start with the CD3 zeta sequence and delete unwanted sequences, or impose mutations, to arrive at the intracellular signaling domain.

[0356] The term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the 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 substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-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.

[0357] In the context of stimulation and / or costimulatory molecule stimulation, the term "stimulation" refers to a response induced by the binding of a stimulatory molecule (e.g., TCR / CD3 complex) and / or a costimulatory molecule with its cognate ligand (e.g., CD28 or 4-1BB), for example, a first or secondary response, thereby mediating a signal transduction event, such as, but not limited to, a signal transduction via a TCR / CD3 complex. Stimulation can mediate the expression of changes in certain molecules, and / or the reconstruction of cytoskeletal structure, etc.

[0358] The term "stimulatory molecule" refers to a molecule expressed by a T cell, which provides one or more primary cytoplasmic signaling sequences for regulating the primary activation of a TCR complex in a stimulating manner for at least some aspects of a T cell signaling pathway. In certain embodiments, the ITAM-containing domain in CAR reproduces the signal transduction of primary TCR independently of endogenous TCR complexes. In certain embodiments, the primary signal is triggered by the combination of, for example, TCR / CD3 complexes and the MHC molecules of load peptides, and it causes the mediation of T cell responses (including but not limited to proliferation, activation, differentiation, etc.). The primary cytoplasmic signaling sequence (also referred to as "primary signaling domain") that works in a stimulating manner can contain a signal transduction motif referred to as an activation motif based on immunoreceptor tyrosine or ITAM. Examples of primary cytoplasmic signaling sequences containing ITAM 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 referred to as "ICOS"), FcεRI and CD66d, DAP10 and DAP12. In the specific CARs of the present invention, the intracellular signaling domain in any one or more CARS of the present invention comprises an intracellular signaling sequence, such as the primary signaling sequence of CD3-ζ. The term "antigen presenting cell" or "APC" refers to immune system cells such as helper cells (e.g., B cells, dendritic cells, etc.) that display foreign antigens complexed with major histocompatibility complexes (MHC) on their surfaces. T cells can recognize these complexes using their T cell receptors (TCRs). APC processes antigens and presents them to T cells.

[0359] As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a molecule. In embodiments, the intracellular signaling domain transduces effector function signals and directs cells to perform specialized functions. Although the entire intracellular signaling domain can be employed, in many cases it is 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 can be used in place of the complete chain, as long as the truncated portion can transduce the effector function signal. Thus, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain that is sufficient to transduce the effector function signal.

[0360] Intracellular signaling domain produces the signal that promotes the immune effector function of the cell (for example, CART cell) containing CAR.The example of immune effector function, for example, in CART cell, includes cytolytic activity and auxiliary activity (including secretion of cytokines).

[0361] In some embodiments, the intracellular signaling domain may include a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules responsible for primary stimulation or antigen-dependent simulation. In some embodiments, the intracellular signaling domain may include a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals or antigen-independent stimulation. For example, in the case of CART, the primary intracellular signaling domain may include the cytoplasmic sequence of the T cell receptor, and the costimulatory intracellular signaling domain may include the cytoplasmic sequence from a co-receptor or a costimulatory molecule.

[0362] The primary intracellular signaling domain can include a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of primary cytoplasmic signaling sequences containing ITAM include, but are not limited to, those derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS"), FcεRI, CD66d, DAP10, and DAP12.

[0363] The term "ζ" or alternatively "ζ chain", "CD3-ζ" or "TCR-ζ" refers to CD247. Swiss-Prot accession number P20963 provides an exemplary human CD3 ζ amino acid sequence. "ζ stimulatory domain" or alternatively "CD3-ζ stimulatory domain" or "TCR-ζ stimulatory domain" refers to the stimulatory domain of CD3-ζ 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 ζ comprises residues 52 to 164 of GenBank accession number 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 "ζ stimulatory domain" or "CD3-ζ stimulatory domain" is the sequence provided in 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).

[0364] 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 required for an effective immune response. Costimulatory molecules include, but are not limited to, 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, 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, CD 8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD 103. ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C , TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD22 9), 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.

[0365] The costimulatory intracellular signaling domain refers to the intracellular portion of a costimulatory molecule.

[0366] The intracellular signaling domain may comprise the entire intracellular portion of the molecule from which it is derived, or the entire native intracellular signaling domain or a functional fragment thereof.

[0367] 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. "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, "4-1BB costimulatory domain" is a sequence provided by 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).

[0368] When the term is used in this article, "immune effector cell" refers to a cell that participates in an immune response (e.g., promotes immune effector responses). Examples of immune effector cells include T cells, such as 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.

[0369] As used herein, "immune effector function or immune effector response" refers to, for example, a function or response of an immune effector cell that enhances or promotes an immune attack on a target cell. For example, an immune effector function or response refers to a property of a T cell or NK cell that promotes killing of a target cell or inhibits its growth or proliferation. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

[0370] The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity (including secretion of cytokines).

[0371] The term "coding" refers to the intrinsic properties of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) as a template for synthesizing other polymers and macromolecules with a definite nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or definite amino acid sequence in a biological process, and the resulting biological properties. Therefore, if transcription and translation of the mRNA corresponding to a gene produce protein in a cell or other biological system, the gene, cDNA, or RNA encodes the protein. Coding strand (its nucleotide sequence is identical to the mRNA sequence and is generally provided in a sequence table) and non-coding strand (transcribed as a template for a gene or cDNA) can be referred to as coded protein or other products of the gene or cDNA.

[0372] Unless otherwise indicated, 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" may also include introns, to the extent that the nucleotide sequence encoding the protein may contain one or more introns in certain versions.

[0373] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material, or composition as described herein that is effective to achieve a specific biological result.

[0374] The term "endogenous" refers to any material derived from or produced within an organism, cell, tissue or system.

[0375] The term "exogenous" refers to any material introduced from or generated outside of an organism, cell, tissue, or system.

[0376] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence. In some embodiments, expression includes translation of mRNA introduced into the cell.

[0377] The term "transfer vector" refers to a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic compounds or amphiphilic compounds, plasmids, and viruses. Therefore, the term "transfer vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted as further including non-plasmids and non-viral compounds that promote the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral transfer vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, etc.

[0378] The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all expression vectors 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 a recombinant polynucleotide.

[0379] The term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver significant amounts of genetic information into the host cell's DNA, making them one of the most effective gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.

[0380] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, and particularly includes the self-inactivating lentiviral vector provided by Milone et al., Mol. Ther. [Molecular Therapy] 17(8): 1453–1464 (2009). Other examples of lentiviral vectors that can be used clinically include, but are not limited to, for example, those from Oxford BioMedica. Gene delivery technology, LENTIMAX from Lentigen TM Vector systems, etc. Non-clinical types of lentiviral vectors are also available and known to those skilled in the art.

[0381] The terms "homologous" or "identity" refer to the subunit sequence identity between two polymeric 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 the two molecules is occupied by the same monomeric subunit; for example, if a position in each of the two DNA molecules is occupied by adenine, then 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 the two sequences (e.g., five positions in a polymer that is ten subunits in length) are homologous, then the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are matched or homologous, then the two sequences are 90% homologous.

[0382] " humanized " form of non-human (such as mouse) antibody is chimeric immunoglobulin, immunoglobulin chain or its fragment (such as Fv, Fab, Fab ', F (ab ') 2 or other antigen binding subsequences of antibody), which contains the minimum sequence from non-human immunoglobulin. In most cases, humanized antibody and antibody fragment thereof are human immunoglobulins (receptor antibody or antibody fragment), in which the residues from the complementary determining region (CDR) of receptor are replaced by the residues of CDR from non-human species (donor antibody) (such as mouse, rat or rabbit with desired specificity, affinity and ability). In some cases, the Fv framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibody / antibody fragment can include residues that are neither found in the receptor antibody nor in the CDR or framework sequence imported. These modifications can further improve and optimize antibody or antibody fragment performance. Typically, a humanized antibody or antibody fragment thereof will comprise substantially all of at least one (typically two) variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or a significant portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment may also comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a constant region 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.

[0383] "Fully human" refers to an immunoglobulin, such as an antibody or antibody fragment, in which the entire molecule is of human origin or consists of the same amino acid sequence as the human form of the antibody or immunoglobulin.

[0384] The term "isolated" means altered or removed from its native state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its native 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).

[0385] In the context of the present invention, the following abbreviations for common nucleic acid bases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0386] The term "operably linked" or "transcriptional control" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence that results in the 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 into a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. DNA sequences that are operably linked can be contiguous to each other and, for example, in the case of linking two protein coding regions, be in the same reading frame.

[0387] The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, intratumoral or infusion techniques.

[0388] The term "nucleic acid", "nucleic acid molecule", "polynucleotide" or "polynucleotide molecule" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known natural nucleotide analogs that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. In some embodiments, "nucleic acid", "nucleic acid molecule", "polynucleotide" or "polynucleotide molecule" includes nucleotide / nucleoside derivatives or analogs. Unless otherwise specified, 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 sequences explicitly noted. In particular, 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)).

[0389] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds comprising amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit on the maximum number of amino acids that can constitute a protein or peptide sequence. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to short chains, such as those also commonly referred to in the art as peptides, oligopeptides, and oligomers; and also to longer chains, such as those commonly referred to in the art as proteins, of which there are many types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include naturally occurring peptides, recombinant peptides, or combinations thereof.

[0390] The term "promoter" refers to a DNA sequence recognized by the cellular synthetic machinery or introduced synthetic machinery required to initiate specific transcription of a polynucleotide sequence.

[0391] The term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, the sequence may be a core promoter sequence, and in other cases, the sequence may also contain enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may be, for example, a promoter / regulatory sequence that expresses the gene product in a tissue-specific manner.

[0392] The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0393] The term "inducible" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell essentially only when an inducer corresponding to the promoter is present in the cell.

[0394] 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 gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0395] As used herein, "B cell antigen" refers to an antigen associated with a B cell. Non-limiting examples of molecules associated with B cells 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.

[0396] As used herein, the term "CD19" refers to cluster of differentiation 19 protein. Human and mouse amino acid and nucleic acid sequences can be found in public databases, such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human CD19 can be found as UniProt / Swiss-Prot accession number P15391, and the nucleic acid sequence encoding human CD19 can be found as accession number NM_001178098. It is also an early marker of B cell progenitor cells. See, for example, Nicholson et al. Mol. Immun. [Molecular Immunology] 34 (16-17): 1157-1165 (1997). On the one hand, the antigen-binding portion of CART recognizes and binds to the antigen in the extracellular domain of CD19 protein. In one aspect, CD19 protein is expressed on autoreactive B cells. As used herein, "CD19" includes proteins containing mutations (e.g., point mutations, fragments, insertions, deletions and splice variants of full-length wild-type CD19).

[0397] As used in the context of scFv, the term "flexible polypeptide linker" or "linker" refers to a peptide linker composed of amino acid residues (such as glycine and / or serine) used alone or in combination to connect the variable heavy chain region and the variable light chain region 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 (SEQ ID NO: 41). For example, n=1, n=2, n=3, n=4, n=5 and n=6, n=7, n=8, n=9 and n=10. In some embodiments, the flexible polypeptide linker includes but is not limited to (Gly4Ser)4 (SEQ ID NO: 27) or (Gly4Ser)3 (SEQ ID NO: 28). In some embodiments, the linker includes multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser) (SEQ ID NO: 25). Also included within the scope of the present invention are linkers described in WO 2012 / 138475, which is incorporated herein by reference.

[0398] As used herein, a 5' cap (also referred to as an RNA cap, an RNA 7-methylguanosine cap, or an RNA m7G cap) is a modified guanine nucleotide that is added to the "front" or 5' end of a eukaryotic messenger RNA shortly after transcription begins. The 5' cap consists of a terminal group attached to the first transcribed nucleotide. Its presence is crucial for being recognized by the ribosome and protected from RNases. Cap addition is coupled to transcription and occurs co-transcriptionally so that each affects the other. Shortly after transcription begins, the 5' end of the synthesized mRNA is bound by a cap synthesis complex associated with RNA polymerase. This enzyme complex catalyzes the chemical reactions required for mRNA capping. Synthesis is performed as a multi-step biochemical reaction. The capping portion can be modified to modulate the function of the mRNA, such as its stability or translation efficiency.

[0399] As used herein, "in vitro transcribed RNA" refers to RNA that has been synthesized in vitro. In some embodiments, the RNA is mRNA. Typically, the in vitro transcribed RNA is produced by an in vitro transcription vector. The in vitro transcription vector comprises a template for producing the in vitro transcribed RNA.

[0400] 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 chemically or enzymatically modified to modulate mRNA function, such as localization, stability, or translation efficiency.

[0401] As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylyl moiety or its modified variant to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. The 3' poly (A) tail is a long sequence of adenine nucleotides (usually hundreds) added to the pre-mRNA by the action of an enzyme (poly (A) polymerase). In higher eukaryotes, the poly (A) tail is added to a transcript containing a specific sequence (polyadenylation signal). The poly (A) tail and the proteins bound thereto help protect the mRNA from being degraded by exonucleases. Polyadenylation is also important for transcription termination, exporting mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after DNA is transcribed into RNA, but may also occur later in the cytoplasm. After transcription has terminated, the mRNA chain is cut 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 at the cleavage site.

[0402] As used herein, "transient" refers to expression of a non-integrated transgene for hours, days, or weeks, where the period of expression is less than the period of expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.

[0403] As used herein, the term "treatment (treat, treatment and treating)" refers to reducing or improving the progress, severity and / or duration of an autoimmune disorder, or improving one or more symptoms (preferably, one or more discernible symptoms) of an autoimmune disorder, which is caused by administering one or more therapies (e.g., one or more therapeutic agents, such as the CAR of the present invention). In a specific embodiment, the term "treatment (treat, treatment and treating)" refers to an improvement in at least one measurable physical parameter (e.g., the level of autoantibodies) of an autoimmune disorder, which is not necessarily discernible to the patient. In other embodiments, the term "treatment (treat, treatment and treating)" refers to physically, for example, by stabilizing discernible symptoms, or physiologically, or by both, suppressing the progress of an autoimmune disorder. The term "signal transduction pathway" refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transfer of signals from one part of a cell to another part of a cell. The phrase "cell surface receptor" includes molecules and molecular complexes that can receive signals and transmit signals across cell membranes.

[0404] The term "subject" is intended to include living organisms (eg, mammals, eg, humans) in which an immune response can be elicited.

[0405] 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 cases, a substantially purified cell population refers to a homogenous cell population. In other cases, the term refers only to cells that have been separated from cells with which they are naturally associated in their naturally occurring state. In some embodiments, the cells are cultured in vitro. In some embodiments, the cells are not cultured in vitro.

[0406] As used herein, the term "therapeutic agent" means to treat. A therapeutic effect is achieved by reducing, inhibiting, alleviating or eradicating a disease state.

[0407] As used herein, the term "prevention" means prophylactic or protective treatment against a disease or disease state.

[0408] The term "transfected" or "transformed" or "transduced" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. Cells include primary host cells and their progeny.

[0409] The term "specifically binds" refers to an antibody or ligand that recognizes and binds to a cognate binding partner (e.g., a stimulatory and / or co-stimulatory molecule present on T cells) protein present in a sample, but wherein the antibody or ligand does not substantially recognize or bind other molecules in the sample.

[0410] As the term is used herein, "membrane anchor" or "membrane tethering domain" refers to a polypeptide or moiety (eg, a myristoyl group) sufficient to anchor an extracellular or intracellular domain to the plasma membrane.

[0411] As used herein, "refractory" refers to an autoimmune disease or disorder that is unresponsive to treatment, such as 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. Refractory autoimmune diseases or disorders are also referred to as resistant autoimmune diseases or disorders.

[0412] As used herein, "severe refractory autoimmune disease" refers to manifestations of an autoimmune disease that has failed to respond (e.g., is still characterized by high disease activity) after at least one standard immunosuppressive therapy or at least one biologic agent. An example of a severe refractory autoimmune disease is severe refractory systemic lupus erythematosus.

[0413] As used herein, "severe refractory systemic lupus erythematosus" or "srSLE" refers to a manifestation of SLE that has failed to respond (e.g., is still characterized by high disease activity) after at least one standard immunosuppressive therapy (e.g., mycophenolate mofetil, cyclophosphamide), glucocorticoids, or at least one biologic agent. In some embodiments, srSLE comprises a manifestation of SLE that has failed to respond to a combination of two or more standard immunosuppressive therapies and glucocorticoids. In some embodiments, srSLE comprises a manifestation of SLE that has failed to respond to at least one biologic agent.

[0414] As used herein, "relapsed" or "recurrence" refers to the return or reappearance of a disease (e.g., an autoimmune disease or disorder) or the signs and symptoms of a disease (e.g., after a period of improvement or response, such as an autoimmune disease or disorder after previous treatment with a therapy (e.g., standard of care therapy). The initial period of response may involve a decrease in autoantibody cell levels below a certain threshold. Recurrence may involve a rise in autoantibody levels above a certain threshold.

[0415] Ranges: Throughout this disclosure, various embodiments of the present invention may be presented in range format. It should be understood that descriptions in range format are merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, descriptions of ranges should be considered to have all possible subranges specifically disclosed, as well as individual numerical values ​​within that range. For example, a description of a range such as from 1 to 6 should be considered to have subranges specifically disclosed, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numerical values ​​within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95%-99% identity includes ranges having 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.

[0416] As used herein, "combination" administration means delivering two (or more) different treatments to a subject during the course of their illness, e.g., after the subject has been diagnosed with a disorder and before the disorder is cured or cleared, or before treatment is terminated for other reasons. In some embodiments, delivery of the first treatment is still ongoing when delivery of the second treatment begins, so there is overlap in administration. This is sometimes referred to herein as "simultaneous delivery" or "parallel delivery." In other embodiments, delivery of one treatment ends before delivery of the other treatment begins. In some embodiments of each case, the treatment is more effective due to being administered in combination. For example, the second treatment is more effective than the results observed when the second treatment is administered in the absence of the first treatment, e.g., an equivalent effect is observed with less of the second treatment, or the second treatment reduces symptoms to a greater extent, or a similar effect is observed for the first treatment. In some embodiments, delivery results in a greater reduction in symptoms or other parameters related to the disorder than the results observed when one treatment is delivered in the absence of the other treatment. The effects of the two treatments may be partially additive, fully additive, or greater than additive. The delivery may be such that the effects of the first treatment delivered are still detectable when the second treatment is delivered.

[0417] The terms "depletion" and "depleting" are used interchangeably herein and refer to a decrease or reduction in the level or amount of cells, proteins, or macromolecules in a sample after a process such as a selection step (e.g., negative selection) is 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 was performed.

[0418] As used herein, "naive T cells" refer to T cells that are antigen-naive. In some embodiments, antigen-naive T cells are in the thymus and do not encounter their cognate antigens in the periphery. In some embodiments, naive T cells are precursors of memory cells. In some embodiments, naive T cells express CD45RA and CCR7, but do not express CD45RO. In some embodiments, naive T cells can be characterized by the 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-α, 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, flow cytometry is used to assess the surface expression level of markers.

[0419] The term "central memory T cells" refers to a subpopulation of T cells in humans that are CD45RO positive and express CCR7. In some embodiments, central memory T cells express CD95. In some embodiments, central memory T cells express IL-2R, IL-7R, and / or IL-15R. In some embodiments, central memory T cells express CD45RO, CD95, IL-2 receptor beta, CCR7, and CD62L. In some embodiments, flow cytometry is used to assess surface expression levels of markers.

[0420] The terms "stem memory T cells," "stem cell memory T cells," "stem cell-like memory T cells," "memory stem cell T cells," "T memory stem cells," "T stem cell memory cells," or "TSCM cells" refer to a subpopulation of memory T cells that have stem cell-like capabilities, e.g., the ability to self-renew and / or reconstitute memory and / or the multipotency of effector T cell subsets. In some embodiments, stem cell 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 cell memory T cells are disclosed in Gattinoni et al., Nat Med. 2017 Jan 06; 23(1): 18–27, which is incorporated herein by reference in its entirety.

