Cellular therapy for treating systemic autoimmune diseases

Abstract

Provided herein are methods and uses of adoptive cell therapy involving administration of doses of T cells expressing CD19-directed chimeric antigen receptors to treat subjects with systemic autoimmune diseases, as well as related methods, compositions, uses and articles of manufacture.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application relates to a method and system for treating systemic autoimmune diseases, each of which is entitled "CELL THERAPY FOR TREATING SYSTEMIC AUTOIMMUNE DISEASES." This application claims priority to U.S. Provisional Patent Application No. 63 / 487,612, filed February 28, 2023; U.S. Provisional Patent Application No. 63 / 466,671, filed May 15, 2023; U.S. Provisional Patent Application No. 63 / 522,085, filed June 20, 2023; U.S. Provisional Patent Application No. 63 / 608,166, filed December 8, 2023; U.S. Provisional Patent Application No. 63 / 618,271, filed January 5, 2024; U.S. Provisional Patent Application No. 63 / 624,745, filed January 24, 2024; and U.S. Provisional Patent Application No. 63 / 553,586, filed February 14, 2024, all of which are incorporated herein by reference in their entireties.

[0002] Reference to the electronic sequence listing This application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042027040SeqList.xml, created on February 23, 2024, which is 200,426 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0003] Field The present disclosure relates, in some embodiments, to adoptive cell therapy involving administration of doses of T cells expressing CD19-directed chimeric antigen receptors to treat subjects with systemic autoimmune diseases, and related methods, compositions, uses, and articles of manufacture. [Background technology]

[0004] Systemic autoimmune diseases are associated with a wide range of diseases and disorders characterized by dysregulation of the immune system. Among these is systemic lupus erythematosus (SLE), an autoimmune disorder that presents with numerous clinical manifestations, most notably renal involvement, i.e., lupus nephritis. Many patients eventually relapse or become refractory to available treatments, limiting second-, third-, and especially fourth-line treatments. There is a need for an effective treatment for patients with SLE who have failed one or more previous treatments, such as severe, refractory SLE. Methods and uses that address such needs are provided. Summary of the Invention

[0005] 1. A method of treating a subject having a systemic autoimmune disease, comprising administering to a subject having or suspected of having a severe systemic autoimmune disease a dose of CD19-directed genetically modified T cells from a composition comprising engineered T cells that express a chimeric antigen receptor (CAR), wherein the dose of T cells is positive for expression of a CAR that binds to CD19, and the dose is greater than or equal to 1 x 10 6 ~50×10 6 provided herein are methods, wherein the CAR-positive T cells are viable CAR-positive T cells.

[0006] 1. A method of treating a subject having a systemic autoimmune disease, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having a moderate systemic autoimmune disease, wherein the dose of T cells is positive for expression of a chimeric antigen receptor (CAR) that binds CD19, and the dose is greater than or equal to 1 x 10 6 ~50×10 6 Also provided herein are methods, wherein the CAR-positive T cells are viable CAR-positive T cells.

[0007] In some embodiments, the systemic autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), Sjogren's syndrome, progressive systemic sclerosis (i.e., scleroderma); idiopathic inflammatory myositis (IIM), including dermatomyositis, polymyositis, and necrotizing myositis; mixed connective tissue disorder (MCTD), highly active relapsing-remitting multiple sclerosis (HSC), and rheumatoid arthritis (RH). sclerosis), primary progressive MS, ANCA-associated vasculitis (AAV), Crohn's disease, myasthenia gravis, Behçet's disease, rheumatoid arthritis, IgA nephropathy, pemphigus vulgaris, myasthenia gravis, autoimmune hemolytic anemia, immune thrombocytopenia, IgG4-related disease, membranous nephropathy, cutaneous lupus erythematosus, sarcoidosis, light-chain amyloidosis, acute respiratory distress syndrome, atopic eczema, hereditary angioedema, hidradenitis suppurativa, inclusion body myositis, inflammatory bowel disease, mastocytosis, multifocal lung disease The disease is selected from the group consisting of motor neuropathy, necrotizing myopathy, neuromyelitis optica spectrum disorder, mixed connective tissue disorder, POEMS syndrome, primary biliary cholangitis, psoriasis, rhesus hemolytic disease, Still's disease, type 1 diabetes, urticaria, capillary leak syndrome, cytokine release syndrome, erythema multiforme, pyoderma gangrenosum, X-linked agammaglobulinemia, antiphospholipid antibody syndrome, and chronic inflammatory demyelinating polyneuropathy (also known as inflammatory demyelinating polyradiculoneuropathy).

[0008] In some embodiments, the systemic autoimmune disease is rheumatoid arthritis. In some embodiments, the systemic autoimmune disease is myositis. In some embodiments, the systemic autoimmune disease is myasthenia gravis. In some embodiments, the systemic autoimmune disease is bullous pemphigoid. In some embodiments, the systemic autoimmune disease is immune thrombocytopenia. In some embodiments, the systemic autoimmune disease is autoimmune hemolytic anemia. In some embodiments, the systemic autoimmune disease is pemphigus vulgaris. In some embodiments, the systemic autoimmune disease is demyelinating polyradiculoneuropathy. In some embodiments, the systemic autoimmune disease is membranous nephropathy.

[0009] In some embodiments, the systemic autoimmune disease is a refractory disease. In some embodiments, the subject is refractory to treatment with one or more previous therapies for the systemic autoimmune disease. In some embodiments, the subject is refractory to treatment with two or more previous therapies for the systemic autoimmune disease. In some embodiments, the systemic autoimmune disease is a severe disease.

[0010] 1. A method of treating a subject with severe systemic lupus erythematosus (SLE), comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having severe systemic lupus erythematosus (SLE), wherein the dose of T cells is positive for expression of a chimeric antigen receptor (CAR) that binds CD19, and the dose is greater than or equal to 1 x 10 6 ~50×10 6 Also provided herein are methods, wherein the CAR-positive T cells are viable CAR-positive T cells.

[0011] 1. A method for reducing systemic lupus erythematosus (SLE) disease activity, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having severe systemic lupus erythematosus (SLE), wherein the dose of T cells is positive for expression of a chimeric antigen receptor (CAR) that binds CD19, and the dose is greater than or equal to 1 x 10 6 ~50×10 6 Also provided herein are methods, wherein the CAR-positive T cells are viable CAR-positive T cells.

[0012] In some embodiments, the SLE in the subject has one or more of the following: renal, central nervous system, or hematological complications.

[0013] In some embodiments, the subject has at least one organ system categorized as Category A ("BILAG A") by the British Isles Lupus Assessment Group 2004 ("BILAG") or at least two organ systems categorized as BILAG B.

[0014] In some embodiments, the subject meets the 2019 American College of Rheumatology (ACR) / European League Against Rheumatism (EULAR) classification criteria for SLE, and / or the subject has detectable anti-dsDNA, anti-histone, anti-chromatin, or anti-Sm antibodies in their blood. In some embodiments, the subject meets the 2019 American College of Rheumatology (ACR) / European League Against Rheumatism (EULAR) classification criteria for SLE. In some embodiments, the subject has detectable anti-dsDNA, anti-histone, anti-chromatin, or anti-Sm antibodies in their blood.

[0015] In some embodiments, the subject has lupus nephritis.

[0016] 1. A method of treating a subject with lupus nephritis, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having lupus nephritis, wherein the dose of T cells is positive for expression of a chimeric antigen receptor (CAR) that binds CD19, and the dose is greater than or equal to 1 x 10 6 ~50×10 6 Also provided herein are methods, wherein the CAR-positive T cells are viable CAR-positive T cells.

[0017] In some embodiments, the subject is refractory to treatment with one or more previous therapies for lupus.

[0018] In some embodiments, the subject has achieved an inadequate response to one or more previous treatments for lupus.

[0019] In some embodiments, the two or more previous treatments for lupus include a glucocorticoid, an antimalarial, an immunosuppressant, an anti-CD20 antibody, or an inhibitor of soluble B-lymphocyte stimulator (BLyS).