[0421] For the sake of clarity, unless otherwise indicated, cells or cell colonies are classified as "not expressing" or having "absence" or being "negative" for a particular marker may not necessarily mean the absolute absence of the marker. Those skilled in the art can easily compare cells with positive and / or negative controls, and / or set a predetermined threshold, and when cells have an expression level lower than a predetermined threshold or cell colonies have a total expression level lower than a predetermined threshold using a conventional detection method (e.g., using flow cytometry, e.g., as described in Examples herein), cells or cell colonies are classified as not expressing or being negative for the marker. As used herein, the term "gene set score (TEM up vs. TSCM down)" for cells refers to a score reflecting the degree to which cells display effector memory T cell (TEM) phenotype vs. stem cell memory T cell (TSCM) phenotype. Higher gene set scores (TEM up vs. TSCM down) indicate that the TEM phenotype increases, while lower gene set scores (TEM up vs. TSCM down) indicate that the TSCM phenotype increases. In some embodiments, a gene set score is determined by measuring the expression of one or more genes that are upregulated in TEM cells and / or downregulated in TSCM cells (TEM up vs. TSCM down), e.g., one or more genes selected from the group consisting of: MXRA7, CLIC1, NAT13, TBC1D2B, GLCCI1, DUSP10, APOBEC3D, CACNB3, ANXA2P2, TPRG1, EOMES, MATK, ARHGAP10, ADAM8, 1A1, SLFN12L, SH2D2A, EIF2C4, CD58, MYO1F, RAB27B, ERN1, NPC1, NBEAL2, APOBEC3G, SYTL2, SLC4A4, PIK3AP1, PTGDR, MAF, PLEKHA5, ADRB2, PLXND1, GNAO1, THBS1, PPP2R2B, CYTH3, KLRF1, FLJ16686, AUTS2, PTPRM, GNLY, and GFPT2. In some embodiments, the gene set score (upward TEM vs. downward TSCM) for each cell is determined using RNA-seq (e.g., single-cell RNA-seq (scRNA-seq)), e.g., as illustrated in Example 10 of WO / 2020 / 047452 in conjunction with Figure 39A, which is hereby incorporated by reference in its entirety. In some embodiments, a gene set score is calculated by taking the mean log-normalized gene expression value of all genes in the gene set (TEM up vs. TSCM down).

[0422] As used herein, the term "gene set score (up Treg vs. down Teff)" of a cell refers to a score reflecting the extent to which a cell displays a regulatory T cell (Treg) phenotype versus an effector T cell (Teff) phenotype. A higher gene set score (up Treg vs. down Teff) indicates an increase in the Treg phenotype, while a lower gene set score (up Treg vs. down Teff) indicates an increase in the Teff phenotype.In some embodiments, a gene set score is determined by measuring the expression of one or more genes that are upregulated in Treg cells and / or downregulated in Teff cells (Treg up vs. Teff down), e.g., one or more genes selected from the group consisting of: C12orf75, SELPLG, SWAP70, RGS1, PRR11, SPATS2L, SPATS2L, TSHR, C14orf145, CASP8, SYT11, ACTN4, ANXA5, GLRX, HLA-DMB, PMCH, RAB11FIP1, IL32, FAM160B1, SHMT2, FRMD4B, CCR3, TNFR SF13B, NTNG2, CLDND1, BARD1, FCER1G, TYMS, ATP1B1, GJB6, FGL2, TK1, SLC2A8, CDKN2A, SKAP2, GPR55, CDCA7, S100A4, GDPD5, PMAIP1, ACOT9, CEP55, S GMS1, ADPRH, AKAP2, HDAC9, IKZF4, CARD17, VAV3, OBFC2A, ITGB1, CIITA, SETD7, HLA-DMA, CCR10, KIAA0101, SLC14A1, PTTG3P, DUSP10, FAM164A, PYHIN 1. MYO1F, SLC1A4, MYBL2, PTTG1, RRM2, TP53INP1, CCR5, ST8SIA6, TOX, BFSP2, ITPRIPL1, NCAPH, HLA-DPB2, SYT4, NINJ2, FAM46C, CCR4, GBP5, C15orf5 3. LMCD1, MKI67, NUSAP1, PDE4A, E2F2, CD58, ARHGEF12, LOC100188949, FAS, HLA-DPB1, SELP, WEE1, HLA-DPA1, FCRL1, ICA1, CNTNAP1, OAS1, METTL7A, C CR6, HLA-DRB4, ANXA2P3, STAM, HLA-DQB2, 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, the gene set score (Treg up vs. Teff down) is determined using RNA-seq (e.g., single-cell RNA-seq (scRNA-seq)), for example, as illustrated in Example 10 of WO / 2020 / 047452 in conjunction with Figure 39B, which is hereby incorporated by reference in its entirety. In some embodiments, the gene set score (Treg up vs. Teff down) is calculated by taking the average log-normalized gene expression value of all genes in the gene set.

[0423] As used herein, the "gene set score (stem cell properties)" of the term cell refers to a score reflecting the degree to which a cell displays a stem cell phenotype. A lower gene set score (stem cell properties) indicates that the stem cell phenotype increases. In certain embodiments, a gene set score (stem cell properties) is determined by measuring the expression of one or more genes that are up-regulated in differentiated stem cells and down-regulated in hematopoietic stem cells, for example, one or more genes selected from the group consisting of ACE, BATF, CDK6, CHD2, ERCC2, HOXB4, MEOX1, SFRP1, SP7, SRF, TAL1, and XRCC5. In certain embodiments, a gene set score (stem cell properties) is determined using RNA-seq (e.g., single cell RNA-seq (scRNA-seq)), for example, as illustrated in Example 10 of WO / 2020 / 047452 in conjunction with Figure 39 C, which is hereby incorporated by reference in its entirety. In certain embodiments, a gene set score (stem cell properties) is calculated by obtaining the average log-normalized gene expression value of all genes in a gene set.

[0424] As used herein, the term "gene set score (hypoxia up)" for a cell refers to a score that reflects the extent to which a cell displays a high hypoxia phenotype. A higher gene set score (hypoxia up) indicates an increased hypoxia phenotype. In some embodiments, the gene set score (hypoxia up) is determined by measuring the expression of one or more genes upregulated in cells experiencing hypoxia, e.g., one or more genes selected from the group consisting of 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, BNIP3L, 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, ELL2, ENG, EN O1, ENO3, ENPEP, EPO, ERRFI1, ETS1, F3, FABP5, FGF3, FKBP4, FLT1, FN1, FOS, FTL, GAPDH, GBE1, GLRX, GPI, GPRC5A, HAP1, HBP1, HDAC1, HDAC9, HERC3, HERPUD1, HGF, HIF1A, HK1, HK2, HLA-DQB1, HMOX1, HMOX2, HSPA5, HSPD1, HSPH1, HYOU1, ICAM1, ID2, IFI27, IGF2, IGFBP1, IGFBP2, IGFB P3, IGFBP5, IL6, IL8, INSIG1, IRF6, ITGA5, JUN, KDR, KRT14, KRT18, KRT19, LDHA, LDHB, LEP, LGALS1, LONP1, LOX, LRP1, MAP4, MET, MIF, MMP13 , MMP2, MMP7, MPI, MT1L, MTL3P, ​​MUC1, MXI1, NDRG1, NFIL3, NFKB1, NFKB2, NOS1, NOS2, NOS2P1, NOS2P2, NOS3, NR3C1, NR4A1, NT5E, ODC1, P4HA1,P4HA2, PAICS, PDGFB, PDK3, PFKFB1, PFKFB3, PFKFB4, PFKL, PGAM1, PGF, PGK1, PGK2, PGM1, PIM1, PIM2, PKM2, PLAU, PLAUR, PLIN2, PLOD2, PNN, PNP, POLM, PPARA, P PAT, PROK1, PSMA3, PSMD9, PTGS1, PTGS2, QSOX1, RBPJ, RELA, RIOK3, RNASEL, RPL36A, RRP9, SAT1, SERPINB2, SERPINE1, SGSM2, SIAH2, SIN3A, SIRPA, SLC16A1, SLC 16A2, SLC20A1, SLC2A1, SLC2A3, SLC3A2, SLC6A10P, SLC6A16, SLC6A6, SLC6A8, SORL1, SPP1, SRSF6, SSSCA1, STC2, STRA13, SYT7, TBPL1, TCEAL1, TEK, TF, TFF3, TF RC, TGFA, TGFB1, TGFB3, TGFBI, TGM2, TH, THBS1, THBS2, TIMM17A, TNFAIP3, TP53, TPBG, TPD52, TPI1, TXN, TXNIP, UMPS, VEGFA, VEGFB, VEGFC, VIM, VPS11, and XRCC6. In some embodiments, the gene set score (upward hypoxia) is determined using RNA-seq (e.g., single-cell RNA-seq (scRNA-seq)), for example, as illustrated in Example 10 of WO / 2020 / 047452 in conjunction with FIG. 39D , which is hereby incorporated by reference in its entirety. In some embodiments, the gene set score (upward hypoxia) is calculated by taking the average log-normalized gene expression value of all genes in the gene set.

[0425] As used herein, the term "gene set score (autophagy up)" for a cell refers to a score that reflects the extent to which a cell displays an autophagic phenotype. A higher gene set score (autophagy up) indicates an increased autophagic phenotype. In some embodiments, the gene set score (autophagy up) is determined by measuring the expression of one or more genes upregulated in cells undergoing autophagy, e.g., one or more genes selected from the group consisting of ABL1, ACBD5, ACIN1, ACTRT1, ADAMTS7, AKR1E2, ALKBH5, ALPK1, AMBRA1, ANXA5, ANXA7, ARSB, ASB2, ATG10, ATG12, ATG13, ATG14, ATG16L1, ATG16L2, ATG2A, ATG2B, ATG3, ATG4A, ATG4B, ATG4C, ATG4D, ATG4E, ATG4F ... TG5, ATG7, ATG9A, ATG9B, ATP13A2, ATP1B1, ATPAF1-AS1, ATPIF1, BECN1, BECN1P1, BLOC1S1, BMP2KL, BNIP1, BNIP3, BOC, C11orf2, C11orf41, C 12orf44, C12orf5, C14orf133, C1orf210, C5, C6orf106, C7orf59, C7orf68, C8orf59, C9orf72, CA7, CALCB, CALCOCO2, CAPS, CCDC36, CD163L1 , CD93, CDC37, CDKN2A, CHAF1B, CHMP2A, CHMP2B, CHMP3, CHMP4A, CHMP4B, CHMP4C, CHMP6, CHST3, CISD2, CLDN7, CLEC16A, CLN3, CLVS1, COX8A, C PA3, CRNKL1, CSPG5, CTSA, CTSB, CTSD, CXCR7, DAP, DKKL1, DNAAF2, DPF3, DRAM1, DRAM2, DYNLL1, DYNLL2, DZANK1, EI24, EIF2S1, EPG5, EPM2A, F ABP1, FAM125A, FAM131B, FAM134B, FAM13B, FAM176A, FAM176B, FAM48A, FANCC, FANCF, FANCL, FBXO7, FCGR3B, FGF14, FGF7, FGFBP1, FIS1, FNBP 1L, 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、KIAA0226、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、MD H1、MEX3C、MFN1、MFN2、MLST8、MRPS10、MRPS2、MSTN、MTERFD1、MTMR14、MTMR 3、MTOR、MTSS1、MYH11、MYLK、MYOM1、NBR1、NDUFB9、NEFM、NHLRC1、NME2、NPC1 、NR2C2、NRBF2、NTHL1、NUP93、OBSCN、OPTN、P2RX5、PACS2、PARK2、PARK7、PD K1、PDK4、PEX13、PEX3、PFKP、PGK2、PHF23、PHYHIP、PI4K2A、PIK3C3、PIK3CA、 PIK3CB、PIK3R4、PINK1、PLEKHM1、PLOD2、PNPO、PPARGC1A、PPY、PRKAA1、PRK AA2、PRKAB1、PRKAB2、PRKAG1、PRKAG2、PRKAG3、PRKD2、PRKG1、PSEN1、PTPN22 、RAB12、RAB1A、RAB1B、RAB23、RAB24、RAB33B、RAB39、RAB7A、RB1CC1、RBM18 、REEP2、REP15、RFWD3、RGS19、RHEB、RIMS3、RNF185、RNF41、RPS27A、RPTOR、R RAGA、RRAGB、RRAGC、RRAGD、S100A8、S100A9、SCN1A、SERPINB10、SESN2、SFRP 4、SH3GLB1、SIRT2、SLC1A3、SLC1A4、SLC22A3、SLC25A19、SLC35B3、SLC35C1、SLC37A4, SLC6A1, SLCO1A2, SMURF1, SNAP29, SNAPIN, SNF8, SNRPB, SNRPB2, SNRPD1, SNRPF, SNTG1, SNX14, S PATA18, SQSTM1, SRPX, STAM, STAM2, STAT2, STBD1, STK11, STK32A, STOM, STX12, STX17, SUPT3H, TBC1D17, TB C1D25, TBC1D5, TCIRG1, TEAD4, TECPR1, TECPR2, TFEB, TM9SF1, TMBIM6, TMEM203, TMEM208, TMEM39A, TMEM3 9B, TMEM59, TMEM74, TMEM93, TNIK, TOLLIP, TOMM20, TOMM22, TOMM40, TOMM5, TOMM6, TOMM7, TOMM70A, TP53IN P1, TP53INP2, TRAPPC8, TREM1, TRIM17, TRIM5, TSG101, TXLNA, UBA52, UBB, UBC, UBQLN1, UBQLN2, UBQLN4, U LK1, ULK2, ULK3, USP10, USP13, USP30, UVRAG, VAMP7, VAMP8, VDAC1, VMP1, VPS11, VPS16, VPS18, VPS25, VPS2 8, VPS33A, VPS33B, VPS36, VPS37A, VPS37B, VPS37C, VPS37D, VPS39, VPS41, VPS4A, VPS4B, VTA1, VTI1A, VTI1B, WDFY3, WDR45, WDR45L, WIPI1, WIPI2, XBP1, YIPF1, ZCCHC17, ZFYVE1, ZKSCAN3, ZNF189, ZNF593, and ZNF681. In some embodiments, the gene set score (autophagy up) is determined using RNA-seq (e.g., single-cell RNA-seq (scRNA-seq)), e.g., as illustrated in Example 10 of WO / 2020 / 047452 in conjunction with Figure 39E, which is hereby incorporated by reference in its entirety. In some embodiments, the gene set score (autophagy up) is calculated by taking the average log-normalized gene expression value of all genes in the gene set.

[0426] As used herein, the term "gene set score (up resting vs. down activated)" for a cell refers to a score that reflects the extent to which a cell displays a resting T cell phenotype versus an activated T cell phenotype. A higher gene set score (up resting vs. down activated) indicates an increase in the resting T cell phenotype, while a lower gene set score (up resting vs. down activated) indicates an increase in the activated T cell phenotype. In some embodiments, the gene set score (up resting vs. down activated) is determined by measuring the expression of one or more genes that are upregulated in resting T cells and / or downregulated in activated T cells, e.g., one or more genes selected from the group consisting of ABCA7, ABCF3, ACAP2, AMT, ANKH, ATF7IP2, ATG14, ATP1A1, ATXN7, ATXN7L3B, BCL7A, BEX4, BSDC1, BTG1, BTG2, BTN3A1, C11orf21, C19orf22, C21orf2, CAMK2G, CA RS2, CCNL2, CD248, CD5, CD55, CEP164, CHKB, CLK1, CLK4, CTSL1, DBP, DCUN1D2, DENND1C, DGKD, DLG1, DUSP1, EAPP, ECE1, ECHDC2, ERB B2IP, FAM117A, FAM134B, FAM134C, FAM169A, FAM190B, FAU, FLJ10038, FOXJ2, FOXJ3, FOXL1, FOXO1, FXYD5, FYB, HLA-E, HSPA1L, HYAL 2. ICAM2, IFIT5, IFITM1, IKBKB, IQSEC1, IRS4, KIAA0664L3, KIAA0748, KLF3, KLF9, KRT18, LEF1, LINC00342, LIPA, LIPT1, LLGL2, LM BR1L, LPAR2, LTBP3, LYPD3, LZTFL1, MANBA, MAP2K6, MAP3K1, MARCH8, MAU2, MGEA5, MMP8, MPO, MSL1, MSL3, MYH3, MYLIP, NAGPA, NDST2 , NISCH, 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, RNF38, 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, SNPH, S P140L, SPATA6, SPG7, SREK1IP1, SRSF5, STAT5B, SVIL, SYF2, SYNJ2BP, TAF1C, TBC1D4, TCF20, TECTA, TES, TMEM127, TMEM159, TMEM30B, TMEM66, TME M8B, TP53TG1, TPCN1, TRIM22, TRIM44, TSC1, TSC22D1, TSC22D3, TSPYL2, TTC9, TTN, UBE2G2, USP33, USP34, VAMP1, VILL, VIPR1, VPS13C, ZBED5, ZBT B25, ZBTB40, ZC3H3, ZFP161, ZFP36L1, ZFP36L2, ZHX2, ZMYM5, ZNF136, ZNF148, ZNF318, ZNF350, ZNF512B, ZNF609, ZNF652, ZNF83, ZNF862, and ZNF91. In some embodiments, the gene set score (up quiescent vs. down activated) is determined using RNA-seq (e.g., single cell RNA-seq (scRNA-seq)), e.g., as illustrated in Example 10 of WO / 2020 / 047452 in conjunction with FIG. 38D , which is hereby incorporated by reference in its entirety. In some embodiments, the gene set score (up quiescent vs. down activated) is calculated by taking the average log-normalized gene expression value of all genes in the gene set.

[0427] As used herein, the term "gene set score (memory differentiation gradually increases)" for a cell refers to a score that reflects the stage of a cell in memory differentiation. A higher gene set score (memory differentiation gradually increases) indicates an increase in late memory T cell phenotype, while a lower gene set score (memory differentiation gradually increases) indicates an increase in early memory T cell phenotype. In some embodiments, the gene set score (upward autophagy) is determined by measuring the expression of one or more genes upregulated during memory differentiation, for example, one or more genes selected from the group consisting of MTCH2, RAB6C, KIAA0195, SETD2, C2orf24, NRD1, GNA13, COPA, SELT, TNIP1, CBFA2T2, LRP10, PRKCI, BRE, ANKS1A, PNPLA6, ARL6IP1, WDFY1, MAPK1, GPR153, SHKBP1, MAP1LC3B2, PIP4K2A, HC N3, GTPBP1, TLN1, C4orf34, KIF3B, TCIRG1, PPP3CA, ATG4D, TYMP, TRAF6, C17orf76, WIPF1, FAM108A1, MYL6, NRM, SPCS2, GGT3P, GALK1, CLIP4, ARL4C, YWHAQ, LPCAT4, ATG2A, IDS, TBC1D5, DMPK, ST6GALNAC6, REEP5, ABHD6, KIAA0247, EMB, TSEN54, SPIRE2, PIWIL4, ZSCAN22 , ICAM1, CHD9, LPIN2, SETD8, ZC3H12A, ULBP3, IL15RA, HLA-DQB2, LCP1, CHP, RUNX3, TMEM43, REEP4, MEF2D, ABL1, TMEM39A, PCBP4, PLCD 1. CHST12, RASGRP1, C1orf58, C11orf63, C6orf129, FHOD1, DKFZp434F142, PIK3CG, ITPR3, BTG3, C4orf50, CNNM3, IFI16, AK1, CDK2AP1 , REL, BCL2L1, MVD, TTC39C, PLEKHA2, FKBP11, EML4, FANCA, CDCA4, FUCA2, MFSD10, TBCD, CAPN2, IQGAP1, CHST11, PIK3R1, MYO5A, KIR2D L3, DLG3, MXD4, RALGDS, S1PR5, WSB2, CCR3, TIPARP, SP140, CD151, SOX13, KRTAP5-2, NF1, PEA15, PARP8, RNF166, UEVLD, LIMK1, CACNB1,TMX4、SLC6A6、LBA1、SV2A、LLGL2、IRF1、PPP2R5C、CD99、RAPGEF1、PPP4R1、O SBPL7、FOXP4、SLA2、TBC1D2B、ST7、JAZF1、GGA2、PI4K2A、CD68、LPGAT1、STX1 1、ZAK、FAM160B1、RORA、C8orf80、APOBEC3F、TGFBI、DNAJC1、GPR114、LRP8、 CD69、CMIP、NAT13、TGFB1、FLJ00049、ANTXR2、NR4A3、IL12RB1、NTNG2、RDX、M LLT4、GPRIN3、ADCY9、CD300A、SCD5、ABI3、PTPN22、LGALS1、SYTL3、BMPR1A、 TBK1、PMAIP1、RASGEF1A、GCNT1、GABARAPL1、STOM、CALHM2、ABCA2、PPP1R16B 、SYNE2、PAM、C12orf75、CLCF1、MXRA7、APOBEC3C、CLSTN3、ACOT9、HIP1、LAG 3、TNFAIP3、DCBLD1、KLF6、CACNB3、RNF19A、RAB27A、FADS3、DLG5、APOBEC3D、 TNFRSF1B、ACTN4、TBKBP1、ATXN1、ARAP2、ARHGEF12、FAM53B、MAN1A1、FAM38 A、PLXNC1、GRLF1、SRGN、HLA-DRB5、B4GALT5、WIPI1、PTPRJ、SLFN11、DUSP2、A NXA5、AHNAK、NEO1、CLIC1、EIF2C4、MAP3K5、IL2RB、PLEKHG1、MYO6、GTDC1、E DARADD、GALM、TARP、ADAM8、MSC、HNRPLL、SYT11、ATP2B4、NHSL2、MATK、ARHGA P18、SLFN12L、SPATS2L、RAB27B、PIK3R3、TP53INP1、MBOAT1、GYG1、KATNAL1 、FAM46C、ZC3HAV1L、ANXA2P2、CTNNA1、NPC1、C3AR1、CRIM1、SH2D2A、ERN1、YP EL1、TBX21、SLC1A4、FASLG、PHACTR2、GALNT3、ADRB2、PIK3AP1、TLR3、PLEKHA 5、DUSP10、GNAO1、PTGDR、FRMD4B、ANXA2、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, S T6GALNAC2, 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, the gene set score (memory differentiation gradually increases) is determined using RNA-seq, for example, as illustrated in Example 10 of WO / 2020 / 047452 in conjunction with Figure 40B, which is hereby incorporated by reference in its entirety. In some embodiments, the gene set score (memory differentiation gradually increases) is calculated by taking the average log-normalized gene expression value of all genes in the gene set.