[0020] In some embodiments, the two or more prior treatments are selected from any two or more of the following: mycophenolate mofetil (MFF), cyclophosphamide (cyc), belimumab, rituximab, anifrolumab, azathioprine, methotrexate, cyclosporine (csp), or voclosporin.

[0021] In some embodiments, the subject does not have drug-induced SLE, clinically significant CNS pathology, associated systemic autoimmune disease, and / or an SLE overlap syndrome.

[0022] In some embodiments, the subject does not have an associated systemic autoimmune disease, including but not limited to multiple sclerosis, psoriasis, and inflammatory bowel disease.

[0023] In some embodiments, the subject does not have an SLE overlap syndrome, including, but not limited to, rheumatoid arthritis, scleroderma, and mixed connective tissue disease. In some embodiments, the subject is at high risk of organ failure.

[0024] In some embodiments, the method reduces systemic autoimmune disease activity in the subject.

[0025] In some embodiments, reducing disease activity in a subject comprises reducing inflammation in the subject.

[0026] In some embodiments, the method reduces SLE disease activity in the subject.

[0027] In some embodiments, reducing SLE disease activity in a subject comprises a BILAG-Based Composite Lupus Assessment (BICLA) response in the subject, a reduction in the subject's Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI) score compared to the subject's CLASI score before treatment, a reduction in the subject's tender and swollen joint count compared to the subject's tender and swollen joint count before treatment, the subject having a BILAG-2004 B score of up to 1 after treatment, the subject having a BILAG-2004 score of C or higher after treatment, the subject having an improvement in at least one patient-reported outcome (PRO) compared to before treatment, and / or a reduction in the subject's SLE flare rate compared to the subject's flare rate before treatment.

[0028] In some embodiments, reducing SLE disease activity in a subject involves the subject achieving clinical remission as defined by the Definitions of Remission in Systemic Lupus Erythematosus (DORIS). In some embodiments, alleviating SLE involves the subject achieving a Lupus Low Disease Activity State (LLDAS).

[0029] In some embodiments, the subject achieves clinical remission of lupus within 3 months or 6 months of administration of the dose of CD19-directed genetically modified T cells.

[0030] In some embodiments, clinical remission is maintained for at least about 6 months, at least about 12 months, at least about 24 months, at least about 3 years, at least about 4 years, or at least about 5 years.

[0031] In some embodiments, the subject achieves long-term remission of lupus.

[0032] 1. A method of treating a subject having idiopathic inflammatory myopathy (IIM), comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having IIM, wherein the dose of T cells is positive for expression of a chimeric antigen receptor (CAR) that binds CD19, and the dose is greater than or equal to 1 x 10 6 ~50×10 6 Also provided herein are methods, wherein the CAR-positive T cells are viable CAR-positive T cells.

[0033] 1. A method for reducing idiopathic inflammatory myopathy (IIM) disease activity, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having idiopathic inflammatory myopathy (IIM), wherein the dose of T cells is positive for expression of a chimeric antigen receptor (CAR) that binds CD19, and wherein the dose is greater than or equal to 1 x 10 6 ~50×10 6 Also provided herein are methods, wherein the CAR-positive T cells are viable CAR-positive T cells.

[0034] In some embodiments, the subject is refractory to treatment with one or more prior therapies for the IIM. In some embodiments, the subject has achieved an inadequate response to one or more prior therapies for the IIM.

[0035] 1. A method of treating a subject with systemic sclerosis (SSc), comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having systemic sclerosis (SSc), wherein the dose of T cells is positive for expression of a chimeric antigen receptor (CAR) that binds CD19, and the dose is greater than or equal to 1 x 10 6 ~50×10 6 Also provided herein are methods, wherein the CAR-positive T cells are viable CAR-positive T cells.

[0036] 1. A method for reducing systemic sclerosis (SSc) disease activity, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having systemic sclerosis (SSc), wherein the dose of T cells is positive for expression of a chimeric antigen receptor (CAR) that binds CD19, and the dose is greater than or equal to 1 x 10 6 ~50×10 6 Also provided herein are methods, wherein the CAR-positive T cells are viable CAR-positive T cells.

[0037] In some embodiments, the subject is refractory to treatment with one or more prior therapies for SSc, hi some embodiments, the subject has achieved an inadequate response to one or more prior therapies for SSc.

[0038] 1. A method of treating a subject with multiple sclerosis (MS), comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having multiple sclerosis (MS), wherein the dose of T cells is positive for expression of a chimeric antigen receptor (CAR) that binds CD19, and the dose is greater than or equal to 1 x 10 6 ~50×10 6 Also provided herein are methods, wherein the CAR-positive T cells are viable CAR-positive T cells.

[0039] 1. A method for reducing multiple sclerosis (MS) disease activity, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having multiple sclerosis (MS), wherein the dose of T cells is positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and the dose is greater than or equal to 1 x 10 6 ~50×10 6 Also provided herein are methods, wherein the CAR-positive T cells are viable CAR-positive T cells.

[0040] In some embodiments, the subject is refractory to treatment with one or more previous therapies for MS. In some embodiments, the subject has achieved an inadequate response to one or more previous therapies for MS. In some embodiments, the subject has or is suspected of having relapsing MS. In some embodiments, the subject has or is suspected of having progressive MS.

[0041] In some embodiments, the subject has or is suspected of having highly active relapsing-remitting MS. In some embodiments, the subject has or is suspected of having primary progressive MS. In some embodiments, the subject has or is suspected of having active secondary progressive MS (aSPMS). In some embodiments, the subject has or is suspected of having inactive secondary progressive MS (iSPMS).

[0042] In some embodiments, the subject has an Expanded Disability Status Scale (EDSS) of ≧3.0 and ≦5.5, or ≧3.0 and ≦6.0. In some embodiments, the subject can complete the 9-Hole Peg Test (9-HPT) for each hand in <240 seconds, and the subject can perform the Timed 25-Foot Walk Test (T25FWT) in <150 seconds. In some embodiments, the subject does not have MS lesions or symptoms that may increase their risk of neurotoxicity.

[0043] In some embodiments, the method reduces autoimmune disease activity in the subject.

[0044] In some embodiments, reducing disease activity in a subject comprises reducing inflammation in the subject.

[0045] In some embodiments, reducing autoimmune disease activity in a subject comprises reducing the subject's IMACS score after treatment compared to the subject's IMACS score before treatment, alleviating the subject's skin lesions, muscle fatigue, and / or weakness compared to the subject's skin lesions, muscle fatigue, and / or weakness before treatment, or the subject having an improvement in at least one patient-reported outcome (PRO) compared to before treatment.

[0046] In some embodiments, reducing autoimmune disease activity in a subject comprises reducing the subject's modified Rodnan skin score, European Scleroderma Study Group (EScSG) index, minimal clinically important difference (MCID), patient reported short-form quality of life assessment (SF-36) Physical Component Summary (PCS) and / or Mental Component Summary (MCS), or a combination thereof, or improving forced vital capacity.

[0047] In some embodiments, reducing autoimmune disease activity in a subject comprises improving the subject's score on any of the following tests: Expanded Disability Status Scale (EDSS), disease stage, Multiple Sclerosis Functional Composite (MSFC), minimal clinically important difference (MCID), Patient-Reported Short Form Quality of Life (SF-36) physical summary score (PCS) and / or mental summary score (MCS), or a combination thereof.

[0048] 1. A method of treating a subject having autoimmune vasculitis (AAV), comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having autoimmune vasculitis (AAV), wherein the dose of T cells is positive for expression of a chimeric antigen receptor (CAR) that binds CD19, and the dose is greater than or equal to 1 x 10 6 ~50×10 6 Also provided herein are methods, wherein the CAR-positive T cells are viable CAR-positive T cells.

[0049] 1. A method for reducing autoimmune vasculitis (AAV) disease activity, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having autoimmune vasculitis (AAV), wherein the dose of T cells is positive for expression of a chimeric antigen receptor (CAR) that binds CD19, and the dose is greater than or equal to 1 x 10 6 ~50×10 6 Also provided herein are methods, wherein the CAR-positive T cells are viable CAR-positive T cells.