[0428] As used herein, the term "gene set score (TEM up vs. TN down)" for a cell refers to a score reflecting the extent to which a cell displays an effector memory T cell (TEM) phenotype vs. a naive T cell (TN) phenotype. A higher gene set score (TEM up vs. TN down) indicates an increase in the TEM phenotype, while a lower gene set score (TEM up vs. TN down) indicates an increase in the TN phenotype.In some embodiments, a gene set score is determined by measuring the expression of one or more genes that are upregulated in TEM cells and / or downregulated in TN cells (TEM up vs. TN down), e.g., one or more genes selected from the group consisting of: 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, OSBPL3, 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, RNF1 9A, RAB27A, FADS3, ACTN4, TBKBP1, FAM53B, MAN1A1, FAM38A, GRLF1, B4GALT5, WIPI1, DUSP2, ANXA5, AHNAK, CLIC1, MAP3K5, ST8SIA1 , TARP, ADAM8, MATK, SLFN12L, PIK3R3, FAM46C, ANXA2P2, CTNNA1, NPC1, SH2D2A, ERN1, YPEL1, TBX21, STOM, PHACTR2, GBP5, ADRB2, PI 1, 2, 3, 4, 5, 7, 8, 9, 10, 15, 16, 18, 19, 21, 22, 23, 24, 27, 28, 36, 37, 38, 59, 70, 71, 72, 73, 74, 77, 78, 79, 81, 82, 83, 71, 72, 73, 74, 77, 79, 81, 82, 73, 74, 77, 79, 81, 82, 73, 74, 77, 79, 81, 82, 73, 74, 77, 79, 81, 82, 73, 74, 79, 81, 82, 73, 74, 79, 81, 82, 73, 74, 79, 81, 82In some embodiments, the gene set score is calculated by taking the mean log-normalized gene expression value of all genes in the gene set (TEM up vs. TN down).

[0429] In the context of a gene set score value (e.g., a median gene set score value), when the positive gene set score decreases by 100%, the value becomes 0. When the negative gene set score increases by 100%, the value becomes 0. For example, as disclosed in WO / 2020 / 047452, the median gene set score for the day 1 sample is -0.084; the median gene set score for the day 9 sample is 0.035; and the median gene set score for the input sample is -0.1. In Figure 39A of WO / 2020 / 047452, an increase of 100% in the median gene set score of the input sample results in a gene set score value of 0; and an increase of 200% in the median gene set score of the input sample results in a gene set score value of 0.1. In Figure 39A of WO / 2020 / 047452, a 100% decrease in the median gene set score for day 9 samples results in a gene set score value of 0; and a 200% decrease in the median gene set score for day 9 samples results in a gene set score value of -0.035.

[0430] As used herein, the term "bead" refers to a discrete particle having a solid surface, ranging in size from about 0.1 μm to several millimeters in diameter. Beads can be spherical (e.g., microspheres) or have an irregular shape. Beads can include a variety of materials, including but not limited to paramagnetic materials, ceramics, plastics, glass, polystyrene, methyl styrene, acrylic polymers, titanium, latex, Sepharose, TM , cellulose, nylon, etc. In some embodiments, the beads are relatively uniform, about 4.5 μm in diameter, spherical, superparamagnetic polystyrene beads, for example, coated with a mixture of anti-CD3 antibodies (e.g., CD3ε) and CD28, for example, covalently coupled. In some embodiments, the beads are In some embodiments, both anti-CD3 and anti-CD28 antibodies are coupled to the same beads, mimicking T cell stimulation by antigen presenting cells. Characteristics and Use for cell isolation and expansion is well known in the art, see, for example, Neurauter et al., Cell isolation and expansion using Dynabeads, Adv Biochem Eng Biotechnol. 2007; 106:41-73, which is incorporated herein by reference in its entirety.

[0431] The term "multispecific binding molecule" refers to a molecule that specifically binds to at least two antigens and comprises two or more antigen binding domains. The antigen binding domains can each independently be an antibody fragment (e.g., scFv, Fab, nanobody), a ligand, or a non-antibody derived binder (e.g., fibronectin, Fynomer, DARPin).

[0432] The term "monovalent" as used herein in the context of 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 in which there is a single antigen binding domain for each antigen of the multispecific binding molecule, antibody (e.g., bispecific antibody), or antibody fragment.

[0433] The term "bivalent" as used herein in the context of 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 in which there are two antigen binding domains for each antigen of the multispecific binding molecule, antibody (e.g., bispecific antibody), or antibody fragment.

[0434] The term "Fc silent" refers to a modified Fc domain that has minimal interaction with effector cells. Silent effector functions can be obtained by mutations in the Fc region of an antibody and have been described in the art, for example, but not limited to LALA and N297A (Strohl, W., 2009, Curr. Opin. Biotechnol. [Current Biotechnology Views] Vol. 20(6): 685-691); and D265A (Baudino et al., 2008, J. Immunol. [Journal of Immunology] 181: 6664-69). See also Heusser et al., WO 2012065950. Examples of Fc silent mutations include the LALA mutant comprising L234A and L235A mutations in the IgG1 Fc amino acid sequence, DAPA (D265A, P329A) (see, e.g., US 6,737,056), N297A, DANAPA (D265A, N297A and P329A) and / or LALADANAPS (L234A, L235A, D265A, N297A and P331S).

[0435] The term "CD3 / TCR complex" refers to a complex comprising a TCR on the surface of a T cell, wherein the TCR includes TCRα and TCRβ chains; CD3 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), P07766 (CD3ε) provide exemplary human sequences of these chains, except for the ζ chain responsible for intracellular signaling, which is discussed in further detail below. Further relevant accession numbers include A0A075B662 (murine TCRα, constant domain), A0A0A6YWV4 and / or A0A075B5J3 (murine TCRβ, constant domain 1), A0A075B5J4 (murine TCRβ, constant domain 2), P11942 (murine CD3γ), P04235 (murine CD3δ), P22646 (murine CD3ε).

[0436] The term "CD28" refers to the T cell-specific glycoprotein CD28, also known as Tp44 and all alternative names thereof, which is used as a co-stimulatory molecule. UniProt accession number P10747 provides an exemplary human CD28 amino acid sequence (also referring to HGNC: 1653, Entrez gene: 940, Ensembl: ENSG00000178562, and OMIM: 186760). Further related CD28 sequences include UniProt accession number P21041 (mouse CD28).

[0437] 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 all alternative names thereof, which acts as a growth factor receptor. UniProt accession number P06729 provides an exemplary human CD2 amino acid sequence (see also HGNC: 1639, Entrez gene: 914, Ensembl: ENSG00000116824, and OMIM: 186990). Further related CD2 sequences include UniProt accession number P08920 (murine CD2).

[0438] As used herein, the term "nanomatrix" refers to a nanostructure comprising a removable polymer chain matrix. The size of the nanomatrix is ​​1 to 500 nm, for example, 10 to 200 nm. In certain embodiments, the matrix of the mobile polymer chain is attached to one or more agonists, which provide activation signals to T cells, such as agonist anti-CD3 antibodies and / or anti-CD28 antibodies. In certain embodiments, the nanomatrix comprises an attached colloidal polymer nanomatrix, such as an agonist covalently attached to one or more stimulatory molecules and / or an agonist of one or more costimulatory molecules. In certain embodiments, the agonist of one or more stimulatory molecules is a CD3 agonist (for example, an anti-CD3 agonist antibody). In certain embodiments, the agonist of one or more costimulatory molecules is a CD28 agonist (for example, an anti-CD28 agonist antibody). In certain embodiments, the nanomatrix is ​​characterized by the absence of a solid surface, for example, as an attachment point for an agonist, such as an anti-CD3 antibody and / or an anti-CD28 antibody. In some embodiments, the nanomatrix is ​​a nanomatrix disclosed in WO 2014 / 048920 A1 or a nanomatrix from Miltenyi Biotcc GmbH. GMP T Cell TransAct TM The nanomatrix provided in the kit is incorporated herein by reference in its entirety. GMP T Cell TransAct TM It consists of a colloidal polymer nanomatrix covalently attached to humanized recombinant agonist antibodies against human CD3 and CD28.

[0439] Various embodiments of the compositions and methods herein are described in further detail below.Additional definitions are set forth throughout the application.

[0440] describe

[0441] Provided herein are methods of making 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 of using such cells for treating a disease in a subject, e.g., an autoimmune disease or disorder, 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 significant lung involvement (e.g., for autologous HSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-associated myositis, Such as anti-synthetase syndrome with ILD), vasculitis (for example, ANCA associated vasculitis), severe refractory Sjögren's syndrome, severe refractory neuroimmune diseases (such as myasthenia gravis (MG), neuromyelitis optica (NMO), MOG related diseases (MOGAD), multiple sclerosis (MS)), severe refractory rheumatoid arthritis, antibody-mediated neuroimmune diseases (for example, AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ neuromyelitis optica (NMO), MOGAD (anti-MOG related diseases), NMDAR+ encephalitis or antibody-related neurological paraneoplastic diseases), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris or amyotrophic lateral sclerosis. In certain embodiments, methods disclosed herein can be manufactured in less than 24 hours through engineered to express CAR immune effector cells. Without wishing to be bound by theory, methods provided herein retain the undifferentiated phenotype of T cells, such as initial T cells, during the manufacturing process. These cells expressing CAR with an undifferentiated phenotype can continue in vivo for a longer time and / or better amplification after infusion. In some embodiments, compared with CART cells produced by a traditional manufacturing process (e.g., as measured using scRNA-seq), the CART cells produced by the manufacturing method provided herein include a higher percentage of stem cell memory T cells. In some embodiments, compared with CART cells produced by a traditional manufacturing process (e.g., as measured using scRNA-seq), the CART cells produced by the manufacturing method provided herein include a higher percentage of effector T cells. In some embodiments, compared with CART cells produced by a traditional manufacturing process, the CART cells produced by the manufacturing method provided herein better preserve the stem cell nature of T cells. In some embodiments, compared with CART cells produced by a traditional manufacturing process (e.g., as measured using scRNA-seq), the CART cells produced by the manufacturing method provided herein show lower hypoxia levels.In some embodiments, CART cells produced by the manufacturing methods provided herein exhibit lower levels of autophagy compared to CART cells produced by traditional manufacturing processes.

[0442] In some embodiments, the methods disclosed herein do not involve the use of beads, e.g. (e.g., CD3 / CD28 ), and does not involve a bead removal step. In some embodiments, the CART cells manufactured by the methods disclosed herein can be administered to a subject with minimal ex vivo expansion, for example, less than 1 day, less than 12 hours, less than 8 hours, less than 6 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, or no ex vivo expansion. Therefore, the methods described herein provide a rapid manufacturing process for preparing improved CAR-expressing cell products for treating a subject's disease. In addition, the present invention provides CAR compositions and their use in medicaments or methods for treating autoimmune diseases or disorders, particularly in diseases, 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 significant lung involvement (e.g., for autologous HSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-related myositis, e.g., antisynthetase syndrome with ILD), vasculitis (e.g., , ANCA-associated vasculitis), severe refractory Sjögren's syndrome, severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-related 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-related disease), NMDAR+ encephalitis, or antibody-related neurological paraneoplastic diseases), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis.

[0443] Activation process

[0444] In some embodiments, the present disclosure provides methods for preparing a population of cells (e.g., T cells) expressing a chimeric antigen receptor (CAR), the methods comprising: (i) subjecting a population of cells (e.g., T cells, e.g., T cells isolated from a frozen or fresh leukapheresis product of a subject with an autoimmune disease or disorder, 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 significant lung involvement (e.g., for autologous HSCT), to a subject with an autoimmune disease or disorder, e.g., ... ), idiopathic inflammatory myopathies (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-related myositis, such as antisynthetase syndrome with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjögren's syndrome, severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-related disease (MOGAD), multiple sclerosis (MS)), severe refractory rheumatoid arthritis, antibody-mediated neuroimmune diseases (e.g., AChR+ and MuSK+ myasthenia gravis (MG), AQP4+ (i) contacting a population of cells (e.g., T cells) with a nucleic acid molecule (e.g., DNA or RNA molecule) encoding a CAR, thereby providing a population of cells (e.g., T cells) comprising the nucleic acid molecule, and (ii) contacting a population of cells (e.g., T cells) with a nucleic acid molecule (e.g., DNA or RNA molecule) encoding a CAR, and (iii) contacting a population of cells (e.g., T cells) with a nucleic acid molecule (e.g., DNA or RNA molecule) encoding a CAR, and (iv) contacting a population of cells (e.g., T cells) with a nucleic acid molecule (e.g., DNA or RNA molecule) encoding a CAR, and (v ... (iii) harvesting the cell (e.g., T cell) population for storage (e.g., reconstituting the cell population in a cryopreservation medium) or administration, wherein: (a) step (ii) is performed together with step (i), or is performed no later than 20 hours after the start of step (i) (e.g., no later than 12, 13, 14, 15, 16, 17, or 18 hours after the start of step (i), for example, no later than 18 hours after the start of step (i)), and step (iii) is performed no later than 26 hours after the start of step (i) (e.g., no later than 22, 23, or 24 hours after the start of step (i), for example, no later than 24 hours after the start of step (i));(b) step (ii) is performed together with step (i), or is performed no later than 20 hours after the start of step (i) (e.g., no later than 12, 13, 14, 15, 16, 17, or 18 hours after the start of step (i), for example, no later than 18 hours after the start of step (i)), and step (iii) is performed no later than 30, 36, or 48 hours after the start of step (ii) (e.g., no later than 22, 23, 24, 25, 26, 27, 28, or (c) the cell population 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 cell population at the start of step (i), e.g., as assessed by the number of viable cells. In some embodiments, the nucleic acid molecule in step (ii) is a DNA molecule. In some embodiments, the nucleic acid molecule in step (ii) is an RNA molecule. In some embodiments, the nucleic acid molecule in step (ii) is on a viral vector (e.g., a viral vector selected from a lentiviral vector, an adenoviral vector, or a retroviral vector). In some embodiments, the nucleic acid molecule in step (ii) is on a non-viral vector. In some embodiments, the nucleic acid molecule in step (ii) is on a plasmid. In some embodiments, the nucleic acid molecule in step (ii) is not on any vector. In some embodiments, step (ii) comprises transducing the cell (e.g., T cell) population with a viral vector comprising a nucleic acid molecule encoding a CAR.

[0445] In some embodiments, a population of cells (e.g., T cells) is collected from an aliquot (e.g., a leukocyte aliquot) of a subject with an autoimmune disease or disorder, 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 significant lung involvement (e.g., for autologous HSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-related myositis, e.g., antisynthetase syndrome with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjögren's syndrome, severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-related 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-related disease), NMDAR+ encephalitis, or antibody-related neurological paraneoplastic diseases), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis.

[0446] In some embodiments, an aliquot sample (e.g., a leukocyte aliquot sample) is collected from a subject and transported to a cell manufacturing facility as a frozen sample (e.g., a cryopreserved sample). The frozen aliquot sample is then thawed, and T cells (e.g., CD4+ T cells and / or CD8+ T cells) are selected from the aliquot sample, for example, using a cell sorter (e.g., Device). The selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are then inoculated using the activation process described herein for CART manufacturing. In some embodiments, the selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) undergo one or more rounds of freeze-thawing before being inoculated for CART manufacturing.

[0447] In some embodiments, an aliquot sample (e.g., a leukocyte aliquot sample) is collected from a subject and shipped to a cell manufacturing facility as a fresh product (e.g., a non-frozen sample). T cells (e.g., CD4+ T cells and / or CD8+ T cells) are selected from the aliquot sample, for example, using a cell sorter (e.g., Device). The selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are then inoculated using the activation process described herein for CART manufacturing. In some embodiments, the selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) undergo one or more rounds of freeze-thawing before being inoculated for CART manufacturing.

[0448] In some embodiments, an aliquot (e.g., a leukocyte aliquot) is collected from a subject. T cells (e.g., CD4+ T cells and / or CD8+ T cells) are selected from the aliquot, e.g., using a cell sorter (e.g., Device). The selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are then transported to a cell manufacturing facility as a frozen sample (e.g., a cryopreserved sample). The selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are subsequently thawed and inoculated for CART manufacturing using the activation process described herein.

[0449] In certain embodiments, cells (e.g., T cells) are contacted with anti-CD3 and anti-CD28 antibodies for example 12 hours, and then transduced with a vector (e.g., a lentiviral vector) encoding CAR. 24 hours after the start of culture, cells are washed and prepared for storage or administration.

[0450] Without wishing to be bound by theory, brief CD3 and CD28 stimulation can promote the effective transduction of self-renewal T cells. Compared with traditional CART manufacturing methods, the activation process provided herein does not involve extended ex vivo expansion. Similar to the cytokine process, the activation process provided herein also retains undifferentiated T cells during CART manufacturing.

[0451] In some embodiments, a population of cells is contacted with a multispecific binding molecule, eg, as described herein.

[0452] In some embodiments, the cell population is contacted with (A) an agent that stimulates the CD3 / TCR complex and / or (B) an agent that stimulates a co-stimulatory molecule and / or a growth factor receptor on the cell surface.

[0453] In certain embodiments, the medicament for stimulating CD3 / TCR complexes is the medicament for stimulating CD3. In certain embodiments, the medicament for stimulating costimulatory molecules and / or growth factor receptors is the medicament for stimulating CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226 or any combination thereof. In certain embodiments, the medicament for stimulating costimulatory molecules and / or growth factor receptors is the medicament for stimulating CD28. In certain embodiments, the medicament for stimulating CD3 / TCR complexes is selected from antibody (such as single domain antibody (such as heavy chain variable domain antibody), peptibody, Fab fragment or scFv), small molecule or part (such as naturally occurring part, recombinant part or chimeric part). In some embodiments, the agent that stimulates co-stimulatory molecules and / or growth factor receptors 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 a 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 beads. In some embodiments, the agent that stimulates co-stimulatory molecules and / or growth factor receptors does not comprise beads. In some embodiments, the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody. In some embodiments, the agent that stimulates co-stimulatory molecules and / or growth factor receptors comprises an anti-CD28 antibody. In some embodiments, the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody covalently attached to a colloidal polymer nanomatrix. In some embodiments, the agent that stimulates CD3 comprises one or more of CD3 or TCR antigen binding domains, such as, but not limited to, anti-CD3 or anti-TCR antibodies or antibody fragments comprising one or more of its CDRs, heavy chains and / or light chains - such as, but not limited to, the anti-CD3 or anti-TCR antibodies provided in Table 27 of WO / 2021 / 173985 (which is hereby incorporated by reference in its entirety). In some embodiments, the agent that stimulates co-stimulatory molecules and / or growth factor receptors comprises an anti-CD28 antibody covalently attached to a colloidal polymer nanomatrix. In some embodiments, the agent that stimulates co-stimulatory molecules and / or growth factor receptors is an agent that stimulates CD28, ICOS, CD27, CD25, 4-1BB, IL6RA, IL6RB, or CD2.In some embodiments, the agent that stimulates co-stimulatory molecules and / or growth factor receptors comprises one or more of CD28, ICOS, CD27, CD25, 4-1BB, IL6RB, and / or CD2 antigen binding domains, such as, but not limited to, anti-CD28, anti-ICOS, anti-CD27, anti-CD25, anti-4-1BB, anti-IL6RA, anti-IL6RB, or anti-CD2 antibodies or antibody fragments comprising one or more of their CDRs, heavy chains, and / or light chains - such as, but not limited to, 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 hereby incorporated by reference in its entirety). In some embodiments, the agent that stimulates the CD3 / TCR complex and the agent that stimulates co-stimulatory molecules and / or growth factor receptors comprise T cell TransAct. TM . In some embodiments, the agent that stimulates the CD3 / TCR complex and the agent that stimulates the co-stimulatory molecule and / or growth factor receptor are included in the 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 are hereby incorporated by reference in their entirety). In some embodiments, the multispecific binding molecule comprises a bispecific antibody. In some embodiments, the bispecific antibody is configured with any of the schemes provided in Figure 50A of WO / 2021 / 173985 (which are hereby incorporated by reference in their entirety). In some embodiments, the bispecific antibody is monovalent or divalent. In some embodiments, the bispecific antibody comprises an Fc region. In some embodiments, the Fc region of the bispecific antibody is silent. 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 are monovalent. In some embodiments, one or more of the multiple bispecific antibodies include an Fc region. In some embodiments, the Fc region of one or more of the multiple bispecific antibodies is silent. In some embodiments, one or more of the multiple bispecific antibodies are conjugated together into a multimer. In some embodiments, the multimer is configured with any of the schemes provided in Figure 50B of WO / 2021 / 173985 (which is hereby incorporated by reference in its entirety).