[0050] 1. A method of treating a subject with IgA nephropathy, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having IgA nephropathy, wherein the dose of T cells is positive for expression of a chimeric antigen receptor (CAR) that binds CD19, and the dose is greater than or equal to 1 x 10 6 ~50×10 6 Also provided herein are methods, wherein the CAR-positive T cells are viable CAR-positive T cells.

[0051] 1. A method for reducing IgA nephropathy disease activity, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having IgA nephropathy, wherein the dose of T cells is positive for expression of a chimeric antigen receptor (CAR) that binds CD19, and the dose is greater than or equal to 1 x 10 6 ~50×10 6 Also provided herein are methods, wherein the CAR-positive T cells are viable CAR-positive T cells.

[0052] 1. A method of treating a subject with pemphigus vulgaris, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having pemphigus vulgaris, wherein the dose of T cells is positive for expression of a chimeric antigen receptor (CAR) that binds CD19, and the dose is greater than or equal to 1 x 10 6 ~50×10 6 Also provided herein are methods, wherein the CAR-positive T cells are viable CAR-positive T cells.

[0053] 1. A method for reducing pemphigus vulgaris disease activity, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having pemphigus vulgaris, wherein the dose of T cells is positive for expression of a chimeric antigen receptor (CAR) that binds CD19, and the dose is greater than or equal to 1 x 10 6 ~50×10 6 Also provided herein are methods, wherein the CAR-positive T cells are viable CAR-positive T cells.

[0054] 1. A method of treating a subject with myasthenia gravis, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having myasthenia gravis, wherein the dose of T cells is positive for expression of a chimeric antigen receptor (CAR) that binds CD19, and the dose is greater than or equal to 1 x 10 6 ~50×10 6 Also provided herein are methods, wherein the CAR-positive T cells are viable CAR-positive T cells.

[0055] 1. A method for reducing myasthenia gravis disease activity, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having myasthenia gravis, wherein the dose of T cells is positive for expression of a chimeric antigen receptor (CAR) that binds CD19, and the dose is greater than or equal to 1 x 10 6 ~50×10 6 Also provided herein are methods, wherein the CAR-positive T cells are viable CAR-positive T cells.

[0056] In some embodiments, the dose is 1×10 6 ~40×10 6or about 1 x 10 6 ~40×10 6 These are CAR-positive viable T cells.

[0057] In some embodiments, the dose is 1×10 6 ~25×10 6 or about 1 x 10 6 ~25×10 6 These are CAR-positive viable T cells.

[0058] In some embodiments, the dose is 5×10 6 or about 5 x 10 6 These are CAR-positive viable T cells.

[0059] In some embodiments, the dose is 10×10 6 or about 10 x 10 6 These are CAR-positive viable T cells.

[0060] In some embodiments, the dose is 25×10 6 or about 25 x 10 6 These are CAR-positive viable T cells.

[0061] In some embodiments, the dose is 50×10 6 or about 50 x 10 6 These are CAR-positive viable T cells.

[0062] In some embodiments, the T cells are autologous to the subject.

[0063] In some embodiments, the method further comprises obtaining a leukapheresis sample from the subject to produce a composition comprising the engineered T cells.

[0064] In some embodiments, prior to administration, the subject has been preconditioned with lymphodepleting therapy.

[0065] In some embodiments, the method further comprises administering to the subject a lymphodepleting therapy immediately prior to administration of the dose of CD19-directed genetically modified T cells, wherein the lymphodepleting therapy comprises administration of fludarabine and / or cyclophosphamide.

[0066] In some embodiments, administration of the dose of CD19-directed genetically modified T cells and / or lymphodepletion therapy is performed by outpatient delivery.

[0067] In some embodiments, lymphodepletion therapy is administered daily to a subject with lymphocytes circulating on 1 m of body surface area. 2 30 mg of fludarabine per m2 of body surface area daily 2 Each dose includes 300 mg of cyclophosphamide per day for 3 days.

[0068] In some embodiments, the dose of CD19-directed genetically modified T cells is administered 48 or about 48 hours to 9 or about 9 days, inclusive, after completion of lymphodepletion therapy.

[0069] In some embodiments, the dose of CD19-directed genetically modified T cells is administered to the subject by intravenous infusion.

[0070] In some embodiments, the CAR comprises an extracellular antigen-binding domain that binds to CD19, a transmembrane domain, and an intracellular signaling domain.

[0071] In some embodiments, the CAR comprises a hinge spacer between the extracellular antigen-binding domain and the transmembrane domain, optionally the hinge spacer is an immunoglobulin hinge or a CD8a hinge.

[0072] In some embodiments, the extracellular antigen-binding domain is a single-chain variable fragment (scFv) derived from the FMC63 monoclonal antibody.

[0073] In some embodiments, the extracellular antigen-binding domain comprises a variable heavy chain set forth in SEQ ID NO:41 and a variable light chain set forth in SEQ ID NO:42.

[0074] In some embodiments, the scFv is set forth as SEQ ID NO:43.

[0075] In some embodiments, the extracellular antigen-binding domain is a Hu19 single-chain variable fragment (scFv).

[0076] In some embodiments, the extracellular antigen-binding domain comprises a variable heavy chain set forth in SEQ ID NO:114 and a variable light chain set forth in SEQ ID NO:112.

[0077] In some embodiments, the extracellular antigen-binding domain comprises, in order, a variable light chain set forth in SEQ ID NO:112, a linker peptide set forth in SEQ ID NO:113, and a variable heavy chain set forth in SEQ ID NO:114.

[0078] In some embodiments, the CAR is a monospecific CAR directed against CD19.

[0079] In some embodiments, the CAR is a tandem bispecific CAR directed against CD19 and at least one other antigen expressed on B cells. In some embodiments, the other antigen expressed on B cells is selected from the group consisting of CD20, CD19, CD22, ROR1, BCMA, CD45, CD21, CD5, CD33, Ig kappa, Ig lambda, CD79a, CD79b, or CD30. In some embodiments, the other antigen expressed on B cells is CD20.

[0080] In some embodiments, the extracellular antigen-binding domain comprises a variable heavy chain and a variable light chain derived from a CD20 antibody selected from the group consisting of Leu16, C2B8, 11B8, 8G6-5, 2.1.2, and GA101.

[0081] In some embodiments, the transmembrane domain is a CD28 transmembrane domain.

[0082] In some embodiments, the transmembrane domain is a transmembrane domain from CD28, optionally comprising the sequence of amino acids set forth in SEQ ID NO:8, or a sequence of amino acids exhibiting at least, or at least about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8.

[0083] In some embodiments, the intracellular signaling domain comprises a 4-1BB costimulatory domain and a CD3 zeta activation domain.

[0084] In some embodiments, the CAR comprises, in order from N-terminus to C-terminus, a single-chain variable fragment (scFv) from the FMC63 monoclonal antibody, an IgG4 hinge region, a CD28 transmembrane domain, a 4-1BB (CD137) costimulatory domain, and a CD3 zeta signaling domain.

[0085] In some embodiments, the 4-1BB costimulatory domain is or comprises the sequence set forth in SEQ ID NO: 12, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12.

[0086] In some embodiments, the CD3 zeta signaling domain is or comprises the sequence set forth in SEQ ID NO: 13, 14 or 15, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0087] In some embodiments, the CAR contains, in order from N-terminus to C-terminus, an extracellular antigen-binding domain that is an scFv set forth in SEQ ID NO: 43, a spacer set forth in SEQ ID NO: 1, a transmembrane domain set forth in SEQ ID NO: 8, a 4-1BB costimulatory signaling domain set forth in SEQ ID NO: 12, and a signaling domain of the CD3-zeta (CD3ζ) chain set forth in SEQ ID NO: 13.

[0088] In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 59, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0089] In some embodiments, the composition is produced by a manufacturing process comprising: (i) generating a stimulated population by stimulating an input composition comprising primary T cells from a subject with an oligomeric stimulating reagent, wherein the oligomeric stimulating reagent comprises a plurality of cross-linked tetramers of streptavidin or a streptavidin mutein, and the streptavidin or streptavidin mutein is reversibly bound to a first agent comprising an anti-CD3 antibody, or antigen-binding fragment thereof, and a second agent comprising an anti-CD28 antibody, or antigen-binding fragment thereof; (ii) generating a population of transduced cells by introducing a heterologous polynucleotide encoding a CD19-targeting CAR into T cells of the stimulated population; (iii) incubating the population of transduced cells for up to 96 hours; and (iv) producing a composition of CD19-directed genetically modified T cells by harvesting T cells of the population of transduced cells, wherein the harvesting occurs 24 to 120 hours, inclusive, after exposure to the stimulating reagent has begun.

[0090] In some embodiments, the anti-CD3 antibody or antigen-binding fragment is a Fab and the anti-CD28 antibody or antigen-binding fragment is a Fab.

[0091] In some embodiments, the first agent and the second agent each comprise a streptavidin-binding peptide that reversibly binds the first agent and the second agent to the oligomeric particle reagent, and optionally the streptavidin-binding peptide comprises a sequence of amino acids set forth in any of SEQ ID NOs: 78-82.

[0092] In some embodiments, the streptavidin mutant protein molecule is a tetramer of streptavidin mutant protein comprising amino acid residues Val44-Thr45-Ala46-Arg47 or Ile44-Gly45-Ala46-Arg47, and optionally the streptavidin mutant protein comprises the sequence set forth in SEQ ID NO: 69, 84, 87, 88, 90, 85 or 59.

[0093] In some embodiments, the oligomeric particle reagent comprises between 1,000 and 5,000 streptavidin mutein tetramers, inclusive.

[0094] In some embodiments, the method further comprises adding biotin or a biotin analog after or during the incubation prior to harvesting the cells.

[0095] In some embodiments, harvesting occurs between 48 and 120 hours, inclusive, after exposure to the stimulating agent has begun.

[0096] In some embodiments, the dose of autologous CD19-directed genetically modified T cells is cryopreserved prior to administration to the subject.

[0097] In some embodiments, a cryopreserved dose of autologous CD19-directed genetically modified T cells is thawed prior to administration to a subject.

[0098] In some embodiments, the dose of autologous CD19-directed genetically modified T cells is administered to the subject within about 2 hours of being thawed.

[0099] In some embodiments, the dose of autologous CD19-directed genetically modified T cells is provided in a formulation that includes a cryoprotectant.

[0100] In some embodiments, the formulation comprises dimethyl sulfoxide (DMSO).

[0101] In some embodiments, the formulation comprises albumin, optionally human albumin.

[0102] In some embodiments, the dose of T cells comprises a CD4 + T cells and CD8 expressing CAR + T cells at a ratio of about 1.5 to about 5:1.

[0103] In some embodiments, the dose of T cells comprises a CD4 + T cells and CD8 expressing CAR + T cells at a ratio of about 1.3 to about 3:1.

[0104] In some embodiments, at least 90% or at least about 90% of the cells in the composition are CD3 + It is a cell.

[0105] In some embodiments, at least 91%, or at least about 91%, at least 92%, or at least about 92%, at least 93%, or at least about 93%, at least 94%, or at least about 94%, at least 95%, or at least about 95%, or at least 95% or at least about 96% of the cells in the composition are CD3 + It is a cell.

[0106] In some embodiments, at least 25% of the T cells in the composition are CAR+ T cells, hi some embodiments, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the T cells in the composition are CAR+ T cells.

[0107] In some embodiments, the CAR in the composition + 5% or about 5% to 30% or about 30% of the T cells, as appropriate, CAR in the composition + 10% or about 10%-15% or about 15% of the T cells express a marker of apoptosis, and further suitably the marker of apoptosis is annexin V or active caspase 3.

[0108] In some embodiments, less than 10% of the T cells in the composition express a marker of apoptosis. In some embodiments, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, or less than 4% of the T cells in the composition express a marker of apoptosis. In some embodiments, less than 10% of the CAR+ T cells in the composition express a marker of apoptosis. In some embodiments, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, or less than 4% of the CAR+ T cells in the composition express a marker of apoptosis. In some of any of the embodiments, the marker of apoptosis is annexin V or active caspase 3.

[0109] In some embodiments, at least 70% of the T cells in the composition are viable T cells. In some embodiments, at least 75% of the T cells in the composition are viable T cells. In some embodiments, at least 80% of the T cells in the composition are viable T cells. In some embodiments, at least 85% of the T cells in the composition are viable T cells. In some embodiments, at least 90% of the T cells in the composition are viable T cells.

[0110] In some of either embodiment, viability is determined by acridine orange (AO) and propidium iodide (PI) staining.

[0111] In some embodiments, the CAR in the composition + At least 80% or at least about 80% of the T cells are of a naive-like or central memory phenotype.

[0112] In some embodiments, the marker expressed on naive-like or central memory T cells is selected from the group consisting of CD45RA, CD27, CD28, and CCR7.

[0113] In some embodiments, at least 70% of the CAR+ T cells in the composition are CCR7+. In some embodiments, at least 75% of the CAR+ T cells in the composition are CCR7+. In some embodiments, at least 80% of the CAR+ T cells in the composition are CCR7+. In some embodiments, at least 85% of the CAR+ T cells in the composition are CCR7+. In some embodiments, at least 90% of the CAR+ T cells in the composition are CCR7+. In some embodiments, at least 95% of the CAR+ T cells in the composition are CCR7+. In some embodiments, at least 85% of the CD8+ CAR+ T cells in the composition are CCR7+, and at least 90% of the CD4+ CAR+ T cells in the composition are CCR7+. In some embodiments, 85%-98% of the CD8+ CAR+ T cells in the composition are CCR7+, and 94%-99% of the CD4+ CAR+ T cells in the composition are CCR7+.

[0114] In some embodiments, the CARs in the composition are of a naive-like or central memory phenotype. + At least 80% or at least about 80% of the T cells are CCR7 + CD45RA + , CCR7+CD45RA - , CD27 + CCR7 + , or CD62L - CCR7 + The phenotype is selected from:

[0115] In some embodiments, at least 40% of the CAR+ T cells in the composition are CD45RA+CCR7+. In some embodiments, at least 50% of the CAR+ T cells in the composition are CD45RA+CCR7+. In some embodiments, at least 60% of the CAR+ T cells in the composition are CD45RA+CCR7+. In some embodiments, at least 70% of the CAR+ T cells in the composition are CD45RA+CCR7+. In some embodiments, at least 80% of the CAR+ T cells in the composition are CD45RA+CCR7+. In some embodiments, at least 20% of the CAR+ T cells in the composition are CD45RA-CCR7+. In some embodiments, at least 30% of the CAR+ T cells in the composition are CD45RA-CCR7+. In some embodiments, at least 40% of the CAR+ T cells in the composition are CD45RA-CCR7+. In some embodiments, at least 50% of the CAR+ T cells in the composition are CD45RA-CCR7+. In some embodiments, at least 60% of the CAR+ T cells in the composition are CD45RA-CCR7+.

[0116] In some embodiments, at least about 50% of the CD4+ CAR+ T cells in the composition are CCR7+CD45RA - In some embodiments, at least about 60% of the CD4+ CAR+ T cells in the composition are CCR7+CD45RA - In some embodiments, at least about 70% of the CD4+ CAR+ T cells in the composition are CCR7+CD45RA - In some embodiments, at least about 30% of the CD8+ CAR+ T cells in the composition are CCR7+CD45RA - In some embodiments, at least about 40% of the CD8+ CAR+ T cells in the composition are CCR7+CD45RA - In some embodiments, at least about 50% of the CD8+ CAR+ T cells in the composition are CCR7+CD45RA - is.

[0117] In some embodiments, greater than or about 50%, greater than or about 60%, greater than or about 70%, or greater than or about 80% of subjects treated according to the method do not exhibit any grade of cytokine release syndrome (CRS).