[0454] In certain embodiments, this matrix comprises polymer or is made up of it, for example, biodegradable or biocompatible inert material, for example, it is nontoxic to cell.In certain embodiments, this matrix is ​​made up of hydrophilic polymer chain, and due to the hydration of chain, it obtains maximum mobility in aqueous solution.In certain embodiments, this mobile matrix can be collagen, purified protein, purified peptide, polysaccharide, glycosaminoglycan or extracellular matrix composition.Polysaccharide can comprise for example cellulose ether, starch, gum arabic, agarose, dextran, chitosan, hyaluronic acid, pectin, xanthan gum, guar gum or alginate.Other polymers can comprise polyester, polyether, polyacrylate, polyacrylamide, polyamine, polyethyleneimine, polyquaternium salt polymer, polyphosphazene, polyvinyl alcohol, polyvinyl acetate, polyvinyl pyrrolidone, block copolymer or polyurethane.In certain embodiments, mobile matrix is ​​the polymer of dextran.

[0455] In certain embodiments, cell colonies are contacted with nucleic acid molecules encoding CAR. In certain embodiments, cell colonies are transduced with DNA molecules encoding CAR.

[0456] In certain embodiments, cell colony is contacted with nucleic acid molecules encoding CAR while cell colony is contacted with the medicament for stimulating CD3 / TCR complex and / or the medicament for stimulating costimulatory molecules and / or growth factor receptors on the cell surface as described above.In certain embodiments, cell colony is contacted with the medicament for stimulating CD3 / TCR complex and / or the medicament for stimulating costimulatory molecules and / or growth factor receptors on the cell surface as described above no later than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0.5 hour after the medicament for stimulating CD3 / TCR complex and / or the medicament for stimulating costimulatory molecules and / or growth factor receptors on the cell surface as described above is contacted. In some embodiments, no later than 20 hours after the cell colony is contacted with the agent that stimulates the CD3 / TCR complex and / or the costimulatory molecules and / or growth factor receptors on the cell surface as described above, the cell colony is contacted with the nucleic acid molecules encoding CAR. In some embodiments, no later than 19 hours after the cell colony is contacted with the agent that stimulates the CD3 / TCR complex and / or the costimulatory molecules and / or growth factor receptors on the cell surface as described above, the cell colony is contacted with the nucleic acid molecules encoding CAR. In some embodiments, no later than 18 hours after the cell colony is contacted with the agent that stimulates the CD3 / TCR complex and / or the costimulatory molecules and / or growth factor receptors on the cell surface as described above, the cell colony is contacted with the nucleic acid molecules encoding CAR. In some embodiments, no later than 17 hours after the cell colony is contacted with the nucleic acid molecules encoding CAR. In some embodiments, no later than 16 hours after the cell colony is contacted with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the costimulatory molecules and / or growth factor receptors on the cell surface as described above, the cell colony is contacted with the nucleic acid molecules encoding CAR. In some embodiments, no later than 15 hours after the cell colony is contacted with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the costimulatory molecules and / or growth factor receptors on the cell surface as described above, the cell colony is contacted with the nucleic acid molecules encoding CAR. In some embodiments, no later than 14 hours after the cell colony is contacted with the agent that stimulates the CD3 / TCR complex and / or the agent that stimulates the costimulatory molecules and / or growth factor receptors on the cell surface as described above, the cell colony is contacted with the nucleic acid molecules encoding CAR.In some embodiments, no later than 14 hours after the cell colony contacts the agent that stimulates the CD3 / TCR complex and / or stimulates the costimulatory molecules and / or growth factor receptors on the cell surface as described above, the cell colony is contacted with the nucleic acid molecules encoding CAR. In some embodiments, no later than 13 hours after the cell colony contacts the agent that stimulates the CD3 / TCR complex and / or stimulates the costimulatory molecules and / or growth factor receptors on the cell surface as described above, the cell colony is contacted with the nucleic acid molecules encoding CAR. In some embodiments, no later than 12 hours after the cell colony contacts the agent that stimulates the CD3 / TCR complex and / or stimulates the costimulatory molecules and / or growth factor receptors on the cell surface as described above, the cell colony is contacted with the nucleic acid molecules encoding CAR. In some embodiments, no later than 11 hours after the cell colony contacts the agent that stimulates the CD3 / TCR complex and / or stimulates the costimulatory molecules and / or growth factor receptors on the cell surface as described above, the cell colony is contacted with the nucleic acid molecules encoding CAR. In some embodiments, no later than 10 hours after the cell colony is contacted with the agent that stimulates the CD3 / TCR complex and / or the costimulatory molecules and / or growth factor receptors on the cell surface as described above, the cell colony is contacted with the nucleic acid molecules encoding CAR. In some embodiments, no later than 9 hours after the cell colony is contacted with the agent that stimulates the CD3 / TCR complex and / or the costimulatory molecules and / or growth factor receptors on the cell surface as described above, the cell colony is contacted with the nucleic acid molecules encoding CAR. In some embodiments, no later than 8 hours after the cell colony is contacted with the agent that stimulates the CD3 / TCR complex and / or the costimulatory molecules and / or growth factor receptors on the cell surface as described above, the cell colony is contacted with the nucleic acid molecules encoding CAR. In some embodiments, no later than 7 hours after the cell colony is contacted with the agent that stimulates the CD3 / TCR complex and / or the costimulatory molecules and / or growth factor receptors on the cell surface as described above, the cell colony is contacted with the nucleic acid molecules encoding CAR. In some embodiments, the cell population is contacted with a nucleic acid molecule encoding CAR no later than 6 hours after the cell population is contacted with an agent that stimulates the CD3 / TCR complex and / or stimulates the costimulatory molecules and / or growth factor receptors on the cell surface as described above. In some embodiments, the cell population is contacted with a nucleic acid molecule encoding CAR no later than 5 hours after the cell population is contacted with an agent that stimulates the CD3 / TCR complex and / or stimulates the costimulatory molecules and / or growth factor receptors on the cell surface as described above.In some embodiments, no later than 4 hours after the cell colony contacts the agent that stimulates the CD3 / TCR complex and / or the co-stimulatory molecules and / or growth factor receptors on the cell surface as described above, the cell colony is contacted with the nucleic acid molecules encoding CAR. In some embodiments, no later than 3 hours after the cell colony contacts the agent that stimulates the CD3 / TCR complex and / or the co-stimulatory molecules and / or growth factor receptors on the cell surface as described above, the cell colony is contacted with the nucleic acid molecules encoding CAR. In some embodiments, no later than 2 hours after the cell colony contacts the agent that stimulates the CD3 / TCR complex and / or the co-stimulatory molecules and / or growth factor receptors on the cell surface as described above, the cell colony is contacted with the nucleic acid molecules encoding CAR. In some embodiments, no later than 1 hour after the cell colony contacts the agent that stimulates the CD3 / TCR complex and / or the co-stimulatory molecules and / or growth factor receptors on the cell surface as described above, the cell colony is contacted with the nucleic acid molecules encoding CAR. In some embodiments, the cell population is contacted with a nucleic acid molecule encoding a CAR no later than 30 minutes after contact of the cell population with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates co-stimulatory molecules and / or growth factor receptors on the cell surface as described above is initiated.

[0457] In some embodiments, a cell population is harvested for storage or administration.

[0458] In certain embodiments, no later than 72,60,48,36,32,31,30,29,28,27,26,25,24,23,22,21,20,19 or 18 hours after cell colony contacts the medicament for stimulating CD3 / TCR compound and / or stimulating costimulatory molecules and / or growth factor receptor on the cell surface as described above, harvesting cell colony is used for storage or application.In certain embodiments, no later than 26 hours after cell colony contacts the medicament for stimulating CD3 / TCR compound and / or stimulating costimulatory molecules and / or growth factor receptor on the cell surface as described above, harvesting cell colony is used for storage or application.In certain embodiments, no later than 25 hours after cell colony contacts the medicament for stimulating CD3 / TCR compound and / or stimulating costimulatory molecules and / or growth factor receptor on the cell surface as described above, harvesting cell colony is used for storage or application. In certain embodiments, no later than 24 hours after cell colony contacts with the medicament that stimulates CD3 / TCR compound and / or stimulates the costimulatory molecules on the cell surface as described above and / or the medicament for growth factor receptor, cell colony is used for storage or administration.In certain embodiments, no later than 23 hours after cell colony contacts with the medicament that stimulates CD3 / TCR compound and / or stimulates the costimulatory molecules on the cell surface as described above and / or the medicament for growth factor receptor, cell colony is used for storage or administration.In certain embodiments, no later than 22 hours after cell colony contacts with the medicament that stimulates CD3 / TCR compound and / or stimulates the costimulatory molecules on the cell surface as described above and / or the medicament for growth factor receptor, cell colony is used for storage or administration.

[0459] In some embodiments, the cell population is not expanded ex vivo.

[0460] In some embodiments, the cell population expands 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% compared to the cell population before contacting the cell population 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 a cell as described above, e.g., as assessed by viable cell number. In some embodiments, the cell population expands by no more than 5% compared to the cell population before contacting the cell population 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 a cell as described above. In some embodiments, for example, as assessed by viable cell number, the cell population is expanded by no more than 10% compared to the cell population before contacting the cell population 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 cell surface as described above. In some embodiments, for example, as assessed by viable cell number, the cell population is expanded by no more than 15% compared to the cell population before contacting the cell population 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 cell surface as described above. In some embodiments, for example, as assessed by viable cell number, the cell population is expanded by no more than 20% compared to the cell population before contacting the cell population 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 cell surface as described above. In some embodiments, for example, as assessed by viable cell number, the cell population is expanded by no more than 25% compared to the cell population before contacting the cell population 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 cell surface as described above. In some embodiments, the cell population expands by no more than 30% compared to the cell population before contacting the cell population with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a co-stimulatory molecule and / or a growth factor receptor on the surface of the cell as described above, e.g., as assessed by viable cell number. In some embodiments, the cell population expands by no more than 35% compared to the cell population before contacting the cell population with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a co-stimulatory molecule and / or a growth factor receptor on the surface of the cell as described above, e.g., as assessed by viable cell number.In some embodiments, the cell population expands by no more than 40% compared to the cell population before contacting the cell population with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a co-stimulatory molecule and / or growth factor receptor on the surface of the cells as described above, e.g., as assessed by viable cell number.

[0461] In some embodiments, the cell population expands for 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 compared to the cell population before contacting the cell population with one or more cytokines as described above, e.g., as assessed by viable cell number.

[0462] In some embodiments, the activation process is performed in a serum-free cell culture medium. In some embodiments, the activation process is performed in a cell culture medium comprising one or more cytokines selected from the group consisting of 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 comprises the following amino acid sequence: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMK CFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQG (SEQ ID NO: 309). In some embodiments, hetIL-15 comprises an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 309. In some embodiments, the activation process is performed in a cell culture medium containing an LSD1 inhibitor. In some embodiments, the activation process is performed in a cell culture medium containing a MALT1 inhibitor. In some embodiments, the serum-free cell culture medium contains a serum replacement. In some embodiments, the serum replacement is CTS. TMImmune Cell Serum Replacement (ICSR). In some embodiments, the level of ICSR can be, for example, up to 5%, for example, about 1%, 2%, 3%, 4%, or 5%. Without wishing to be bound by theory, using a cell culture medium, for example, Rapid Media as shown in Table 21 or Table 25, containing ICSR, for example, 2% ICSR, can improve cell viability during the manufacturing processes described herein.

[0463] In some embodiments, the present disclosure provides methods for preparing a population of cells (e.g., T cells) expressing a chimeric antigen receptor (CAR), the methods comprising: (a) providing an aliquot sample (e.g., a fresh or cryopreserved leukocyte aliquot sample) collected from a subject having, 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 significant pulmonary involvement (e.g., for autologous HSCT)), 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), or a combination of the following: ), severe refractory Sjögren's syndrome, severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-related diseases (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-related diseases), NMDAR+ encephalitis, or antibody-related neurological paraneoplastic diseases), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis; (b) selecting T cells from a single sample (e.g., using negative selection, positive selection, or bead-free selection); (c) seeding the isolated T cells, e.g., 1×10 6 to 1x 10 7cells / mL; (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 co-stimulatory molecules and / or growth factor receptors on the cell surface (e.g., contacting the T cells with anti-CD3 and / or anti-CD28 antibodies, e.g., contacting the T cells with TransAct); (e.g., 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) for, e.g., 6-48 hours, e.g., 20-28 hours; and (f) washing and harvesting the T cells for storage (e.g., reconstituting the T cells in a cryopreservation medium) or administration. In some embodiments, step (f) is performed no later than 30, 36, or 48 hours after the start of step (d) or (e), for example, no later than 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours after the start of step (d) or (e).

[0464] In some embodiments of the aforementioned methods, these methods are performed in a closed system. In some embodiments, T cell isolation, activation, transduction, incubation, and washing are all performed in a closed system. In some embodiments of the aforementioned methods, these methods are performed in separate devices. In some embodiments, T cell isolation, activation, transduction, incubation, and washing are performed in separate devices.

[0465] In some embodiments of the aforementioned methods, the methods further comprise adding an adjuvant or transduction enhancing agent to the cell culture medium to enhance transduction efficiency. In some embodiments, the adjuvant or transduction enhancing agent comprises a cationic polymer. In some embodiments, the adjuvant or transduction enhancing agent is selected from the group consisting of: LentiBOOST TM (Sirion Biotech), vectofusin-1, F108 (Poloxamer 338 or F-38), protamine sulfate, hexamethylenetetramine (polybrene), PEA, Pluronic F68, Pluronic F127, poloxamer, or LentiTrans TM In some embodiments, the transduction enhancing agent is LentiBOOST TM (Sirion Biotech). In some embodiments, the transduction enhancing agent is F108 (Poloxamer 338 or F-38).

[0466] In some embodiments of the aforementioned methods, transducing a cell (e.g., T cell) population with a viral vector comprises subjecting the cell population and the viral vector to centrifugal force under conditions that enhance transduction efficiency. In embodiments, the cells are transduced by spinoculation.

[0467] In some embodiments of the foregoing methods, cells (e.g., T cells) are activated and transduced in a cell culture flask comprising a gas permeable membrane at the base that supports large culture medium volumes without substantially impairing gas exchange. In some embodiments, cell growth is achieved by providing access to nutrients via convection, e.g., substantially uninterrupted access.

[0468] Multispecific binding molecules

[0469] The method for preparing cells expressing CAR can use the medicament for stimulating CD3 / TCR complex and the medicament for stimulating costimulatory molecules and / or growth factor receptors.In certain embodiments, the medicament for stimulating CD3 / TCR complex and the medicament for stimulating costimulatory molecules and / or growth factor receptors are included in multispecific binding molecules.In certain embodiments, the multispecific binding molecules of the present disclosure are WO 2021 / 173985 (incorporated by reference in its entirety), WO 2022 / 040586 (incorporated by reference in its entirety) and PCT / IB2022 / 057799 (incorporated by reference in its entirety) any one of the multispecific binding molecules described.

[0470] In certain embodiments, the multispecific binding molecule includes 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 certain embodiments, the costimulatory molecule binding domain is combined with CD2, CD28, CD25, CD27, IL6Rb, ICOS or 41BB. As described above, non-limiting examples of such binding domains are provided in Table 27 of WO 2021 / 173985 (which contents are hereby incorporated by reference in their entirety). In certain embodiments, the multispecific binding molecule is configured with any of the schemes provided in Figures 50A-50B, Figures 51A-51B and Figures 61A-61B and Figures 63A-63B of WO 2021 / 173985 (incorporated by reference in their entirety).

[0471] 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) provided in Table 27 of WO 2021 / 173985 (the contents of which are hereby incorporated by reference in their entirety), or an antibody fragment comprising one or more of its CDRs, VHs, and / or VLs. In some embodiments, the CD28 antigen binding domain is an anti-CD28 antibody, optionally anti-CD28 (1), or anti-CD28 (2) provided in Table 27 of WO 2021 / 173985 (the contents of which are hereby incorporated by reference in their entirety), or an antibody fragment comprising one or more of its CDRs, VHs, heavy chains, VLs, and / or light chains. 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 of its CDRs, VH, heavy chains, VL and / or light chains. 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.

[0472] In some embodiments, the multispecific binding molecules include one or more heavy chains 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 molecules include one or more heavy and / or light chain sequences disclosed in Table 20 of WO 2022 / 040586 (incorporated by reference in its entirety), or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% homogeneity thereto.

[0473] In some embodiments, the multispecific binding molecules described herein comprise an Fc region, e.g., wherein the Fc region is Fc-silent, e.g., such as those 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, which are 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), which are 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), which are numbered according to the EU numbering system. In some embodiments, the Fc region comprises one or more mutations as described in, e.g., WO 2021 / 173985 (incorporated by reference in its entirety) or WO 2022 / 040586 (incorporated by reference in its entirety).

[0474] In some embodiments, the 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 at the N-terminus 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 at the C-terminus of the costimulatory molecule binding domain, e.g., an anti-CD2 Fab or an anti-CD28 Fab.

[0475] 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 at the C-terminus of the costimulatory molecule binding domain, wherein the Fc region is located between the anti-CD3 binding domain and the costimulatory molecule binding domain.

[0476] In some embodiments, the multispecific binding molecule comprises a CH2, and the anti-CD3 binding domain is located at the N-terminus of the CH2. In some embodiments, the anti-CD3 binding domain is connected to the CH2 via a peptide linker (e.g., a glycine-serine linker, such as a (G4S)4 linker).

[0477] 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 connected to the CH1, for example, via a disulfide bridge.

[0478] In some embodiments, the multispecific binding molecule comprises: (i) a first polypeptide comprising the following from N-terminus to C-terminus: VH, CH1, CH2, CH3 of the costimulatory molecule binding domain, VH of the anti-CD3 binding domain, and VL of the anti-CD3 binding domain; and (ii) a second polypeptide comprising the following from N-terminus to C-terminus: VL and CL of the costimulatory molecule binding domain. In some embodiments, the anti-CD3 binding domain comprises an scFv. In some embodiments, the costimulatory molecule binding domain is part of a Fab fragment, for example, the Fab fragment is part of a polypeptide sequence comprising an Fc domain. In some embodiments, the anti-CD3 binding domain is connected to CH3 via a peptide linker (e.g., a glycine-serine linker, such as a (G4S)4 linker).

[0479] In some embodiments, the multispecific binding molecule comprises: (i) a first polypeptide comprising, from N-terminus to C-terminus, the VH of the anti-CD3 binding domain, the VL of the anti-CD3 binding domain, the VH, CH1, CH2, and CH3 of the costimulatory molecule binding domain; and (ii) a second polypeptide comprising, from N-terminus to C-terminus, the VL and CL of the costimulatory molecule binding domain. In some embodiments, the anti-CD3 binding domain and the costimulatory molecule binding domain are connected by a peptide linker (e.g., a glycine-serine linker, such as a (G4S)4 linker).

[0480] In some embodiments, the multispecific binding molecule comprises: (i) a first polypeptide comprising, from N-terminus to C-terminus, the VH and CHI of the costimulatory molecule binding domain, the VH and CHI of the anti-CD3 binding domain, the VL and CHI of the anti-CD3 binding domain, and (ii) a second polypeptide comprising, from N-terminus to C-terminus, the VL and CHI of the costimulatory molecule binding domain. In some embodiments, the anti-CD3 binding domain is connected to CHI via a peptide linker (e.g., a glycine-serine linker, such as a (G4S)2 linker).

[0481] 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.

[0482] It will be understood that in many embodiments herein, multispecific binding molecules comprise two or more polypeptide chains that are covalently linked to each other, for example, via disulfide bridges. However, in some embodiments, two or more polypeptide chains of a multispecific binding molecule may be non-covalently linked to each other.

[0483] It will also be appreciated that a Fab fragment may exist as part of a larger protein, for example, a Fab fragment may be fused to CH2 and CH3 and thus be part of a full-length antibody.

[0484] It is contemplated that multispecific binding molecules comprising an agent that stimulates the CD3 / TCR complex and an agent that stimulates a co-stimulatory molecule and / or a growth factor receptor as disclosed herein can be used in the manufacturing embodiments disclosed herein, such as traditional manufacturing or activated rapid manufacturing.

[0485] CAR-expressing cell populations produced by the processes disclosed herein

[0486] In some embodiments, the present disclosure is characterized by being engineered to express immune effector cells (e.g., T cells or NK cells) of CAR (e.g., B cell antigens, e.g., CD19), for example, prepared by any manufacturing method described herein, wherein the engineered immune effector cells exhibit immunosuppressive properties. In some embodiments, CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain. Exemplary antigens are B cell antigens as described herein. In some embodiments, cells (e.g., T cells or NK cells) are transformed with CAR, and CAR is expressed on the cell surface. In some embodiments, cells (e.g., T cells or NK cells) are transduced with a viral vector encoding CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, cells can stably express CAR. In some embodiments, cells (e.g., T cells or NK cells) are transfected with nucleic acids (e.g., mRNA, cDNA, or DNA) encoding CAR. In some such embodiments, cells can transiently express CAR.

[0487] In some embodiments, the cell populations provided herein are populations of cells (e.g., immune effector cells, such as T cells or NK cells) prepared by any of the manufacturing processes described herein (e.g., the activation processes described herein) that are engineered to express a CAR.

[0488] In some embodiments, the percentage of naive cells (e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells) in the cell population 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., no more than 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, or (3) increases by, e.g., 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+ cells) in the cell population at the beginning of the manufacturing process (e.g., at the beginning of an activation process described herein). In some embodiments, the cell population at the end of the manufacturing process (e.g., at the end of the activation process described herein) exhibits a higher percentage of naive cells (e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells) (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) compared to cells prepared by an otherwise similar method except that the method lasts, e.g., for more than 26 hours (e.g., for more than 5, 6, 7, 8, 9, 10, 11, or 12 days) or involves expanding the cell population in vitro for, e.g., more than 3 days (e.g., expanding the cell population in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days).

[0489] In some embodiments, the percentage of naive cells (e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells) in the cell population at the end of the manufacturing process (e.g., at the end of the activation process described herein) is no less than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%.

[0490] In some embodiments, the percentage of central memory cells (e.g., central memory T cells, e.g., CD95+ central memory T cells) in the cell population at the end of the manufacturing process (e.g., at the end of the activation process described herein) is (1) the same, (2) differs by, e.g., no more than 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, or (3) is reduced by, e.g., 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 cell population at the beginning of the manufacturing process (e.g., at the beginning of the activation process described herein). In some embodiments, the cell population at the end of the manufacturing process (e.g., at the end of the activation process described herein) 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 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% lower) compared to cells prepared by an otherwise similar method except that the method continues, e.g., for more than 26 hours (e.g., for more than 5, 6, 7, 8, 9, 10, 11, or 12 days) or involves expanding the cell population in vitro, e.g., for more than 3 days (e.g., expanding the cell population in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days).

[0491] In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells in the cell population at the end of the manufacturing process (e.g., at the end of the activation process described herein) is no more than 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%.

[0492] In some embodiments, the cell population at the end of the manufacturing process (e.g., at the end of the activation process described herein) persists longer or expands at a higher level (e.g., at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% higher) after in vivo administration compared to cells manufactured by an otherwise similar method except that the method lasts, e.g., for more than 26 hours (e.g., lasts for more than 5, 6, 7, 8, 9, 10, 11, or 12 days) or involves expanding the cell population in vitro for, e.g., more than 3 days (e.g., expanding the cell population in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days).

[0493] In some embodiments, before the manufacturing process begins (e.g., before the activation process described herein begins), the cell population is enriched for cells expressing IL6R (e.g., cells that are positive for IL6R α and / or IL6R β). In some embodiments, at the beginning of the manufacturing process (e.g., at the beginning of the activation process described herein), the cell population comprises, for example, no less than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% of cells that express IL6R (e.g., cells that are positive for IL6R α and / or IL6R β).

[0494] Pharmaceutical composition

[0495] In addition, the present disclosure provides cell compositions expressing CARs and their use in medicaments or methods for treating, in particular, autoimmune diseases involving cells or tissues expressing antigens as described herein, 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 significant lung involvement (e.g., for autologous HSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-related myositis, e.g., antisynthetase syndrome with IL-1β, IL-23, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-50, IL-61, IL-72, IL-83, IL-19, IL-19, IL-11 D), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjögren's syndrome, severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-related diseases (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-related diseases), NMDAR+ encephalitis, or antibody-related neurological paraneoplastic diseases), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis. In some embodiments, provided herein are pharmaceutical compositions comprising cells expressing CAR, for example, a plurality of CAR-expressing cells prepared 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.

[0496] Chimeric Antigen Receptor (CAR)

[0497] The present invention provides immune effector cells (e.g., T cells, NK cells), which are engineered to contain one or more CARs that guide immune effector cells to cells associated with autoimmune disorders. This is achieved by the antigen binding domains on CAR, which are specific to B cell-associated antigens. There are two types of B cell antigens that can be targeted by CAR as described herein: (1) B cell antigens expressed on the surface of B cells; and (2) B cell antigens themselves in cells, however, such antigen fragments (peptides) are presented on the surface of B cells by MHC (major histocompatibility complex).

[0498] 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.

[0499] Sequences of non-limiting examples of various components that can 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 encoding the corresponding peptide.

[0500] Table 1. Sequences of various components of CAR

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508] Bispecific CAR

[0509] In certain embodiments, multispecific antibody molecules are bispecific antibody molecules. Bispecific antibodies have specificity for no more than two antigens. The bispecific antibody molecule is characterized by having a first immunoglobulin variable domain sequence with binding specificity to a first epitope and a second immunoglobulin variable domain sequence with binding specificity to a second epitope. In certain embodiments, the first epitope and the second epitope are on the same antigen, such as the same protein (or subunit of a multimeric protein). In certain embodiments, the first epitope and the second epitope overlap. In certain embodiments, the first epitope and the second epitope do not overlap. In certain embodiments, the first epitope and the second epitope are on different antigens, such as different proteins (or different subunits of a multimeric protein). In certain embodiments, the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence with binding specificity to the first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence with binding specificity to the second epitope. In certain embodiments, the bispecific antibody molecule comprises a half antibody with binding specificity to the first epitope and a half antibody with binding specificity to the second epitope. In some embodiments, the bispecific antibody molecule comprises a half antibody or fragment thereof having binding specificity for a first epitope, and a half antibody or fragment thereof having binding specificity for a second epitope. In some embodiments, the bispecific antibody molecule comprises a scFv or fragment thereof having binding specificity for a first epitope, and a scFv or fragment thereof having binding specificity for a second epitope.

[0510] In certain embodiments, antibody molecule is multispecific (such as bispecific or trispecific) antibody molecule.For the generation of the scheme of bispecific or heterodimer antibody molecule and the various configurations of bispecific antibody molecule are described in the 455th-458th paragraph of WO2015 / 142675 submitted to for example on March 13, 2015, it is incorporated to with its entirety by reference.

[0511] In some embodiments, the bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence (e.g., a scFv that has binding specificity for CD19, e.g., comprising a scFv as described herein, or comprising light chain CDRs and / or heavy chain CDRs from a scFv described herein) and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope on a different antigen.

[0512] Chimeric TCR

[0513] In some embodiments, the antibodies and antibody fragments of the present invention (e.g., CD19 antibodies and fragments) can be grafted to one or more constant domains of a T cell receptor ("TCR") chain (e.g., TCR α or TCR β chain) to produce a chimeric TCR. Without wishing to be bound by theory, it is believed that the chimeric TCR will signal through the TCR complex upon antigen binding. For example, scFv as disclosed herein can be grafted to a constant domain (e.g., at least a portion of an extracellular constant domain), a transmembrane domain, and a cytoplasmic domain of a TCR chain (e.g., a TCR α chain and / or a TCR β chain). As another example, an antibody fragment (e.g., a VL domain as described herein) can be grafted to the constant domain of a TCR α chain, and an antibody fragment (e.g., a VH domain as described herein) can be grafted to the constant domain of a TCR β chain (or alternatively, a VL domain can be grafted to the constant domain of a TCR β chain, and a VH domain can be grafted to a TCR α chain). As another example, the CDRs of an antibody or antibody fragment can be transplanted into TCR α and / or β chains to produce a chimeric TCR. For example, the LCDR disclosed herein can be grafted into the variable domains of the TCR α chain, and the HCDR disclosed herein can be grafted into the variable domains of the TCR β chain, or vice versa. Such chimeric TCRs can be produced by methods known in the art, for example (e.g., Willemsen RA et al., Gene Therapy [gene therapy] 2000; 7: 1369–1377; Zhang T et al., Cancer Gene Ther [cancer gene therapy] 2004; 11: 487–496; Aggen et al., Gene Ther. [gene therapy] April 2012; 19 (4): 365-74).

[0514] Non-antibody scaffolds

[0515] In an embodiment, the antigen binding domain comprises a non-antibody scaffold, for example, fibronectin, ankyrin, a domain antibody, a lipocalin, a small modular immunopharmaceutical, a maxibody, protein A, or an affilin. The non-antibody scaffold has the ability to bind to a target antigen on a cell. In an embodiment, 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, as long as the resulting polypeptide comprises at least one binding region that specifically binds to a target antigen on a target cell.

[0516] Non-antibody scaffolds include: fibronectin (Novartis, Massachusetts), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd., Cambridge, Massachusetts, and Ablynx nv, Zimvinarad, Belgium), lipocalin (Pieris Proteolab AG, Freising, Germany), small modular immunopharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, Washington), large antibodies (Avidia, Inc., Mountain View, California), protein A (Affibody AG, Sweden), and affilin (γ-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany).

[0517] In some embodiments, the antigen binding domain comprises an extracellular domain of a molecule that binds an opposing ligand on the surface of a target cell, or an opposing ligand-binding fragment thereof.

[0518] Immune effector cell may include a recombinant DNA construct comprising a sequence encoding CAR, wherein CAR includes an antigen binding domain (e.g., antibody or antibody fragment, TCR or TCR fragment) specifically bound to a B cell antigen (e.g., B cell antigen as described herein), and an intracellular signaling domain. The intracellular signaling domain may include a costimulatory signaling domain and / or a primary signaling domain, such as a ζ chain. As described elsewhere, the methods described herein may include transducing a cell from a cell population depleted of, for example, T regulatory cells using a nucleic acid encoding CAR (e.g., CAR as described herein).

[0519] In some embodiments, the CAR comprises a scFv domain, wherein the scFv may be preceded by an optional leader sequence (as provided in SEQ ID NO: 1) and followed by an optional hinge sequence (as provided in SEQ ID NO: 2 or SEQ ID NO: 36 or SEQ ID NO: 38), a transmembrane region (as provided in SEQ ID NO: 6), an intracellular signaling domain comprising SEQ ID NO: 7 or SEQ ID NO: 16, and a CD3 zeta sequence comprising SEQ ID NO: 9 or SEQ ID NO: 10, e.g., wherein these domains are contiguous and in the same reading frame to form a single fusion protein.

[0520] In some embodiments, exemplary CAR constructs include 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 include 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).

[0521] 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 sequence of the intracellular signaling domain of the 4-1BB protein is provided as SEQ ID NO: 7. An exemplary sequence of the intracellular signaling domain of CD27 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.

[0522] In some embodiments, the immune effector cell includes 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, wherein the sequence is continuous and in the same reading frame with the nucleic acid sequence encoding the intracellular signaling domain. Exemplary intracellular signaling domains that can be used in CAR include, but are not limited to, one or more intracellular signaling domains such as CD3-ζ, CD28, CD27, 4-1BB, etc. In some cases, CAR can include any combination of CD3-ζ, CD28, 4-1BB, etc.

[0523] The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, for example, by screening a library from cells expressing the nucleic acid molecule, by obtaining the nucleic acid molecule from a vector known to include the nucleic acid molecule, or by isolating it directly from cells and tissues containing the gene using standard techniques. Alternatively, the nucleic acid of interest can be produced synthetically rather than cloned.

[0524] Nucleic acid encoding a CAR can be introduced into immune effector cells using, for example, retroviral or lentiviral vector constructs.

[0525] It is also possible to use, for example, an RNA construct that can be directly transfected into a cell to introduce the nucleic acid encoding CAR into immune effector cells. The method for producing mRNA for transfection is related to in vitro transcription (IVT) of the template with specially designed primers, then adding poly (A) to produce a construct containing 3' and 5' untranslated sequences ("UTR") (for example, 3' and / or 5'UTR as described herein), 5' caps (for example, 5' caps as described herein) and / or internal ribosome entry sites (IRES) (for example, IRES as described herein), nucleic acids to be expressed and poly (A) tails, typically 50-2000 bases in length (for example, herein, for example, SEQ ID NO:35). The RNA produced in this way can effectively transfect different types of cells. In certain embodiments, the template includes a sequence for CAR. In certain embodiments, RNA CAR vectors are transduced into cells, for example, in T cells, by electroporation.

[0526] Antigen binding domain

[0527] In some embodiments, multiple immune effector cells (e.g., cell colonies depleted of T regulatory cells) include nucleic acids encoding CARs that include target-specific binding elements (otherwise referred to as antigen binding domains). The selection of binding elements depends on the type and number of the ligands limiting the target cell surface. For example, antigen binding domains can be selected to identify the ligands acting as cell surface markers on target cells associated with a particular disease state. Therefore, the example of cell surface markers that can serve as the ligands of the antigen binding domains in CARs described herein include those related to autoimmune diseases.

[0528] In some embodiments, the portion of the CAR that comprises an antigen binding domain comprises an antigen binding domain that targets a B cell antigen (e.g., a B cell antigen described herein).

[0529] The antigen binding domain can be any domain that is combined with an antigen, including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and functional fragments thereof, including but not limited to single domain antibodies (heavy chain variable domains (VH), light chain variable domains (VL), and variable domains (VHH) of nanobodies such as camel sources), and alternative scaffolds (such as recombinant fibronectin domains, etc.), T cell receptors (TCR) or fragments thereof (e.g., single chain TCR) known in the art 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 use in humans, it can be beneficial for the antigen binding domains of the CAR to comprise people or humanized residues of the antigen binding domains of an antibody or antibody fragment.

[0530] CD19 CAR

[0531] In some embodiments, the CAR-expressing cell described herein is a CD19 CAR-expressing cell (e.g., a cell expressing a CAR that binds to human CD19).

[0532] In some embodiments, the antigen binding domain of CD19 CAR has the same or similar binding specificity as the FMC63 scFv fragment described in Nicholson et al. Mol. Immun. [Molecular Immunology] 34 (16-17): 1157-1165 (1997). In some embodiments, the antigen binding domain of CD19 CAR includes the scFv fragment described in Nicholson et al. Mol. Immun. [Molecular Immunology] 34 (16-17): 1157-1165 (1997).

[0533] 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 determining the binding and efficacy of various CAR constructs.

[0534] In certain embodiments, the parent mouse scFv sequence is the CAR19 construct provided in PCT Publication WO 2012 / 079000 (incorporated herein by reference). In certain embodiments, the anti-CD19 binding domain is the scFv described in WO 2012 / 079000.

[0535] In some embodiments, the CAR molecule comprises a fusion polypeptide sequence provided as SEQ ID NO: 12 in PCT Publication WO 2012 / 079000, which provides a murine scFv fragment that specifically binds to human CD19.

[0536] In some embodiments, the CD19 CAR comprises the amino acid sequence provided as SEQ ID NO: 12 in PCT Publication WO 2012 / 079000.

[0537] In some embodiments, the amino acid sequence is:

[0538] Diqmtqttsslsaslgdrvtiscrasqdiskylnwyqqkpdgtvklliyhtsrlhsgvpsrfsgsgsgtdysltisnleqediatyfcqqgntlpytfgggtkleitggggsggggs gggsevklqesgpglvapsqslsvtctvsgvslpdygvswirqpprkglewlgviwgsettyynsalksrltiikdnsksqvflkmnslqtddtaiyycakhyyyggsyamdywgq gtsvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggc elrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr(SEQ ID NO:292), or a sequence substantially homologous thereto.

[0539] In some embodiments, the CD19 CAR has the USAN name TISAGENLECLEUCEL-T. In an embodiment, CTL019 is prepared by genetic modification of T cells, and CTL019 is mediated by stable insertion of a self-inactivating replication-defective lentiviral (LV) vector containing a CTL019 transgene under the control of an EF-1α promoter. CTL019 can be a mixture of transgenic positive and negative T cells, which are delivered to the subject based on the percentage of transgenic positive T cells.

[0540] In some embodiments, the colony of CAR T cells specifically bound to CD19 includes radakiolunsai. Redakiolunsai is prepared using autologous T cells obtained from peripheral blood mononuclear cells (e.g., from subjects with autoimmune diseases or disorders) by leukocyte apheresis, and is subsequently transduced with a self-inactivating non-replicating lentiviral vector encoding a T cell chimeric antigen receptor targeting CD19. The expressed transgene includes a CD8α leader sequence, a mouse anti-CD19 single-chain variable region fragment (scFv) derived from mouse hybridoma FMC63, CD8α hinge and transmembrane region, and 4-1BB (CD137) and CD3ζ (TCRζ) signaling domains, and is controlled by an elongation factor 1α (EF1α) promoter. The construct is flanked by 5' and 3' long terminal repeats (LTRs) and also contains ψ packaging signals, Rev response elements (RREs), central polypurine region (cPPT) sequences, and optimized woodchuck hepatitis virus post-transcriptional regulatory elements (WPREs). Leukocyte apheresis material is enriched for CD4 / CD8 T cells by positive immune selection, activated by CD3 and CD28 agonists and transduced with a vector. In the absence of further cell propagation, the T cells are washed, formulated for infusion, and cryopreserved. Redaquilansel consists of a mixture of ≥80% T cells and ≤1% B cells, as well as transgenic positive T cells (>3.4%) and negative T cells. The CD4+ and CD8+ initial T cell subsets (CD45RA+CCR7+) present in the leukocyte apheresis material are retained in large quantities.

[0541] In some embodiments, the CAR-expressing cells or CAR-positive cells described herein (e.g., cells expressing CD19 CAR or CD19 CAR-positive cells) are radakinumab. In some embodiments, the population of ARM-CD19 CAR T cells is radakinumab.

[0542] In some embodiments, radakinumab is prepared from autologous T cells obtained from a subject 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 significant lung involvement (e.g., for autologous HSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-related myositis, e.g., antisynthetase syndrome with ILD), vasculitis (e.g., ANC A-related vasculitis), severe refractory Sjögren's syndrome, severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-related 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-related disease), NMDAR+ encephalitis, or antibody-related neurological paraneoplastic diseases), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis.

[0543] In other 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).

[0544] For clinical settings, the humanization of mouse CD19 antibodies may be desirable, wherein mouse-specific residues can induce human-anti-mouse antigen (HAMA) responses in patients receiving CART19 treatment (i.e., T cell therapy transduced with CAR19 constructs). The generation, characterization, and efficacy of humanized CD19CAR sequences are described in International Application WO 2014 / 153270, which is incorporated herein by reference in its entirety, including Examples 1-5 (pp. 115-159).

[0545] In some embodiments, the CAR molecule is a humanized CD19 CAR comprising the following amino acid sequence: EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS (SEQ ID NO: 293)

[0546] In some embodiments, the CAR molecule is a humanized CD19 CAR comprising the following amino acid sequence: EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGS YAMDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:294)

[0547] Any CD19 CAR known in the art can be used according to the present disclosure, such as the CD19 antigen binding domain of any known CD19 CAR. For example, LG-740; CD19 CAR is described in the following: U.S. Patent No. 8,399,645; U.S. Patent No. 7,446,190; Xu et al., Leuk Lymphoma. 2013 54(2):255-260 (2012); Cruz et al., Blood 122(17):2965-2973 (2013); Brentjens et 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 SocGen Cell Ther(ASGCT) [American Society for Gene and Cell Therapy (ASGCT) 16th Annual Meeting] (May 15–18, Salt Lake City) 2013, abstract 10.

[0548] Exemplary CD19 CARs include the CD19 CARs described herein, or the anti-CD19 CARs 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, NC T02546739, NCT02656147, NCT02772198, NCT00709033, NCT02081937, NCT00924326, NCT02735083, NCT02794246, NCT02746952, NCT0159369 6. NCT02134262, NCT01853631, NCT02443831, NCT02277522, NCT02348216, NCT02614066, NCT02030834, NCT02624258, NCT02625480, NCT02 030847, NCT02644655, NCT02349698, NCT02813837, NCT02050347, NCT01683279, NCT02529813, NCT02537977, NCT02799550, NCT02672501, N CT02819583, NCT02028455, NCT01840566, NCT01318317, NCT01864889, NCT02706405, NCT01475058, NCT01430390, NCT02146924, NCT02051 257. NCT02431988, NCT01815749, NCT02153580, NCT01865617, NCT02208362, NCT02685670, NCT02535364, NCT02631044, NCT02728882, NCT0 2735291, 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.

[0549] In some embodiments, the CD19 CAR comprises a sequence, e.g., a CDR, VH, VL, scFv, or full-length CAR sequence disclosed in Table 2, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto.