[0118] In some embodiments, greater than or greater than about 40%, greater than or greater than about 50%, or greater than or greater than about 60% of subjects treated according to the method do not exhibit any grade of neurotoxicity.

[0119] In some embodiments, the subject is a human.

[0120] In some embodiments, at least 60% of the T cells in the composition are viable; at least 25% of the T cells in the composition are CAR+ T cells; less than 10% of the cells in the composition are positive for a marker of apoptosis, where optionally the marker of apoptosis is annexin V or active caspase 3; at least 85% of the CD8+ CAR+ T cells in the composition are CCR7+; and / or at least 90% of the CD4+ CAR+ T cells in the composition are CCR7+.

[0121] In some embodiments, at least 80% of the T cells in the composition are viable; at least 45% of the T cells in the composition are CAR+; less than 4% of the cells in the composition are positive for a marker of apoptosis, where optionally the marker of apoptosis is annexin V or active caspase 3; at least 85% of the CD8+ CAR+ T cells in the composition are CCR7+; and / or at least 90% of the CD4+ CAR+ T cells in the composition are CCR7+.

[0122] In some embodiments, at least 60% of the T cells in the composition are viable; at least 25% of the T cells in the composition are CAR+; less than 10% of the cells in the composition are positive for a marker of apoptosis, optionally the marker of apoptosis is annexin V or active caspase 3; and / or greater than or greater than about 40% of the CAR+ T cells in the composition are CCR7+CD45RA+.

[0123] In some embodiments, at least 80% of the T cells in the composition are viable; at least 45% of the T cells in the composition are CAR+; less than 4% of the cells in the composition are positive for a marker of apoptosis, optionally the marker of apoptosis is annexin V or active caspase 3; and / or at least 40% of the CAR+ T cells in the composition are CCR7+CD45RA+.

[0124] In some embodiments, at least 60% of the T cells in the composition are viable; at least 25% of the T cells in the composition are CAR+; less than 10% of the cells in the composition are positive for a marker of apoptosis, optionally the marker of apoptosis is annexin V or active caspase 3; and / or more than 20% of the CAR+ T cells in the composition are CCR7+CD45RA-.

[0125] In some embodiments, at least 80% of the T cells in the composition are viable; at least 45% of the T cells in the composition are CAR+; less than 4% of the cells in the composition are positive for a marker of apoptosis, optionally the marker of apoptosis is annexin V or active caspase 3; and / or at least 20% of the CAR+ T cells in the composition are CCR7+CD45RA-. [Brief explanation of the drawings]

[0126] [Figure 1]Figures 1A and 1B depict T cell memory subtypes in CAR+CD4+ and CAR+CD8+ for non-expansion and expansion processes, respectively. [Figure 2] Figures 2A-2C depict the fold expansion of T cells in T cell compositions produced by the expanded and non-expanded process in a long-term stimulation assay after CAR stimulation with anti-idiotypic antibodies as an indicator of persistence and expandability. After 10 days of stimulation with CAR anti-idiotypic antibodies, the fold expansion of T cell compositions from different donors was calculated over time and depicted as the fold cell number expansion (Figure 2A), the area under the curve for the fold expansion (Figure 2B), and the fold expansion of CAR T cells produced by the non-expanded process divided by the fold expansion of CAR T cells from the same donor produced by the expanded process (Figure 2C). [Figure 3] Figures 3A-3D depict cytokine production of CAR+ CD4+ and CD8+ T cells in T cell compositions generated by the expanded and non-expanded processes in long-term stimulation assays after 10 days of CAR stimulation with the anti-idiotypic antibodies depicted in Figures 2A-2C. Results show the percent of CAR+ CD4+ or CAR+ CD8+ T cells positive for IL-2 (Figure 3A), IFN-γ (Figure 3B), TNFα (Figure 3C), or IL-2, IFNγ, and TNFα (Figure 3D). [Figure 4] Figures 4A and 4B depict CD19+ target cell-specific lysis over time by CAR+ T cells generated from both the expanded and non-expanded processes (Figure 4A), and the area under the curve of cell lysis over time (Figure 4B). [Figure 5] Figures 5A-5F depict CAR transgene levels (Figure 5A), serum IgG (Figure 5B), serum IgA (Figure 5C), neutrophil count (Figure 5D), total lymphocyte count (Figure 5E), and platelet count (Figure 5F) in human patients after treatment with 10x10 or 25x10 anti-CD19 CARs produced by a non-expanded process. [Figure 6]Figure 6A depicts the cumulative population doublings (PDL) of engineered cell compositions at harvest time points after pre-transduction stimulation in a process for producing anti-CD19 from donor human subjects, including SLE subjects. Figure 6B depicts the cell viability of engineered cell compositions at harvest time points after pre-transduction stimulation in a process for producing anti-CD19 from donor human subjects, including SLE subjects. DETAILED DESCRIPTION OF THE INVENTION

[0127] Generally, provided herein are methods for treating subjects having a disease or condition that is or includes a severe or moderate systemic autoimmune disease, and the use of engineered cells (e.g., T cells) and / or compositions thereof for such treatment. In embodiments of the provided methods, a therapeutic T cell composition containing engineered cells is administered to a subject having a severe or moderate systemic autoimmune disease, e.g., by adoptive cell therapy, e.g., adoptive T cell therapy. In some embodiments, the disease or condition is a systemic autoimmune disease. In some embodiments, the disease or condition is a severe or moderate systemic autoimmune disease. Systemic autoimmune diseases are a class of abnormal immune disorders, which share similar clinical symptoms and are generally treatable by similar approaches. In addition to systemic lupus erythematosus (SLE), other systemic autoimmune diseases include, for example, Sjögren's syndrome, progressive systemic sclerosis (i.e., scleroderma), idiopathic inflammatory myositis (IIM, including dermatomyositis, polymyositis, and necrotizing myositis), mixed connective tissue disease (MCTD), relapsing-remitting multiple sclerosis, ANCA-associated vasculitis (AAV), Crohn's disease, myasthenia gravis, Behçet's disease, rheumatoid arthritis, multiple sclerosis (MS), IgA nephropathy, pemphigus vulgaris, myasthenia gravis, autoimmune hemolytic anemia, immune thrombocytopenia, IgG4-related disease, membranous nephropathy, cutaneous lupus erythematosus, sarcoidosis, light-chain amyloidosis, rheumatoid arthritis, bullous pemphigoid, and chronic inflammatory demyelinating polyneuropathy. In particular embodiments of any of the provided methods and uses, the T cells are engineered with a chimeric antigen receptor (CAR) directed against cluster of differentiation 19 (CD19).

[0128] In some embodiments, the methods and uses involve administering to a subject in adoptive cell therapy T cells that express a genetically engineered (recombinant) cell surface receptor, typically a chimeric receptor that recognizes CD19, e.g., a chimeric antigen receptor (CAR). In some embodiments, CD19 is expressed by cells (e.g., B cells) that contribute to the symptoms of systemic autoimmune disease. In some embodiments, CD19 is associated with SLE, IIM, SSc, AAV, systemic sclerosis, highly active relapsing-remitting multiple sclerosis (MS), primary progressive MS, IgA nephropathy, pemphigus vulgaris, myasthenia gravis, demyelinating polyneuropathy, autoimmune hemolytic anemia, immune thrombocytopenia, IgG4-related disease, membranous nephropathy, primary Sjogren's syndrome, cutaneous lupus erythematosus, sarcoidosis, light chain amyloidosis, rheumatoid arthritis, bullous pemphigoid, acute respiratory distress syndrome, atopic eczema, hereditary vascular In some embodiments, CD19 is expressed by cells associated with and / or specific for the symptoms of: chronic edema, hidradenitis suppurativa, inclusion body myositis, inflammatory bowel disease, mastocytosis, multifocal motor neuropathy, necrotizing myopathy, neuromyelitis optica spectrum disorder, mixed connective tissue disorder, POEMS syndrome, primary biliary cholangitis, psoriasis, rhesus hemolytic disease, Still's disease, type 1 diabetes, urticaria, capillary leak syndrome, cytokine release syndrome, erythema multiforme, pyoderma gangrenosum, antiphospholipid syndrome, or X-linked agammaglobulinemia. In some embodiments, CD19 is expressed by cells associated with and / or specific for the symptoms of: SLE, IIM, AAV, systemic sclerosis, highly active relapsing-remitting multiple sclerosis (MS), primary progressive MS, IgA nephropathy, pemphigus vulgaris, or myasthenia gravis.