[0550] Table 2. Amino acid sequences of exemplary anti-CD19 molecules

[0551]

[0552]

[0553]

[0554]

[0555]

[0556]

[0557]

[0558]

[0559]

[0560]

[0561] Other exemplary CAR properties

[0562] In some embodiments, the B cell antigen binding domain is a fragment, e.g., a single chain variable region fragment (scFv). In some embodiments, the B cell antigen binding domain is a Fv, Fab, (Fab')2, or a bifunctional (e.g., bispecific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. [European Journal of Immunology] 17, 105 (1987)). In some embodiments, the antibodies and fragments thereof of the present invention bind to B cell antigen proteins as described herein with wild-type or enhanced affinity.

[0563] In some cases, scFv can be prepared according to methods known in the art (see, for example, 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 generated by linking the VH and VL regions together using a flexible polypeptide linker. ScFv molecules contain a linker (e.g., a Ser-Gly linker) of optimized length and / or amino acid composition. The linker length can greatly affect how the variable regions of the scFv fold and interact. In fact, if a short polypeptide linker (e.g., between 5-10 amino acids) is used, intrachain folding can be prevented. Interchain folding is also required to bring the two variable regions together to form a functional epitope binding site. For examples of linker orientation and size, 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 PCT Publication Nos. WO 2006 / 020258 and WO 2007 / 024715 (incorporated herein by reference).

[0564] scFv can comprise a linker having 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 can comprise any naturally occurring amino acid. In certain embodiments, the linker sequence comprises amino acid glycine and serine. In certain embodiments, the linker sequence comprises multiple groups of glycine and serine repeats, such as (Gly4Ser)n, wherein n is a positive integer equal to or greater than 1 (SEQ ID NO:25). In certain embodiments, the linker can be (Gly4Ser)4 (SEQ ID NO:27) or (Gly4Ser)3 (SEQ ID NO:28). Variations in linker length can retain or enhance activity, thereby producing excellent efficacy in activity studies.

[0565] 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 preparing such TCRs are known in the art. See, for example, Willemsen RA et al., Gene Therapy [gene therapy] 7: 1369–1377 (2000); Zhang T et al., Cancer Gene Ther [cancer gene therapy] 11: 487–496 (2004); Aggen et al., Gene Ther. [gene therapy] 19 (4): 365-74 (2012) (the references are incorporated herein in their entirety). For example, a scTCR can be engineered to contain Vα and Vβ genes from a T cell clone connected by a linker (e.g., a flexible peptide).

[0566] transmembrane domain

[0567] About transmembrane domain, in various embodiments, CAR can be designed to include the transmembrane domain of the extracellular domain attached to CAR.The transmembrane domain may include one or more other amino acids adjacent to the transmembrane region, such as one or more amino acids related to the extracellular region of the protein cell derived across the membrane (such as 1,2,3,4,5,6,7,8,9,10 to 15 amino acids in the extracellular region) and / or one or more other amino acids related to the intracellular region of the protein derived transmembrane protein (such as 1,2,3,4,5,6,7,8,9,10 to 15 amino acids in the intracellular region). In certain embodiments, the transmembrane domain is a transmembrane domain related to one of the other domains of the CAR used. In some cases, it is possible to select or modify the transmembrane domain by amino acid substitution to avoid such a domain from being combined with the transmembrane domain of the same or different surface membrane proteins, such as to minimize the interaction with other members of the receptor complex. In certain embodiments, the transmembrane domain can be homodimerized with another CAR on the surface of the cell (for example, CART cells) expressing CAR. In some embodiments, the amino acid sequence of the transmembrane domain can 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., CART).

[0568] The transmembrane domain can be derived from a natural source or from a recombinant source. In the case where the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. In some embodiments, whenever CAR binds to a target, the transmembrane domain can conduct the signal to one or more intracellular domains. The transmembrane domain used in particular in the present invention may include at least one or more transmembrane regions: for example, α, β or ζ chains of T cell receptors, CD28, CD3ε, CD45, CD4, CD5, CD8 (e.g., CD8α, CD8β), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, the transmembrane domain may include at least one or more transmembrane regions of a costimulatory molecule, which is, for example, an 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, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (C D278), 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, CD1 1d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB 7. NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM 1. 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 ligands that specifically bind to CD83.

[0569] In some cases, the transmembrane domain can be attached to the extracellular region of CAR (e.g., the antigen binding domain of 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.

[0570] 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.

[0571] In some embodiments, the hinge or spacer comprises an IgD hinge. For example, in some embodiments, the hinge or spacer comprises a hinge having an amino acid sequence of SEQ ID NO: 4. In some embodiments, the hinge or spacer comprises a hinge encoded by a nucleotide sequence of SEQ ID NO: 15.

[0572] In some embodiments, the transmembrane domain can be recombinant, in which case it will primarily comprise hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine can be found at each end of the recombinant transmembrane domain.

[0573] Optionally, a short oligopeptide or polypeptide linker between 2 and 10 amino acids in length can form a linkage between the transmembrane domain and the cytoplasmic region of the CAR. Glycine-serine doublets provide particularly suitable linkers. For example, in some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the linker is encoded by the nucleotide sequence of SEQ ID NO: 16.

[0574] In some embodiments, the hinge or spacer comprises a KIR2DS2 hinge.

[0575] cytoplasmic domain

[0576] The cytoplasmic domain or region of the CAR of the present invention includes an intracellular signaling domain. The intracellular signaling domain is generally responsible for activating at least one normal effector function of the immune cell into which the CAR has been introduced.

[0577] Examples of intracellular signaling domains for use in the CARs of the present invention include the cytoplasmic sequences of T cell receptors (TCRs) and co-receptors (which act synergistically to initiate signal transduction following antigen receptor engagement), as well as any derivatives or variants of these sequences and any recombinant sequences with the same functional capacity.

[0578] It is known that the signal generated by TCR alone is insufficient to fully activate T cells and that secondary and / or co-stimulatory signals are also required. Thus, T cell activation can be considered to be mediated by two different classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic domains, such as co-stimulatory domains).

[0579] The primary signaling domain regulates the primary activation of the TCR complex in a stimulatory or inhibitory manner. The primary intracellular signaling domain that acts in a stimulatory manner can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.

[0580] Examples of primary intracellular signaling domains containing ITAMs 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, the CAR of the present invention comprises an intracellular signaling domain, such as a primary signaling domain of CD3-ζ.

[0581] In some embodiments, the primary signaling domain comprises a modified ITAM domain, e.g., a mutant ITAM domain having altered (e.g., increased or decreased) activity compared to a native ITAM domain. In some embodiments, the primary signaling domain comprises a primary intracellular signaling domain comprising a modified ITAM, e.g., a primary intracellular signaling domain comprising an optimized and / or truncated ITAM. In some embodiments, the primary signaling domain comprises one, two, three, four, or more ITAM motifs.

[0582] Additional examples of molecules containing a primary intracellular signaling domain that are particularly useful in the present invention include those of DAP10, DAP12, and CD32.

[0583] The intracellular signaling domain of CAR can include a primary signaling domain (e.g., CD3-ζ signaling domain) alone, or it can be combined with any other desired one or more intracellular signaling domains useful in the context of the CAR of the present invention. For example, the intracellular signaling domain of CAR can include a primary signaling domain (e.g., CD3ζ chain portion) and a costimulatory signaling domain. The costimulatory signaling domain refers to the portion of the intracellular domain of CAR that includes a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for the effective 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, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD 278), 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, CD11 d. ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, 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 ligands that specifically bind to CD83, etc.For example, it has been shown that CD27 costimulation can enhance the expansion, effector function and survival of human CART cells in vitro, and increase the persistence and anti-tumor activity of human T cells in vivo (Song et al. Blood. [blood] 2012; 119 (3): 696-706). The intracellular signaling sequences in the cytoplasmic portion of the CAR of the present invention can be connected to each other in a random or specified order. Optionally, a short oligopeptide or polypeptide linker, such as a length between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids), can form a connection 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.

[0584] 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, 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 comprises two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

[0585] In some embodiments, the intracellular signaling domain is designed to include the signaling domain of CD3-ζ and the signaling domain of CD28. In some embodiments, the intracellular signaling domain is designed to include the signaling domain of CD3-ζ 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 CD3-ζ is the signaling domain of SEQ ID NO: 9 (mutant CD3ζ) or SEQ ID NO: 10 (wild-type human CD3ζ).

[0586] In some embodiments, the intracellular signaling domain is designed to include the signaling domain of CD3-ζ 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.

[0587] In some embodiments, the cell is designed to include a signaling domain of CD3-ζ and a 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 cell is designed to include a signaling domain of CD3-ζ and a 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.

[0588] Co-expression of CAR with other molecules or agents

[0589] Co-expression of a second CAR

[0590] In some embodiments, the CAR-expressing cells described herein can further comprise a second CAR, e.g., a second CAR comprising a different antigen binding domain (e.g., to the same target (e.g., CD19) or a different target (e.g., a target other than CD19, e.g., a target described herein)).

[0591] In some embodiments, the cell expressing CAR comprises a first CAR and a second CAR, the first CAR targeting a first antigen and comprising an intracellular signaling domain having a costimulatory signaling domain but not having a primary signaling domain, the second CAR targeting a second different antigen and comprising an intracellular signaling domain having a primary signaling domain but not having a costimulatory signaling domain. The costimulatory signaling domain (eg, 4-1BB, CD28, CD27, OX-40, or ICOS) is placed on the first CAR, and the primary signaling domain (eg, CD3 ζ) is placed on the second CAR to limit the activity of CAR to cells expressing two targets. In some embodiments, the cell expressing CAR comprises a first CAR (which includes an antigen binding domain, a transmembrane domain, and a costimulatory domain), and a second CAR (which targets other antigens and includes an antigen binding domain, a transmembrane domain, and a primary signaling domain). In some embodiments, the CAR-expressing cell comprises a first CAR (which includes an antigen binding domain, a transmembrane domain, and a primary signaling domain), and a second CAR (which targets another antigen and includes an antigen binding domain for the antigen, a transmembrane domain, and a co-stimulatory signaling domain).

[0592] In certain embodiments, when the cell expressing CAR includes two or more different CARs, the antigen binding domains of different CARs can make the antigen binding domains not interact with each other.For example, the cell expressing the first and second CARs can have the antigen binding domains of the first CAR (for example, as a fragment, such as scFv), which does not form an association with the antigen binding domains of the second CAR, such as the antigen binding domains of the second CAR are VHH.

[0593] In some embodiments, the antigen binding domain comprises a single domain antigen binding (SDAB) molecule, which includes a molecule whose complementary determining region is a part of a single domain polypeptide. Examples include, but are not limited to, heavy chain variable domains, binding molecules naturally lacking light chains, single domains derived from conventional 4-chain antibodies, engineered domains, and single domain scaffolds other than those derived from antibodies. The SDAB molecule can be any prior art or any future single domain molecule. The SDAB molecule can be derived from any species, including but not limited to mice, humans, camels, llamas, lampreys, fish, sharks, goats, rabbits, and cattle. The term also includes naturally occurring single domain antibody molecules from species other than camelids and sharks.

[0594] In some embodiments, the SDAB molecule can be derived from the variable region of an immunoglobulin found in fish, such as, for example, the variable region of an immunoglobulin isotype found in shark serum called the novel antigen receptor (NAR). Methods for generating single domain molecules derived from NAR variable regions ("IgNARs") are described in WO 03 / 014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

[0595] In some embodiments, the SDAB molecule is a naturally occurring single domain antigen binding molecule, referred to as a heavy chain lacking a light chain. Such single domain molecules are disclosed, for example, in WO 9404678 and Hamers-Casterman, C. et al. (1993) Nature 363: 446-448. For clarity, such variable domains derived from naturally occurring heavy chain molecules lacking light chains are referred to herein as VHHs or nanobodies to distinguish them from conventional VHs of four-chain immunoglobulins. Such VHH molecules can be derived from species of the Camelidae family, such as camels, llamas, dromedaries, alpacas, and guanacos. Other species besides the Camelidae family can produce heavy chain molecules that naturally lack light chains; such VHHs are within the scope of the present invention.

[0596] SDAB molecules can be recombinant, CDR-grafted, humanized, camelized, deimmunized, and / or generated in vitro (e.g., selected by phage display).

[0597] It has also been found that cells with multiple chimeric membrane-embedded receptors (these chimeric membrane-embedded receptors comprise antigen binding domains, and the antigen binding domains of the receptors interact with each other) may be undesirable, for example, because it inhibits the ability of one or more antigen binding domains to bind to their cognate antigens. Therefore, disclosed herein are cells with first and second non-naturally occurring chimeric membrane-embedded receptors, which comprise antigen binding domains that minimize such interactions. Also disclosed herein are nucleic acids encoding a first non-naturally occurring chimeric membrane-embedded receptor and a second non-naturally occurring chimeric membrane-embedded receptor (the chimeric membrane-embedded receptor comprises antigen binding domains that minimize such interactions) and methods for preparing and using such cells and nucleic acids. In some embodiments, the antigen binding domain of one of the first and second non-naturally occurring chimeric membrane-embedded receptors 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.

[0598] In some embodiments, the compositions herein include the first and second CARs, wherein the antigen binding domains of one of the first and second CARs do not include a variable light domain and a variable heavy domain. In some embodiments, the antigen binding domains of one of the first and second CARs are scFv, and the other is not scFv. In some embodiments, the antigen binding domains of one of the first and second CARs include 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 some embodiments, the antigen binding domains of one of the first and second CARs include nano antibodies. In some embodiments, the antigen binding domains of one of the first and second CARs include camelid VHH domains.

[0599] In some embodiments, 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 some embodiments, the antigen binding domain of one of the first and second CARs comprises an scFv, and the other comprises a nanobody. In some embodiments, the antigen binding domain of one of the first and second CARs comprises an scFv, and the other comprises a camelid VHH domain.

[0600] In some embodiments, when present on the cell surface, the 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. In some embodiments, the 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% of the binding of the antigen binding domain of the first CAR to its cognate antigen in the absence of the second CAR, for example, 85%, 90%, 95%, 96%, 97%, 98% or 99%.

[0601] In some embodiments, when present on the cell surface, the antigen binding domains of the first and second CARs are less associated with each other than when both are scFv antigen binding domains. In some embodiments, the antigen binding domains of the first and second CARs are less associated with each other than when both are scFv antigen binding domains by at least 85%, 90%, 95%, 96%, 97%, 98% or 99%, for example, 85%, 90%, 95%, 96%, 97%, 98% or 99%.

[0602] Co-expression of agents that enhance CAR activity

[0603] In some embodiments, the CAR-expressing cells described herein can further express another agent, such as an agent that enhances the activity or fitness of the CAR-expressing cell.

[0604] For example, in some embodiments, the agent can be an agent that inhibits modulation or regulates (e.g., inhibits) a molecule of T cell function. In certain embodiments, the molecule that modulates or regulates T cell function is an inhibitory molecule. In certai...

Claims

1. A method of treating a subject with an autoimmune disease or disorder, wherein the autoimmune disease or disorder is, 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 significant pulmonary involvement (e.g., for autologous HSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-related myositis, e.g., antisynthetase syndrome with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjögren's syndrome, severe refractory neuroimmune myopathy, Disease (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-related 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-related disease), NMDAR+ encephalitis, or antibody-related neurological paraneoplastic disease), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis, the method comprising administering to the subject a population of cells (e.g., T cells) expressing a CD19 chimeric antigen receptor (CAR) or comprising a nucleic acid configured to express a CD19 CAR, wherein the cell population is prepared by a method 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 co-stimulatory molecule on the surface of the cell; (ii) contacting the cell (e.g., T cell) population with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding a CAR, thereby providing a cell (e.g., T cell) population comprising the nucleic acid molecule, wherein the CAR comprises a CD19 antigen binding domain ("CD19 CAR"); and (iii) harvesting the cell (eg, T cell) population for storage (eg, reconstituting the cell population in a cryopreservation medium) or administration, wherein: (a) step (ii) is performed together with step (i) or not later than 20 hours after the start of step (i), for example not later than 12, 13, 14, 15, 16, 17, or 18 hours after the start of step (i), for example not later than 18 hours after the start of step (i), and Step (iii) is performed no later than 30 (e.g. 26) hours after the start of step (i), for example no later than 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the start of step (i), for example no later than 24 hours after the start of step (i), (b) step (ii) is performed together with step (i) or not later than 20 hours after the start of step (i), for example not later than 12, 13, 14, 15, 16, 17, or 18 hours after the start of step (i), for example not later than 18 hours after the start of step (i), and step (iii) is performed no later than 30 hours after the start of step (ii), for example no later than 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the start of step (ii), or (c) the cell population from step (iii) does not expand, or expands by no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40%, e.g., by no more than 10%, compared to the cell population at the start of step (i), e.g., as assessed by viable cell number, Optionally, the nucleic acid molecule in step (ii) is on a viral vector, optionally, the nucleic acid molecule in step (ii) is an RNA molecule on a viral vector, optionally, step (ii) comprises transducing the cell (e.g., T cell) population with a viral vector, the viral vector comprising a nucleic acid molecule encoding the CAR.

2. The method of claim 1, wherein the agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3 (e.g., an anti-CD3 antibody), and wherein the agent that stimulates a co-stimulatory 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 wherein the agent that stimulates the CD3 / TCR complex or the agent that stimulates a co-stimulatory molecule 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 a scFv), a small molecule, or a ligand (e.g., a naturally occurring wherein the agent that stimulates the CD3 / TCR complex or the agent that stimulates the co-stimulatory molecule does not comprise beads; optionally wherein the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody and the agent that stimulates the co-stimulatory molecule comprises an anti-CD28 antibody; optionally wherein the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody covalently attached to a colloidal polymer nanomatrix and the agent that stimulates the co-stimulatory molecule comprises an anti-CD28 antibody covalently attached to a colloidal polymer nanomatrix; optionally wherein the agent that stimulates the CD3 / TCR complex and the agent that stimulates the co-stimulatory molecule comprise T cell TransAct TM .

3. A method as claimed in claim 1 or 2, wherein step (i) increases the percentage of cells expressing CAR in the cell population from step (iii) compared to cells prepared by a method that is otherwise similar except that step (i) is not included, for example, the cell population from step (iii) shows a higher percentage (e.g., at least 10%, 20%, 30%, 40%, 50%, or 60%) of cells expressing CAR.

4. The method according to any one of claims 1 to 3, wherein (a) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the cell population 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 cell population 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 cell population 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 as compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells in the cell population at the start of step (i); (c) the percentage of naive T cells expressing CAR, such as CD45RA+CD45RO-CCR7+ T cells expressing CAR, in the cell population increases during the duration of step (ii), such as by at least 30%, 35%, 40%, 45%, 50%, 55%, or 60% between 18 and 24 hours after the start of step (ii); or (d) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the cell population from step (iii) does not decrease, or decreases by no more than 5% or 10%, as compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells in the cell population at the start of step (i).

5. The method according to any one of claims 1 to 4, wherein (a) the cell population 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 prepared by an otherwise 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 cell population 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 prepared by an otherwise 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 naive T cells expressing CAR, e.g., CD45RA+CD45RO-CCR7+ T cells expressing CAR, in the cell population from step (iii) is higher (e.g., at least 4, 6, 8, 10, or 12 times higher) than the percentage of naive T cells expressing CAR, e.g., CD45RA+CD45RO-CCR7+ T cells expressing CAR, in cells prepared by an otherwise 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 cell population from step (iii) shows 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 prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii); (e) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells in the cell population 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 prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population 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 naive T cells expressing CAR, e.g., CD45RA+CD45RO-CCR7+ T cells expressing CAR, in the cell population from step (iii) is higher (e.g., at least 4, 6, 8, 10, or 12 times higher) than the percentage of naive T cells expressing CAR, e.g., CD45RA+CD45RO-CCR7+ T cells expressing CAR, in cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii).

6. The method according to any one of claims 1 to 5, wherein (a) the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells in the cell population from step (iii) is the same as the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells in the cell population at the beginning of step (i) or differs by no more than 5% or 10%; (b) the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the cell population from step (iii) is reduced by at least 20%, 25%, 30%, 35%, 40%, 45%, or 50% as compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells in the cell population at the start of step (i); (c) the percentage of central memory T cells expressing CAR, such as CCR7+CD45RO+ cells expressing CAR, decreases during the duration of step (ii), such as by at least 8%, 10%, 12%, 14%, 16%, 18%, or 20% between 18-24 hours after the start of step (ii); or (d) the percentage of central memory cells, such as central memory T cells, such as CCR7+CD45RO+ T cells, in the cell population from step (iii) does not increase or increases by no more than 5% or 10% as compared to the percentage of central memory cells, such as central memory T cells, such as CCR7+CD45RO+ T cells in the cell population at the beginning of step (i).