[0129] In certain embodiments, CD19 is expressed by cells associated with and / or specific for symptoms of SLE, e.g., severe, refractory SLE. In certain embodiments, CD19 is expressed by cells associated with and / or specific for symptoms of idiopathic inflammatory myopathy (IIM). In certain embodiments, CD19 is expressed by cells associated with and / or specific for symptoms of systemic sclerosis (SSc). In certain embodiments, CD19 is expressed by cells associated with and / or specific for symptoms of multiple sclerosis (MS). In certain embodiments, CD19 is expressed by cells associated with and / or specific for symptoms of rheumatoid arthritis (RA). In certain embodiments, CD19 is expressed by cells associated with and / or specific for symptoms of active secondary progressive MS (aSPMS). In certain embodiments, CD19 is expressed by cells associated with and / or specific for symptoms of myositis. In certain embodiments, CD19 is expressed by cells associated with and / or specific for symptoms of myasthenia gravis. In certain embodiments, CD19 is expressed by cells associated with and / or specific for symptoms of bullous pemphigoid. In certain embodiments, CD19 is expressed by cells associated with and / or specific for symptoms of immune thrombocytopenia. In certain embodiments, CD19 is expressed by cells associated with and / or specific for symptoms of autoimmune hemolytic anemia. In certain embodiments, CD19 is expressed by cells associated with and / or specific for symptoms of pemphigus vulgaris. In certain embodiments, CD19 is expressed by cells associated with and / or specific for symptoms of demyelinating polyradiculoneuropathy. In certain embodiments, CD19 is expressed by cells associated with and / or specific for symptoms of membranous nephropathy.

[0130] In some embodiments, the systemic autoimmune disease is SLE, IIM, MS, or SSc. In some embodiments, the disease or condition is moderate SLE. In some embodiments, the disease or condition is severe refractory SLE. In particular, provided herein are methods for treating subjects with severe refractory SLE, and the use of engineered cells (e.g., T cells) and / or compositions thereof for such treatment. In embodiments of the provided methods, a therapeutic T cell composition containing engineered cells is administered to a subject with severe refractory SLE, for example, by adoptive cell therapy, e.g., adoptive T cell therapy. In certain embodiments of such methods and uses, T cells are engineered with a chimeric antigen receptor (CAR) directed against cluster of differentiation 19 (CD19).

[0131] In some aspects, the methods and uses result in or achieve improved response, and / or more durable response or efficacy, and / or reduced risk of toxicity or other side effects, e.g., in particular groups of treated subjects, compared to certain alternative methods. In some embodiments, the methods are advantageous by virtue of administering a specified or relative number of engineered cells, administering a defined ratio of particular types of cells, administering cells in which a particular high percentage are less differentiated cells (e.g., naive-like or central memory cells or cells in an early differentiation state, e.g., CCR7+CD27+ cells), treating particular patient populations, e.g., those with particular risk profiles, disease staging, and / or prior treatment history, and / or combinations thereof.

[0132] The engineered T cells are generally administered in a composition formulated for administration, and the methods generally involve administering one or more doses of cells to a subject, which dose(s) may contain a specific or relative number of cells or engineered cells. In some cases, the CD19-directed CAR+ engineered cells in the composition include a defined ratio or composition of two or more subtypes, e.g., CD4 T cells to CD8 T cells, within the composition.

[0133] In certain embodiments, compositions of cells for use or administration in the provided methods comprise primary T cells engineered to express a CD19-directed CAR that (i) contain a low percentage (e.g., less than 40%, less than 30%, less than 20%, or less than 10%) of exhausted cells and / or cells displaying markers or phenotypes associated with exhaustion; and / or (ii) contain a relatively high percentage (e.g., greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%) of memory-like T cells, e.g., naive-like T cells, central memory T cells, or long-lived memory T cells.

[0134] In provided embodiments, the features of the compositions and provided methods result in improved or enhanced immune activity compared to methods involving the administration of other CD19-directed CAR T-cell therapies that contain a higher percentage of exhausted cells and / or a greater number of cells displaying an exhaustion-associated phenotype, and / or a lower percentage of certain T cells, e.g., naive-like T cells, central memory T cells, or long-lived memory T cells. In provided embodiments, the features of the compositions and provided methods result in improved therapeutic efficacy, e.g., a higher percentage of patients achieving a complete response (CR), compared to methods involving the administration of other CD19-directed CAR T-cell therapies that contain a higher percentage of exhausted cells and / or a greater number of cells displaying an exhaustion-associated phenotype, and / or a lower percentage of certain T cells, e.g., naive-like T cells, central memory T cells, or long-lived memory T cells. In provided embodiments, the features of the compositions and provided methods result in improved clinical durability of the therapeutic response, such as a CR, e.g., a response that persists for a period of time from the initiation of treatment, compared to methods involving the administration of other CD19-directed CAR T cell therapies that contain a higher percentage of exhausted cells and / or a greater number of cells displaying an exhaustion-associated phenotype, and / or that contain a lower percentage of memory-like T cells, e.g., naive-like T cells, central memory T cells, or long-lived memory T cells.

[0135] In certain embodiments, use or administration of the provided CD19-directed CAR T-cell compositions in the provided methods can be achieved with a dose of cells that is less than half, e.g., five or ten times lower, than the dose of a reference CD19-directed CAR T-cell composition (e.g., one engineered with the same or similar CAR, such as one with the same antigen-binding domain), but containing a higher percentage of exhausted cells, and / or a greater number of cells displaying a phenotype associated with exhaustion, and / or a lower percentage of memory-like T cells, e.g., naive-like T cells, central memory T cells or long-lived memory T cells. In some embodiments, the reference CD19-directed CAR T-cell composition is a composition produced ex vivo by a process that involves culturing cells under conditions for expansion, e.g., cell proliferation or population doubling of cells (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doublings of cells in the population compared to the start of the process), during the process to produce the cells.

[0136] In some embodiments, the CD19-directed CAR T cell compositions for use in the provided methods and uses are produced by a relatively short process that does not include steps for culturing cells under expansion conditions designed to expand or grow the cells. Different processes, which typically include steps designed or intended to culture cells to expand or increase cell proliferation, can be used to produce compositions containing engineered T cell populations, including for producing engineered T cells expressing CARs. However, in certain embodiments, some of these processes may require a long or relatively long time to produce engineered cells. In addition, in various embodiments, some existing processes require different amounts of time to successfully produce engineered T cells suitable for cell therapy, making it difficult to adjust the administration of the cell therapy. In certain embodiments, some of these processes may produce populations of cells that contain a relatively high percentage or amount of exhausted cells, differentiated cells, or cells with low potency. The provided CD19-directed CAR T cell compositions for use in the provided methods address one or more of these issues.

[0137] In certain embodiments, the provided methods are used in connection with a process for efficiently producing or generating engineered cells suitable for use in cell therapy. In some embodiments, the provided compositions containing CD19-directed CAR engineered T cells are produced by a process that does not require any additional steps for expanding the cells, e.g., no expansion unit operation and / or no steps aimed at causing cell expansion. In embodiments of a process for producing a CD19-directed CAR T cell composition, the process includes one or more steps for stimulating and genetically engineering (e.g., transforming, transducing, or transfecting) T cells to produce a population of engineered T cells that can be collected or formulated for use as a cell therapy composition. In certain embodiments, the process includes transducing cells with a viral vector (e.g., a lentiviral vector) containing a nucleic acid encoding a CD19-directed CAR. In some embodiments, the provided process results in stable integration of the heterologous nucleic acid (expressed from the viral vector) into the genome of the cell. In some embodiments, the provided process produces engineered CD19-directed CAR T cells with enhanced potency compared to engineered T cell compositions produced from alternative processes, e.g., processes involving cell expansion.