7. The method of any one of claims 1 to 6, wherein (a) the cell population from step (iii) exhibits a lower percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells (e.g., at least 10%, 20%, 30%, or 40% lower) compared to cells prepared by an otherwise similar method except that step (iii) is performed 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); (b) the percentage of central memory cells, such as central memory T cells, such as CCR7+CD45RO+ T cells in the cell population from step (iii) is lower (e.g., at least 20%, 30%, 40%, or 50% lower) than the percentage of central memory cells, such as central memory T cells, such as CCR7+CD45RO+ T cells in cells prepared by an otherwise similar method except that step (iii) is performed 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); (c) the percentage of central memory T cells expressing CAR, such as CCR7+CD45RO+ T cells expressing CAR, in the cell population from step (iii) is lower (e.g., at least 10%, 20%, 30%, or 40%) than the percentage of central memory T cells expressing CAR, such as CCR7+CD45RO+ T cells expressing CAR, in cells prepared by an otherwise similar method except that step (iii) is performed 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); (d) the cell population from step (iii) shows a lower percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells (e.g., at least 10%, 20%, 30%, or 40%) compared to cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, such as 5, 6, 7, 8, or 9 days after step (ii) and before step (iii); (e) the percentage of central memory cells, such as central memory T cells, such as CCR7+CD45RO+ T cells in the cell population from step (iii) is lower (e.g., at least 20%, 30%, 40%, or 50%) than the percentage of central memory cells, such as central memory T cells, such as CCR7+CD45RO+ T cells in cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, such as 5, 6, 7, 8, or 9 days after step (ii) and before step (iii); or (f) the percentage of central memory T cells expressing CAR, such as CCR7+CD45RO+ T cells expressing CAR, in the cell population from step (iii) is lower (e.g., at least 10%, 20%, 30%, or 40%) than the percentage of central memory T cells expressing CAR, such as CCR7+CD45RO+ T cells expressing CAR, in cells prepared by a method otherwise similar to that described in step (ii) but further comprising amplifying the cell (e.g., T cell) population in vitro for more than 3 days, such as 5, 6, 7, 8, or 9 days before step (iii).

8. The method of any one of claims 1 to 7, wherein (a) the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in the cell population from step (iii) is increased as compared to the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in the cell population at the start of step (i); (b) the percentage of CAR-expressing stem cell memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells expressing CAR, in the cell population from step (iii) is increased as compared to the percentage of CAR-expressing stem cell memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells expressing CAR, in the cell population at the beginning of step (i); (c) the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in the cell population from step (iii) is higher than the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in cells prepared by an otherwise 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) the percentage of CAR-expressing stem cell memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in the cell population from step (iii) is higher than the percentage of CAR-expressing stem cell memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in cells prepared by an otherwise 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 cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in the cell population from step (iii) is higher than the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population 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 cell memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in the cell population from step (iii) is higher than the percentage of CAR-expressing stem cell memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in cells prepared by a method otherwise similar except further comprising amplifying the cell (e.g., T cell) population in vitro for more than 3 days, such as 5, 6, 7, 8, or 9 days after step (ii) and before step (iii).

9. The method of any one of claims 1 to 8, wherein (a) the median gene set score (TEM up vs. TSCM down) of the cell population from step (iii) is about the same as or differs by no more than (e.g., increases by no more than) about 25%, 50%, 75%, 100%, or 125% from the median gene set score (TEM up vs. TSCM down) of the cell population at the start of step (i); (b) the median gene set score (TEM up vs. TSCM down) of the population of cells from step (iii) is lower (e.g., at least about 100%, 150%, 200%, 250%, or 300% lower) than the median gene set score (TEM up vs. TSCM down): Cells prepared by an otherwise similar method except that step (iii) is performed 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 prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii); (c) the median gene set score (up Treg vs. down Teff) of the cell population from step (iii) is about the same as or differs by no more than (e.g., increases by no more than) about 25%, 50%, 100%, 150%, or 200% from the median gene set score (up Treg vs. down Teff) of the cell population at the beginning of step (i); (d) the median gene set score (Treg Up vs. Teff Down) of the cell population from step (iii) is lower (e.g., at least about 50%, 100%, 125%, 150%, or 175% lower) than the median gene set score (Treg Up vs. Teff Down) of: Cells prepared by an otherwise similar method except that step (iii) is performed 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 prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii); (e) the median gene set score (downward stemness) of the cell population from step (iii) is about the same as or differs by no more than (e.g., increases by no more than) about 25%, 50%, 100%, 150%, 200%, or 250% from the median gene set score (downward stemness) of the cell population at the beginning of step (i); (f) the median gene set score (downward stemness) of the population of cells from step (iii) is lower (e.g., at least about 50%, 100%, or 125% lower) than the median gene set score (downward stemness) of: Cells prepared by an otherwise similar method except that step (iii) is performed 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 prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii); (g) the median gene set score (upward hypoxia) of the cell population from step (iii) is about the same as or differs by no more than (e.g., increases by no more than) about 125%, 150%, 175%, or 200% from the median gene set score (upward hypoxia) of the cell population at the start of step (i); (h) the median gene set score (hypoxia up) of the population of cells from step (iii) is lower (e.g., at least about 40%, 50%, 60%, 70%, or 80% lower) than the median gene set score (hypoxia up) of: Cells prepared by an otherwise similar method except that step (iii) is performed 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 prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii); (j) the median gene set score (autophagy up) of the cell population from step (iii) is about the same as or differs by no more than (e.g., increases by no more than) about 180%, 190%, 200%, or 210% from the median gene set score (autophagy up) of the cell population at the start of step (i); or (k) the median gene set score (autophagy up) of the population of cells from step (iii) is lower (e.g., at least about 20%, 30%, or 40% lower) than the median gene set score (autophagy up) of: Cells prepared by an otherwise similar method except that step (iii) is performed 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 prepared by an otherwise similar method except further comprising expanding the cell (eg, T cell) population in vitro for more than 3 days, such as 5, 6, 7, 8, or 9 days after step (ii) and before step (iii).

10. The method of any one of claims 1-9, wherein the cell population from step (iii) secretes IL-2 at a higher level (e.g., at least 2, 4, 6, 8, 10, 12, or 14 times higher) after incubation with cells expressing the antigen recognized by the CAR than cells prepared by an otherwise similar method except that step (iii) is performed 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 prepared by an otherwise similar method except that the cell (e.g., T cell) population is expanded in vitro for more than 3 days, such as 5, 6, 7, 8, or 9 days after step (ii) and before step (iii).

11. The method of any one of claims 1-10, wherein the cell population from step (iii) persists for a longer time or expands at a higher level after in vivo administration to the subject compared to cells prepared by an otherwise similar method except that step (iii) is performed 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 prepared by an otherwise similar method except that further comprising expanding the cell (e.g., T cell) population 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 method of any one of claims 1 to 11, wherein the cell population from step (iii) exhibits greater activity (e.g., greater activity at a low dose, e.g., no more than 0.15 x 10 T cells) after in vivo administration to the subject compared to cells prepared by an otherwise 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 compared to cells prepared by an otherwise similar method except that the cell (e.g., T cell) population is expanded in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii). 6 , 0.2x 10 6 , 0.25x 10 6 , or 0.3x 10 6 The dose of viable cells expressing CAR).

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

14. The method of any one of claims 1 to 13, wherein the cell population from step (iii) does not expand or expands for less than 2 hours, such as less than 1 or 1.5 hours, compared to the cell population at the beginning of step (i).

15. The method of any one of claims 1-14, wherein steps (i) and / or (ii) are performed in a cell culture medium (e.g., serum-free culture 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), a 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 performed in a serum-free cell culture medium comprising a serum replacement.

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

18. The method of any one of claims 1 to 17, further comprising, 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 removal (e.g., a fresh product from a thymectomy)) from an entity, such as a laboratory, hospital, or health care 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 removal (e.g., a fresh product from a thymectomy)), optionally wherein: step (iii) is performed no later than 35 hours after the start of step (v), for example no later than 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the start of step (v), for example no later than 30 hours after the start of step (v), or For example, the cell population 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 cell population at the end of step (v), as assessed by viable cell number.

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

20. The method of any one of claims 1 to 17, further comprising, 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 removal (e.g., a cryopreserved product from a thymectomy)) from an entity, such as a laboratory, hospital, or health care 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, e.g., a cryopreserved whole blood product, a cryopreserved bone marrow product, or a cryopreserved organ biopsy or ablation (e.g., a cryopreserved product from a thymectomy)), optionally wherein: step (iii) is performed no later than 35 hours after the start of step (v), for example no later than 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the start of step (v), for example no later than 30 hours after the start of step (v), or For example, the cell population 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 cell population at the end of step (v), as assessed by viable cell number.

21. The method of any one of claims 1 to 20, further comprising step (vi): Culturing a portion of the cell population from step (iii) 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 no more than 7 days, and measuring the level of CAR expression in the portion (e.g., measuring the percentage of live cells expressing CAR in the portion), optionally wherein: Step (iii) comprises harvesting and freezing the cell (e.g., T cell) population, and step (vi) comprises thawing a portion of the cell population 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 no more than 7 days, and measuring the level of CAR expression in the portion (e.g., measuring the percentage of live cells expressing CAR in the portion).

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

23. The method of any one of claims 1-22, wherein the cell population at the start of step (i) or step (1) comprises no less than 50%, 60%, or 70% of cells expressing IL6R (e.g., cells positive for IL6Rα and / or IL6Rβ).

24. The method of any one of claims 1-23, wherein steps (i) and (ii) or steps (1) and (2) are performed in a cell culture medium comprising IL-15 (eg, hetIL-15 (IL15 / sIL-15Ra)).

25. The method of claim 24, wherein IL-15 increases the ability of the cell population to expand, e.g., after 10, 15, 20, or 25 days.

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

27. A method of treating a subject 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 significant pulmonary involvement (e.g., for autologous HSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-related myositis, e.g., antisynthetase syndrome with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjögren's syndrome, severe refractory neuroimmune disease (e.g., Myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-related 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-related disease), NMDAR+ encephalitis, or antibody-related neurological paraneoplastic disease), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis, the method comprising administering to the subject a cell population engineered to express a CD19 CAR ("CAR-expressing cell population"), the population comprising: (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 cell population before being engineered to express the CAR; (b) initial cells, e.g., initial T cells, e.g., CD45RO-CCR7+ T cells, that vary within about 5% to about 10%, e.g., as compared to the percentage of initial cells, e.g., initial T cells, e.g., CD45RO-CCR7+ cells in the same cell population before being engineered to express the CAR; (c) an increase in the percentage of initial cells, e.g., initial T cells, e.g., CD45RO-CCR7+ T cells, as compared to the percentage of initial cells, e.g., initial T cells, e.g., CD45RO-CCR7+ cells in the same cell population before being engineered to express the CAR, e.g., an increase of at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3 times; (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 cell population before being engineered to express the CAR; (e) 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 cell population before being engineered to express the CAR, which varies within about 5% to about 10%; (f) a decrease in the 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 cell population before being engineered to express the CAR, e.g., a decrease of at least 20%, 25%, 30%, 35%, 40%, 45%, or 50%; (g) about the same percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, as compared to the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in the same cell population before being engineered to express the CAR; (h) stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, that vary within about 5% to about 10% as compared to the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in the same cell population before being engineered to express the CAR; or (i) increasing the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, as compared to the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in the same cell population before being engineered to express the CAR.

28. A method of treating a subject 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 significant pulmonary involvement (e.g., for autologous HSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-related myositis, e.g., antisynthetase syndrome with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjögren's syndrome, severe refractory neuroimmune disease ( For example, myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-related 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-related disease), NMDAR+ encephalitis, or antibody-related neurological paraneoplastic disease), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis, the method comprising administering to the subject a cell population engineered to express a CD19 CAR ("CAR-expressing cell population"), wherein: (a) the median gene set score (TEM up vs. TSCM down) of the cell population is about the same as or differs by no more than (e.g., increases by no more than) about 25%, 50%, 75%, 100%, or 125% from the median gene set score (TEM up vs. TSCM down) of the same cell population before being engineered to express the CAR; (b) the median gene set score (upward Treg vs. downward Teff) of the cell population is about the same as or differs by no more than (e.g., increases by no more than) about 25%, 50%, 100%, 150%, or 200% from the median gene set score (upward Treg vs. downward Teff) of the cell population before being engineered to express the CAR; (c) the median gene set score (downward stemness) of the cell population is about the same as or differs by no more than (e.g., increases by no more than) about 25%, 50%, 100%, 150%, 200%, or 250% from the median gene set score (downward stemness) of the cell population before being engineered to express the CAR; (d) the median gene set score (upward hypoxia) of the cell population is about the same as or differs by no more than (e.g., increases by no more than) about 125%, 150%, 175%, or 200% from the median gene set score (upward hypoxia) of the cell population prior to being engineered to express the CAR; or (e) the median gene set score (up autophagy) of the cell population is approximately the same as or differs by no more than (e.g., increases by no more than) about 180%, 190%, 200%, or 210% from the median gene set score (up autophagy) of the cell population before being engineered to express the CAR.

29. A method of treating a subject 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 significant pulmonary involvement (e.g., for autologous HSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-related myositis, e.g., antisynthetase syndrome with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjögren's syndrome The method is for a subject having a neuroimmune disease (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-related 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-related disease), NMDAR+ encephalitis, or antibody-related neurological paraneoplastic disease), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis, the method comprising administering radakinumab to the subject.

30. A method of treating a subject having a severe refractory autoimmune disease, the method comprising administering radakinumab to the subject.

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

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

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

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

35. The method of claim 34, wherein (a) the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, (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 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 thereto.

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

37. The method of claim 36, wherein (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 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 thereto.

38. The method of any one of claims 1-28, 36 or 37, wherein the population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is approximately 0.5 x 10 6 Up to 50x 10 6 viable cells expressing CAR, for example, about 5 x 10 6 The method comprises administering a dose of 5 x 10 viable CAR-expressing cells to a subject, optionally wherein the CAR-expressing cells (e.g., CD19 CAR-expressing cells) are divided into a population of 5 x 10 6 A dose of viable cells expressing CAR is administered.

39. The method of any one of claims 1-28, 36 or 37, wherein the population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is approximately 2.5 x 10 6 Up to 2.5x 10 8 viable cells expressing CAR, for example, about 1.25 x 10 7 The CAR-expressing cells (e.g., CD19 CAR-expressing cells) are administered at a dose of 1.25 x 10 7 A dose of viable cells expressing CAR is administered.

40. The method of any one of claims 1-28, 36 or 37, wherein the population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is approximately 1.25 x 10 7 Up to 1.25x 10 9 viable cells expressing CAR, for example, about 1.25 x 10 8 The CAR-expressing cells (e.g., CD19 CAR-expressing cells) are administered at a dose of 1.25 x 10 8 A dose of viable cells expressing CAR is administered.

41. The method of any one of claims 1-28, 36 or 37, wherein the population of CAR-expressing cells (e.g., CD19 CAR-expressing cells) is approximately 2.5 x 10 6 Up to 2.5x 10 8 viable cells expressing CAR, for example, about 1 x 10 7 or 5x10 7 A dose of viable cells expressing CAR is administered.

42. A method of treating a subject having severe refractory systemic lupus erythematosus (srSLE), the method comprising administering to the subject radakinumab in an amount sufficient to treat the srSLE, The srSLE is thereby treated.

43. The method of any one of claims 29-33 or 42, wherein the amount of Radix Cialis is about 0.5 x 10 6 Up to 50x 10 6 CAR-positive live cells, for example, about 5 x 10 6 CAR-positive living cells, optionally wherein radakinumab is administered at a dose of 5 x 10 6 The dose of CAR-positive live cells was administered.

44. The method of any one of claims 29-33 or 42, wherein the amount of Radix Acetophenone is about 2.5 x 10 6 Up to 2.5x 10 8 viable cells expressing CAR, for example, about 1.25 x 10 7 CAR-positive live cells, optionally wherein radaxolamine is administered at a dose of 1.25 x 10 7 The dose of CAR-positive live cells was administered.

45. The method of any one of claims 29-33 or 42, wherein the amount of Radix Acetocillin is about 1.25 x 10 7 Up to 1.25x 10 9 viable cells expressing CAR, for example, about 1.25 x 10 8 CAR-positive live cells, optionally wherein radaxolamine is administered at a dose of 1.25 x 10 8 The dose of CAR-positive live cells was administered.

46. ​​The method of any one of claims 29-33 or 42, wherein the amount of Radix Acetophenone is about 2.5 x 10 6 Up to 2.5x 10 8 CAR-positive live cells, for example, about 1 x 10 7 or 5x 10 7 The dose of CAR-positive live cells was administered.

47. 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), the method comprising administering to the subject a population of cells expressing a CD19 chimeric antigen receptor (CD19 CAR) or comprising a nucleic acid configured to express a CD19 CAR, wherein the cells are cultured at a rate of 0.5-50 x 10 6 CAR+T cells (e.g., 5-12.5 x 10 6 The dose of CAR+T cells (viable cells) was administered.

48. 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), the method comprising administering to the subject radaxylamine, wherein radaxylamine is administered at a concentration of 0.5-50 x 10 6 CAR+T cells (e.g., 5-12.5 x 10 6 The dose of CAR+T cells (viable cells) was administered.

49. The method of any one of claims 47 or 48, wherein the lupus is systemic lupus erythematosus.

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

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. The method of any one of claims 47 or 49-51, wherein (a) the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, (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 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 thereto.

53. The method of any one of claims 1-28, 32-41, 47, or 49-52, wherein the CD19 binding domain comprises a heavy chain complementary determining region 1 (HC CDR1), HC CDR2, HC CDR3, a light chain complementary determining region 1 (LC CDR1), LC CDR2, and LC CDR3, wherein: (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. The method of any one of claims 1-26, 34-41, 47, 49-53, wherein the CD 19 binding domain comprises a VH and a VL, wherein the VH and VL are connected by a linker, optionally wherein the linker comprises the amino acid sequence of SEQ ID NO: 63 or 104.

55. The method of any one of claims 1-26, 34-41, 47, 49-54, wherein the CD19 binding domain is connected to the transmembrane domain via a hinge region, optionally wherein: (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 thereto.

56. The method of any one of claims 34-41, 47, 49-55, wherein the intracellular signaling domain comprises a primary signaling domain, optionally wherein the primary signaling domain comprises a functional signaling domain derived from CD3 zeta, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (ICOS), Fc epsilon RI, DAP10, DAP12, or CD66d, optionally wherein: (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 thereto, 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 thereto.

57. The method of any one of claims 34-41, 47, 49-56, wherein the intracellular signaling domain comprises a co-stimulatory signaling domain, optionally wherein the co-stimulatory signaling domain comprises a protein derived from 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, 4-1BB (CD137), B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LI GHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, I TGA4, 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, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM 1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, C D100 (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 functional signaling domain of a ligand that specifically binds to CD83, optionally wherein: (a) the co-stimulatory 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 the co-stimulatory 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 thereto.

58. The method of any one of claims 34-41, 47, 49-57, wherein the intracellular signaling domain comprises a functional signaling domain derived from 4-1BB and a functional signaling domain derived from CD3 zeta, optionally wherein the intracellular 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) and 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), optionally wherein 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 1-28, 32-41, 47 or 49-58, wherein the CAR further comprises a leader sequence comprising the amino acid sequence of SEQ ID NO:

1.

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

61. The method of any one of claims 1-28, 32-41, 47, 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 thereto.

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

63. The method of any one of claims 1-62, wherein the subject has been identified as having no response to treatment comprising two or more immunosuppressive therapies (e.g., mycophenolate mofetil or cyclophosphamide) in combination with a glucocorticoid) and a biologic agent.

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., radaxylamine), adoptive T cell therapy, or a gene therapy product.

65. The method of any one of claims 1-64, wherein the subject receives lymphocyte depletion therapy prior to administering the CD19CAR (e.g., radaxylamine).

66. The method of claim 65, wherein the subject receives lymphocyte depletion therapy approximately two weeks prior to administration of the CD19 CAR (e.g., radaxylamine).

67. The method of claim 65 or 66, wherein the lymphocyte depletion therapy comprises fludarabine (e.g., 25 mg / m 2 IV, three times a day) and cyclophosphamide (eg, 250 mg / m 2 IV, three doses per day).

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

69. The method of claim 68, wherein the second therapeutic agent is administered before, simultaneously with, or after administration of the CAR-expressing cell population or radaxapol.

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

71. The method of any one of claims 1-70, wherein (i) before administration of a corticosteroid and / or (ii) when the absolute T cell count is ≥ 300 / mm 3 Leukocyte apheresis was performed.