[0138] In certain aspects, the duration of the process for producing a provided composition can be measured from when the input cell population or cells, e.g., T cells, of the input composition are first contacted or exposed to stimulating conditions (e.g., as described herein, e.g., in Section II-C), which initial contact or exposure is referred to herein as stimulation or initiation of stimulating, and also referred to herein as exposure to a stimulating reagent, e.g., when exposure to a stimulating reagent is initiated. In some embodiments, the duration of time required to harvest or collect the output population containing engineered cells (also referred to herein as the output composition or the composition of engineered cells, e.g., engineered T cells) is measured from the initiation of stimulation. In certain embodiments, the duration of the process is, or is about, or is less than 120, 108, 96, 84, 72, 60, 48, 36, or 30 hours. In certain embodiments, the duration of the process is, or is about, or is less than 5, 4, 3, 2, or 1 day. In certain embodiments, the engineered cells, e.g., cells of an output composition or population, are more potent, persistent, or naive-like than cells engineered using processes requiring longer times. In some embodiments, the duration of the provided processes, e.g., the time required to generate or produce an engineered population of T cells, is 2, 3, 4, 5, 6, 7, or more than 7 days, about 2, 3, 4, 5, 6, 7, or more than 7 days, or at least 2, 3, 4, 5, 6, 7, or more than 7 days shorter than some existing processes.In some embodiments, the duration of the provided process is, is about, or is less than 75%, 60%, 50%, 40%, 30%, 25%, 15%, or 10% of an alternative or existing process.

[0139] In certain embodiments, the provided processes are performed using cells isolated, enriched, or selected from a biological sample, e.g., a population of CD3+, CD4+, and / or CD8+ T cells. In some aspects, the provided methods can produce or generate compositions of engineered T cells in a reduced time from the time the biological sample is obtained from the subject compared to other methods or processes. In some embodiments, the provided methods are capable of producing or generating engineered T cells, including when the biological sample, or enriched, isolated, or selected cells, are cryopreserved and stored, they can be produced or generated at any time or every time within, or about, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days, or within, or about, 120, 96, 72, or 48 hours, of when the biological sample is taken from the subject, until the engineered T cells are collected, recovered, or formulated (e.g., for cryopreservation or administration).

[0140] In certain embodiments, a process for producing or manipulating a T cell population includes stimulating the cells, e.g., prior to transduction with a viral vector. In an embodiment of the provided process, stimulation is carried out using an oligomeric stimulating reagent, e.g., a streptavidin mutein oligomer, to which a stimulatory binding agent, e.g., anti-CD3 / anti-CD28, is immobilized or bound. Existing reagents for use in stimulating T cells in vitro, e.g., in the absence of exogenous growth factors or in the presence of low amounts of exogenous growth factors, are known (see, e.g., U.S. Pat. No. 6,352,694 B1 and European Patent EP 0 700 430 B1). Generally, such reagents may utilize beads, e.g., magnetic beads, greater than 1 μm in diameter to which various binding agents (e.g., anti-CD3 and / or anti-CD28 antibodies) are immobilized. However, in some cases, such magnetic beads are difficult to incorporate into methods for stimulating cells under conditions required for clinical trials or therapeutic purposes, for example, because it must be ensured that these magnetic beads are completely removed before administering the expanded T cells to a subject. In some embodiments, such removal, for example, by exposing the cells to a magnetic field, can reduce the yield of viable cells available for cell therapy. In certain cases, such reagents, for example, stimulating reagents containing magnetic beads, must be incubated with cells for a minimum time to allow a sufficient amount of T cells to detach from the stimulating reagent.

[0141] The provided processes utilizing oligomeric stimulating reagents, e.g., streptavidin mutein polymers, overcome such potential limitations. For example, in some embodiments, the provided processes avoid or reduce the risk of residual stimulating reagents, e.g., reagents containing magnetic beads, in the output cells generated or produced by the process. In some embodiments, this also means that processes that comply with GMP standards can be more easily established compared to other methods in which additional measures must be taken to ensure that the final engineered T cell population is bead-free. In some embodiments, this can be easily accomplished in this embodiment by, for example, simply rinsing the cells or washing them, e.g., by centrifugation, with the addition of a substance, e.g., a competing reagent, that dissociates the oligomeric stimulating reagent from the cells. Thus, in some embodiments, removal or separation of the oligomeric stimulating reagent from the cells, e.g., by addition of a substance or competing reagent, results in little or no cell loss compared to removal or separation of bead-based stimulating reagents. In some embodiments, the timing of removal or separation of the oligomeric stimulating reagent is not limited or is less restrictive than removal or separation of bead-based stimulating reagents. Thus, in some embodiments, the oligomeric stimulatory reagents may be removed or separated from the cells at any time or stage during the process provided.

[0142] In some embodiments, the use of oligomeric stimulating reagents (e.g., anti-CD3 / anti-CD28 streptavidin mutein oligomers) can result in an overall reduced stimulatory signal compared to alternative stimulating reagents, e.g., anti-CD3 / anti-CD28 paramagnetic beads. The provided processes, which may involve weaker or reduced stimulation, can produce engineered CAR+ T cells that are similar to or even more potent, persistent, or effective than CAR+ T cells that may be produced by stronger stimulating conditions or processes involving higher amounts or concentrations of stimulating reagents, e.g., after stimulation with anti-CD3 / anti-CD28 paramagnetic beads. Additionally, in some embodiments, stimulating cells with lower or relatively lower amounts of oligomeric stimulating reagents can increase the potency, efficacy, or persistence of the resulting engineered cell population compared to processes using higher amounts of oligomeric stimulating reagents. Such embodiments contemplate that such effects may persist even at doses low enough to reduce expression of activation markers or the proportion of cells positive for activation markers during and after the process.

[0143] In certain embodiments, T cell output compositions or populations containing engineered T cells, e.g., T cells expressing a recombinant receptor such as a chimeric antigen receptor, produced or generated by the provided processes are particularly effective or potent when utilized as cells for cell therapy. For example, in some embodiments, output compositions containing engineered T cells, e.g., CAR+ T cells, produced from the provided processes have a much greater degree of potency and / or proliferative capacity than engineered T cells produced or generated by alternative, existing processes. In some embodiments, output compositions containing engineered T cells, e.g., CAR+ T cells, produced by the provided processes have enhanced immune activity than engineered T cells, e.g., CAR+ T cells, produced by alternative or existing methods.

[0144] In certain embodiments, a process for producing the provided CD19-directed T cell compositions that does not include a step in which cells are expanded to a threshold amount or concentration is further advantageous. In some embodiments, protocols for increasing the number or concentration of cells from a starting cell population (e.g., an input population) without relying on cell expansion do not require incubation or culturing, which may vary depending on the cell population. For example, some embodiments contemplate that cell populations obtained from different subjects, such as subjects with different diseases or disease subtypes, may divide or expand at different rates, particularly in the case of patients with high-risk, aggressive, and / or severe, refractory SLE. In certain embodiments, eliminating the potentially variable step of cell expansion allows for tight control over the duration of the overall process. In certain embodiments, variability in process duration is reduced or eliminated, which, in some embodiments, may allow for improved coordination of appointments and procedures between donors, patients, and technicians to facilitate autologous cell therapy.

[0145] In some embodiments, provided methods involve treating a specific group or subset of subjects, e.g., subjects identified as having a high-risk disease, e.g., a systemic autoimmune disease, e.g., a severe systemic autoimmune disease. In some embodiments, subjects to be treated for a systemic autoimmune disease, e.g., any described herein, are relapsing or refractory (R / R) to standard therapy for treating the systemic autoimmune disease and / or have a poor prognosis. In some embodiments, the methods treat subjects with severe disease that is relapsing or refractory (R / R) to standard therapy. In some embodiments, provided methods involve treating a specific group or subset of subjects, e.g., subjects identified as having a high-risk disease, e.g., SLE, e.g., severe refractory SLE. In some embodiments, the methods treat subjects with certain aggressive and / or poor-prognosis SLE, e.g., SLE that is relapsing or refractory (R / R) to standard therapy and / or has a poor prognosis. In some embodiments, the methods treat subjects with severe SLE that is relapsing or refractory (R / R) to standard therapy.