72. A method for preparing a population of cells (e.g., T cells) expressing a chimeric antigen receptor (CAR), the method comprising: (i) contacting (e.g., binding) a cell population (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 co-stimulatory molecule on the surface of the cell, wherein the cell population is from a subject suffering from an autoimmune disease or disorder, 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 significant pulmonary involvement (e.g., for autologous HSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlapping Myositis, cancer-related myositis (e.g., antisynthetase syndrome with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjögren's syndrome, severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-related diseases (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-related diseases), NMDAR+ encephalitis, or antibody-related neurological paraneoplastic diseases), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis; (ii) contacting the cell (eg, T cell) population with a nucleic acid molecule (eg, DNA or RNA molecule) encoding the CAR, thereby providing a cell (eg, T cell) population comprising the nucleic acid molecule, wherein optionally the CAR comprises a CD19 antigen binding domain; as well as (iii) harvesting the cell (eg, T cell) population for storage (eg, reconstituting the cell population in a cryopreservation medium) or administration, wherein: (a) step (ii) is performed together with step (i) or not later than 20 hours after the start of step (i), for example not later than 12, 13, 14, 15, 16, 17, or 18 hours after the start of step (i), for example not later than 18 hours after the start of step (i), and Step (iii) is performed no later than 30 (e.g. 26) hours after the start of step (i), for example no later than 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the start of step (i), for example no later than 24 hours after the start of step (i), (b) step (ii) is performed together with step (i) or not later than 20 hours after the start of step (i), for example not later than 12, 13, 14, 15, 16, 17, or 18 hours after the start of step (i), for example not later than 18 hours after the start of step (i), and step (iii) is performed no later than 30 hours after the start of step (ii), for example no later than 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the start of step (ii), or (c) the cell population from step (iii) does not expand, or expands by no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40%, e.g., by no more than 10%, compared to the cell population at the start of step (i), e.g., as assessed by viable cell number, Optionally, the nucleic acid molecule in step (ii) is on a viral vector, optionally, the nucleic acid molecule in step (ii) is an RNA molecule on a viral vector, optionally, step (ii) comprises transducing the cell (e.g., T cell) population with a viral vector, the viral vector comprising a nucleic acid molecule encoding the CAR.

73. A method as claimed in claim 72, wherein the agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3 (e.g., an anti-CD3 antibody), and wherein the agent that stimulates a co-stimulatory 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 wherein the agent that stimulates the CD3 / TCR complex or the agent that stimulates a co-stimulatory molecule 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 a scFv), a small molecule, or a ligand (e.g., a natural ligand, recombinant ligand, or chimeric ligand present); optionally wherein the agent that stimulates the CD3 / TCR complex or the agent that stimulates the co-stimulatory molecule does not comprise beads; optionally wherein the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody and the agent that stimulates the co-stimulatory molecule comprises an anti-CD28 antibody; optionally wherein the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody covalently attached to a colloidal polymer nanomatrix and the agent that stimulates the co-stimulatory molecule comprises an anti-CD28 antibody covalently attached to a colloidal polymer nanomatrix; optionally wherein the agent that stimulates the CD3 / TCR complex and the agent that stimulates the co-stimulatory molecule comprise T cell TransAct TM .

74. A method as described in claim 72 or 73, wherein step (i) increases the percentage of cells expressing CAR in the cell population from step (iii) compared to cells prepared by a method that is otherwise similar except that step (i) is not included, for example, the cell population from step (iii) shows a higher percentage (e.g., at least 10%, 20%, 30%, 40%, 50%, or 60%) of cells expressing CAR.

75. The method of any one of claims 72-74, wherein (a) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the cell population 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 cell population 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 cell population 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 as compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells in the cell population at the start of step (i); (c) the percentage of naive T cells expressing CAR, such as CD45RA+CD45RO-CCR7+ T cells expressing CAR, in the cell population increases during the duration of step (ii), such as by at least 30%, 35%, 40%, 45%, 50%, 55%, or 60% between 18 and 24 hours after the start of step (ii); or (d) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the cell population from step (iii) does not decrease, or decreases by no more than 5% or 10%, as compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells in the cell population at the start of step (i).

76. The method of any one of claims 72-74, wherein (a) the cell population 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 prepared by an otherwise 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 cell population 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 prepared by an otherwise 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 naive T cells expressing CAR, e.g., CD45RA+CD45RO-CCR7+ T cells expressing CAR, in the cell population from step (iii) is higher (e.g., at least 4, 6, 8, 10, or 12 times higher) than the percentage of naive T cells expressing CAR, e.g., CD45RA+CD45RO-CCR7+ T cells expressing CAR, in cells prepared by an otherwise 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 cell population from step (iii) shows 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 prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii); (e) the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells in the cell population 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 prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population 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 naive T cells expressing CAR, e.g., CD45RA+CD45RO-CCR7+ T cells expressing CAR, in the cell population from step (iii) is higher (e.g., at least 4, 6, 8, 10, or 12 times higher) than the percentage of naive T cells expressing CAR, e.g., CD45RA+CD45RO-CCR7+ T cells expressing CAR, in cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii).

77. The method of any one of claims 72-76, wherein (a) the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells in the cell population from step (iii) is the same as the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells in the cell population at the beginning of step (i) or differs by no more than 5% or 10%; (b) the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the cell population from step (iii) is reduced by at least 20%, 25%, 30%, 35%, 40%, 45%, or 50% as compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells in the cell population at the start of step (i); (c) the percentage of central memory T cells expressing CAR, such as CCR7+CD45RO+ cells expressing CAR, decreases during the duration of step (ii), such as by at least 8%, 10%, 12%, 14%, 16%, 18%, or 20% between 18-24 hours after the start of step (ii); or (d) the percentage of central memory cells, such as central memory T cells, such as CCR7+CD45RO+ T cells, in the cell population from step (iii) does not increase or increases by no more than 5% or 10% as compared to the percentage of central memory cells, such as central memory T cells, such as CCR7+CD45RO+ T cells in the cell population at the beginning of step (i).

78. The method of any one of claims 72-77, wherein (a) the cell population from step (iii) exhibits a lower percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells (e.g., at least 10%, 20%, 30%, or 40% lower) compared to cells prepared by an otherwise similar method except that step (iii) is performed 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); (b) the percentage of central memory cells, such as central memory T cells, such as CCR7+CD45RO+ T cells in the cell population from step (iii) is lower (e.g., at least 20%, 30%, 40%, or 50% lower) than the percentage of central memory cells, such as central memory T cells, such as CCR7+CD45RO+ T cells in cells prepared by an otherwise similar method except that step (iii) is performed 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); (c) the percentage of central memory T cells expressing CAR, such as CCR7+CD45RO+ T cells expressing CAR, in the cell population from step (iii) is lower (e.g., at least 10%, 20%, 30%, or 40%) than the percentage of central memory T cells expressing CAR, such as CCR7+CD45RO+ T cells expressing CAR, in cells prepared by an otherwise similar method except that step (iii) is performed 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); (d) the cell population from step (iii) shows a lower percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells (e.g., at least 10%, 20%, 30%, or 40%) compared to cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, such as 5, 6, 7, 8, or 9 days after step (ii) and before step (iii); (e) the percentage of central memory cells, such as central memory T cells, such as CCR7+CD45RO+ T cells in the cell population from step (iii) is lower (e.g., at least 20%, 30%, 40%, or 50%) than the percentage of central memory cells, such as central memory T cells, such as CCR7+CD45RO+ T cells in cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, such as 5, 6, 7, 8, or 9 days after step (ii) and before step (iii); or (f) the percentage of central memory T cells expressing CAR, such as CCR7+CD45RO+ T cells expressing CAR, in the cell population from step (iii) is lower (e.g., at least 10%, 20%, 30%, or 40%) than the percentage of central memory T cells expressing CAR, such as CCR7+CD45RO+ T cells expressing CAR, in cells prepared by a method otherwise similar to that described in step (ii) but further comprising amplifying the cell (e.g., T cell) population in vitro for more than 3 days, such as 5, 6, 7, 8, or 9 days before step (iii).

79. The method of any one of claims 72-78, wherein (a) the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in the cell population from step (iii) is increased as compared to the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in the cell population at the start of step (i); (b) the percentage of CAR-expressing stem cell memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells expressing CAR, in the cell population from step (iii) is increased as compared to the percentage of CAR-expressing stem cell memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells expressing CAR, in the cell population at the beginning of step (i); (c) the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in the cell population from step (iii) is higher than the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in cells prepared by an otherwise 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) the percentage of CAR-expressing stem cell memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in the cell population from step (iii) is higher than the percentage of CAR-expressing stem cell memory T cells, e.g., CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in cells prepared by an otherwise 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 cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in the cell population from step (iii) is higher than the percentage of stem cell memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in cells prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population 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 cell memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in the cell population from step (iii) is higher than the percentage of CAR-expressing stem cell memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+T cells, in cells prepared by a method otherwise similar except further comprising amplifying the cell (e.g., T cell) population in vitro for more than 3 days, such as 5, 6, 7, 8, or 9 days after step (ii) and before step (iii).

80. The method of any one of claims 72-79, wherein (a) the median gene set score (TEM up vs. TSCM down) of the cell population from step (iii) is about the same as or differs by no more than (e.g., increases by no more than) about 25%, 50%, 75%, 100%, or 125% from the median gene set score (TEM up vs. TSCM down) of the cell population at the start of step (i); (b) the median gene set score (TEM up vs. TSCM down) of the population of cells from step (iii) is lower (e.g., at least about 100%, 150%, 200%, 250%, or 300% lower) than the median gene set score (TEM up vs. TSCM down): Cells prepared by an otherwise similar method except that step (iii) is performed 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 prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii); (c) the median gene set score (up Treg vs. down Teff) of the cell population from step (iii) is about the same as or differs by no more than (e.g., increases by no more than) about 25%, 50%, 100%, 150%, or 200% from the median gene set score (up Treg vs. down Teff) of the cell population at the beginning of step (i); (d) the median gene set score (Treg Up vs. Teff Down) of the cell population from step (iii) is lower (e.g., at least about 50%, 100%, 125%, 150%, or 175% lower) than the median gene set score (Treg Up vs. Teff Down) of: Cells prepared by an otherwise similar method except that step (iii) is performed 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 prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii); (e) the median gene set score (downward stemness) of the cell population from step (iii) is about the same as or differs by no more than (e.g., increases by no more than) about 25%, 50%, 100%, 150%, 200%, or 250% from the median gene set score (downward stemness) of the cell population at the beginning of step (i); (f) the median gene set score (downward stemness) of the population of cells from step (iii) is lower (e.g., at least about 50%, 100%, or 125% lower) than the median gene set score (downward stemness) of: Cells prepared by an otherwise similar method except that step (iii) is performed 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 prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii); (g) the median gene set score (upward hypoxia) of the cell population from step (iii) is about the same as or differs by no more than (e.g., increases by no more than) about 125%, 150%, 175%, or 200% from the median gene set score (upward hypoxia) of the cell population at the start of step (i); (h) the median gene set score (hypoxia up) of the population of cells from step (iii) is lower (e.g., at least about 40%, 50%, 60%, 70%, or 80% lower) than the median gene set score (hypoxia up) of: Cells prepared by an otherwise similar method except that step (iii) is performed 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 prepared by an otherwise similar method except further comprising expanding the cell (e.g., T cell) population in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii); (j) the median gene set score (autophagy up) of the cell population from step (iii) is about the same as or differs by no more than (e.g., increases by no more than) about 180%, 190%, 200%, or 210% from the median gene set score (autophagy up) of the cell population at the start of step (i); or (k) the median gene set score (autophagy up) of the population of cells from step (iii) is lower (e.g., at least about 20%, 30%, or 40% lower) than the median gene set score (autophagy up) of: Cells prepared by an otherwise similar method except that step (iii) is performed 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 prepared by an otherwise similar method except further comprising expanding the cell (eg, T cell) population in vitro for more than 3 days, such as 5, 6, 7, 8, or 9 days after step (ii) and before step (iii).

81. A method as described in any of claims 72-80, wherein the cell population from step (iii) secretes IL-2 at a higher level (e.g., at least 2, 4, 6, 8, 10, 12, or 14 times higher) after incubation with cells expressing the antigen recognized by the CAR than cells prepared by an otherwise similar method except that step (iii) is performed 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 prepared by an otherwise similar method except that the cell (e.g., T cell) population is expanded in vitro for more than 3 days, such as 5, 6, 7, 8, or 9 days after step (ii) and before step (iii).

82. The method of any one of claims 72-81, wherein the cell population from step (iii) persists for a longer time or expands at a higher level after in vivo administration to the subject compared to cells prepared by an otherwise similar method except that step (iii) is performed more than 26 hours after initiation of step (i), such as more than 5, 6, 7, 8, 9, 10, 11, or 12 days after initiation of step (i), or compared to cells prepared by an otherwise similar method except that further comprises expanding the cell (e.g., T cell) population 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 method of any one of claims 72-82, wherein the cell population from step (iii) exhibits greater activity (e.g., greater activity at a low dose, e.g., no more than 0.15 x 10 T cells) after in vivo administration to the subject compared to cells prepared by an otherwise 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 compared to cells prepared by an otherwise similar method except that the cell (e.g., T cell) population is expanded in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). 6 , 0.2x 10 6 , 0.25x 10 6 , or 0.3x 10 6 The dose of viable cells expressing CAR).

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

85. The method of any one of claims 72-84, wherein the cell population from step (iii) does not expand or expands for less than 2 hours, e.g., less than 1 or 1.5 hours, compared to the cell population at the start of step (i).

86. The method of any one of claims 72-85, wherein steps (i) and / or (ii) are performed in a cell culture medium (e.g., serum-free culture 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), a LSD1 inhibitor, a MALT1 inhibitor, or a combination thereof.

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

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

89. The method of any one of claims 72-88, further comprising, 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 removal (e.g., a fresh product from a thymectomy)) from an entity, such as a laboratory, hospital, or health care 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 removal (e.g., a fresh product from a thymectomy)), optionally wherein: step (iii) is performed no later than 35 hours after the start of step (v), for example no later than 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the start of step (v), for example no later than 30 hours after the start of step (v), or For example, the cell population 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 cell population at the end of step (v), as assessed by viable cell number.

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

91. The method of any one of claims 72-88, further comprising, 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 removal (e.g., a cryopreserved product from a thymectomy)) from an entity, such as a laboratory, hospital, or health care 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, e.g., a cryopreserved whole blood product, a cryopreserved bone marrow product, or a cryopreserved organ biopsy or ablation (e.g., a cryopreserved product from a thymectomy)), optionally wherein: step (iii) is performed no later than 35 hours after the start of step (v), for example no later than 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the start of step (v), for example no later than 30 hours after the start of step (v), or For example, the cell population 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 cell population at the end of step (v), as assessed by viable cell number.

92. The method of any one of claims 72-91, further comprising step (vi): Culturing a portion of the cell population from step (iii) 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 no more than 7 days, and measuring the level of CAR expression in the portion (e.g., measuring the percentage of live cells expressing CAR in the portion), optionally wherein: Step (iii) comprises harvesting and freezing the cell (e.g., T cell) population, and step (vi) comprises thawing a portion of the cell population 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 no more than 7 days, and measuring the level of CAR expression in the portion (e.g., measuring the percentage of live cells expressing CAR in the portion).

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

94. The method of any one of claims 72-93, wherein the cell population at the start of step (i) or step (1) comprises no less than 50%, 60%, or 70% of cells expressing IL6R (e.g., cells positive for IL6Rα and / or IL6Rβ).

95. The method of any one of claims 72-94, wherein steps (i) and (ii) or steps (1) and (2) are performed in a cell culture medium comprising IL-15 (eg, hetIL-15 (IL15 / sIL-15Ra)).

96. The method of claim 95, wherein IL-15 increases the ability of the cell population to expand, e.g., after 10, 15, 20, or 25 days.

97. The method of claim 95, wherein IL-15 increases the percentage of cells in the cell population that express IL6Rβ.

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

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

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

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

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 method of any one of claims 100 or 101, wherein the antigen binding domain comprises a CD19 binding domain, the CD19 binding domain comprising a heavy chain complementary determining region 1 (HC CDR1), HC CDR2, HC CDR3, a light chain complementary determining region 1 (LC CDR1), LC CDR2, and LC CDR3, wherein: (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. The method of any one of claims 100-103, wherein the antigen binding domain comprises a VH and a VL, wherein the VH and VL are connected by a linker, optionally wherein the linker comprises the amino acid sequence of SEQ ID NO: 63 or 104.

105. The method of any one of claims 100-104, wherein (a) the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, (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 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 thereto.

106. The method of any one of claims 100-105, wherein the antigen binding domain is connected to the transmembrane domain via a hinge region, optionally wherein: (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 thereto.

107. The method of any one of claims 100-106, wherein the intracellular signaling domain comprises a primary signaling domain, optionally wherein the primary signaling domain comprises a functional signaling domain derived from CD3 zeta, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (ICOS), Fc epsilon RI, DAP10, DAP12, or CD66d, optionally wherein: (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 thereto, 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 thereto.

108. The method of any one of claims 100-107, wherein the intracellular signaling domain comprises a co-stimulatory signaling domain, optionally wherein the co-stimulatory signaling domain comprises a protein derived from 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, 4-1BB (CD137), B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LI GHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, I TGA4, 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, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM 1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, C D100 (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 functional signaling domain of a ligand that specifically binds to CD83, optionally wherein: (a) the co-stimulatory 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 the co-stimulatory 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 thereto.

109. The method of any one of claims 100 to 108, wherein the intracellular signaling domain comprises a functional signaling domain derived from 4-1BB and a functional signaling domain derived from CD3 zeta, optionally wherein the intracellular 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) and 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), optionally wherein 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 100-109, wherein the CAR further comprises a leader sequence comprising the amino acid sequence of SEQ ID NO:

1.

111. 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 thereto.

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

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

114. The method of any one of claims 72-113, wherein the subject has been identified as having no response to treatment including two or more immunosuppressive therapies (e.g., mycophenolate mofetil or cyclophosphamide) in combination with a glucocorticoid) and a biologic agent.

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

116. The method of any one of claims 72-115, wherein (i) before administration of a corticosteroid and / or (ii) when the absolute T cell count is ≥ 300 / mm 3 Leukocyte apheresis was performed.

117. A population of cells expressing CAR (e.g., autologous or allogeneic T cells or NK cells expressing CAR), wherein the cell population is prepared by the method described in any one of claims 72-116.

118. A CAR-expressing cell population as described in claim 117, wherein the population comprises autoreactive B cells (e.g., autoreactive B cells that do not express CAR).

119. A pharmaceutical composition comprising a CAR-expressing cell population as described in claim 117 or 118, and a pharmaceutically acceptable carrier.

120. A CAR-expressing cell population as described in claim 117 or 118, or a pharmaceutical composition as described in claim 119, for use in a method for regulating 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 CAR-expressing cell population or an effective amount of the pharmaceutical composition.

121. 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 cell population expressing a CD19 chimeric antigen receptor (CD19 CAR) or comprising a nucleic acid configured to express a CD19 CAR, and a second therapy selected from an antimalarial agent or a stable immunosuppressant, wherein the second therapy and the CD19 CAR cells are present in the subject simultaneously, e.g., wherein the second therapy is administered while the CD19 CAR cells are present in the subject.

122. 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: Reda Kiolon, and a second therapy selected from an antimalarial agent or a stable immunosuppressant, wherein the second therapy and redaxolotl are present in the subject simultaneously, e.g., wherein the second therapy is administered while redaxolotl is present in the subject.

123. Radakiorencel, which is prepared from autologous cells of a subject suffering from an autoimmune disease or disorder, such as lupus (e.g., systemic lupus erythematosus (SLE), such as severe refractory systemic lupus erythematosus (srSLE), or lupus nephritis), systemic sclerosis (e.g., rapidly progressive systemic sclerosis (SSc) with significant pulmonary involvement (e.g., for autologous HSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-related myositis, such as antisynthetase syndrome with ILD), vasculitis (e.g., ANCA-associated myositis), severe refractory Sjögren's syndrome, severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-related diseases (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-related diseases), NMDAR+ encephalitis, or antibody-related neurological paraneoplastic 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 radakinumab as claimed in claim 123, and a pharmaceutically acceptable carrier.

125. The radakiolancept of claim 123, the pharmaceutical composition of claim 124, for use in a method of modulating an immune response in a subject 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 significant pulmonary involvement (e.g., for autologous HSCT)), idiopathic inflammatory myopathy (e.g., polymyositis, dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy, inclusion body myositis, overlap myositis, cancer-related myositis, e.g., antisynthetase syndrome with ILD), vasculitis (e.g., ANCA-associated vasculitis), severe refractory Sjögren's syndrome, severe refractory neuroimmune diseases (e.g., myasthenia gravis (MG), neuromyelitis optica (NMO), MOG-related diseases (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-related diseases), NMDAR+ encephalitis, or antibody-related neurological paraneoplastic diseases), Addison's disease, Goodpasture's syndrome, thyrotoxicosis, chronic active hepatitis, relapsing polychondritis, pemphigus vulgaris, or amyotrophic lateral sclerosis, the method comprising administering to the subject an effective amount of radakinumab or an effective amount of the pharmaceutical composition.

126. The redaxolomide as described in claim 123 and the pharmaceutical composition as described in claim 124 are used in a method for regulating the immune response of a subject having 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 redaxolomide or an effective amount of the pharmaceutical composition.

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