[0146] In certain embodiments, the engineered cells are autologous to the subject and administered after production by an ex vivo process that is shorter than existing methods, does not include or does not involve a culturing step for cell expansion during the process of producing the engineered cells, and / or can produce a less differentiated CAR engineered T cell composition, thus allowing for lower dose administration. As a result, the provided methods are advantageous compared to existing methods because they can shorten the time it takes for a therapy using engineered T cells to be available to patients, particularly those in need of treatment, such as those who have relapsed or are refractory to treatment after one or more previous therapies for treating a disease or condition. In some embodiments, the provided methods, compositions, uses, and articles of manufacture achieve an improved or superior response to available therapies. In some embodiments, the improved or superior response is relative to the current standard of care (SOC).

[0147] CD19 is a member of the immunoglobulin superfamily and a component of the B cell surface signaling complex that positively regulates signal transduction by the B cell receptor. CD19 is expressed by most B cell malignancies from early development to differentiation into plasma cells [Stamenkovic et al., J Exp Med. 1988; 168(3):1205-10]. CD19 is an attractive therapeutic target because CAR-T therapy has the unique potential to provide transformative treatment for severe, refractory lupus and other related conditions. CD19 CAR T cell therapy provides transformative efficacy and a favorable safety profile in severe SLE.

[0148] In certain embodiments, the methods provided herein are based on the administration of CD19-directed CAR T cell therapy, in which the CAR contains a CD19-directed scFv antigen-binding domain (e.g., from FMC63). The CAR further contains an intracellular signaling domain containing a signaling domain from CD3 zeta, and also incorporates a 4-1BB costimulatory domain, which is associated with a lower incidence of cytokine release syndrome (CRS) and neurotoxicity (NE) compared to CD28-containing constructs (Lu et al. J Clin Oncol. 2018;36:3041).

[0149] The provided methods are based on research findings that the less differentiated state of adoptively transferred T cells can affect their ability to sustain and promote durable immune activity. In some embodiments, the provided CD19-directed CAR+ engineered T cell compositions are produced by methods in which cells are not cultured under expansion conditions, thereby limiting or reducing the number of population doublings in the final engineered output composition and resulting in a less differentiated product. However, the provided compositions are also produced by processes that result in a stably integrated vector copy number (iVCN) to ensure consistent and reliable expression of the CAR, thereby resulting in a consistent cell product for administration to subjects and reducing variability between CAR-expressing cells in an administered dose. Meanwhile, most protocols for T cell engineering routinely expand T cells ex vivo for 9-14 days or longer. The provided data exemplified herein support that CAR T cell products with an increased composition of less differentiated memory T cells can exhibit enhanced durable immune activity. These findings demonstrate that strategies aimed at minimizing effector differentiation in CAR T cell products can result in improved clinical efficacy. Embodiments that can achieve such aims are provided herein.

[0150] In particular, the results herein demonstrate the beneficial effect that CD19-directed CAR T cells can induce immune reset following targeted cytotoxic killing of CD19-expressing B cells. In some embodiments, as demonstrated in Example 2 for relapsed or refractory (R / R) non-Hodgkin's lymphoma (NHL), compositions comprising anti-CD19 CAR T cells can suppress B cell hyperactivation, resulting in immune reset and restoration of homeostatic immune system function. Thus, these results support the use of CD19-directed CAR-expressing T cells to achieve the same effect: to reset the immune system in autoimmune diseases by removing hyperactive B cells, and to enable a reduction in autoimmune disease activity and the achievement of clinical remission. Other treatments have attempted to deplete B cells or reset the immune system, such as the use of HSCT or antibody therapy against B cell surface proteins (e.g., Tyndall et al. Ann Rheum Dis 2001, 60:702-707; Sullivan et al. N Engl J Med 2018, 378:35-47; Wise and Stohl, Front. Med., 2020, &:303), but none have been successful in efficiently reducing circulating B cells to reduce disease activity as observed herein through the cytotoxic activity of CD19-directed CAR-expressing T cells and / or in doing so while also minimizing toxicity to the subject from the treatment.

[0151] In some embodiments, the results herein surprisingly demonstrate a reduction in disease activity in subjects with autoimmune or inflammatory diseases, as shown by the results of treated subjects with SLE. The reduction in disease activity was as little as 10×10 6This was observed using a relatively low dose of CD19-directed CAR T cells, with viable CAR+ T cells (including CD4+ and CD8+ CAR+ T cells). This dose is several orders of magnitude lower than the doses administered with other CD19-directed CAR T cell products. Furthermore, the doses for administration herein are generally administered as a flat dose (not a weight-based dose based on the subject's body weight), which has the added benefit of improving dosing consistency and reducing the risk of toxic side effects that may result in the administration of too many cells in some subjects from a weight-based dosing strategy. The results herein show that no severe toxicity was observed, thus demonstrating the safety of the provided T cell therapy. Of note, while administering a relatively low dose of cells (e.g., CAR-expressing T cells) may reduce the risk of toxic adverse events, relatively low doses of cells produced from other methods may not be fully effective in treating a disease or condition. The ability to deliver CAR-T cell products at low doses while maintaining high disease efficacy is a unique advantage of the provided methods and compositions.

[0152] The provided embodiments also support that subjects can be successfully treated without any further immunosuppression. Typically, successful treatment of autoimmune indications generally requires continued immunosuppression. However, as described herein, increased hospitalization and side effects of medications such as long-term oral corticosteroids (OCS or glucocorticoids and other immunosuppressive treatments) can increase the disease burden in subjects with autoimmune indications such as SLE. The results herein support that remission of disease activity is possible with a single infusion of a dose of CD19-CAR-directed T cells, without further administration of immunosuppressants (e.g., corticosteroids, e.g., glucocorticoids or other immunosuppressive treatments) after administration of the dose of T cells. In some embodiments, subjects achieve long-term remission by treatment according to the provided methods. In some embodiments, no further treatment of the disease is required, and the subject remains in remission after the dose of CD19-CAR-directed T cells. For example, in provided embodiments, after administration of the CD19-CAR-directed T cells, the subject remains in remission and the subject is not administered another treatment (e.g., methotrexate, mycophenolate, cyclophosphamide, tocilizumab, IVIg, rituximab, nintedanib, or an immunosuppressant).

[0153] The observations herein support the use of CD19-directed CAR T cell therapy to treat subjects with high-risk disease according to the provided methods. For example, subjects with systemic autoimmune disease, such as severe or moderate systemic autoimmune disease, are treated according to the provided methods. In some embodiments, subjects with SLE, including those with severe SLE or certain high-risk characteristics, such as those with relapsing / refractory (R / R) severe SLE, can be treated according to the provided methods. In some embodiments, the provided methods can be used to treat subjects who have undergone multiple prior treatments (e.g., one, two, three, four, or more treatments to treat the disease). Any reference herein to a method for treating a human or animal body by surgery or therapy refers to a compound, composition, or drug for use in the method.

[0154] All publications, including patent documents, scientific articles, and databases, referenced in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. To the extent that a definition set forth herein contradicts or otherwise conflicts with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein takes precedence over the definition incorporated herein by reference.

[0155] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0156] I. Methods and Uses of CD19-Targeted Cellular Therapy in Systemic Autoimmune Disease Provided herein are treatment methods involving administering engineered cells or compositions containing engineered cells, such as engineered T cells. Provided herein are methods and uses for CD19-directed CAR engineered cells (e.g., T cells) and / or compositions thereof, including methods for treating subjects with systemic autoimmune diseases, including severe or moderate systemic autoimmune diseases for which at least two or more previous treatments have failed. In certain embodiments, the method comprises administering to a subject a dose of T cells, including CD4+ and CD8+ T cells, wherein the T cells comprise a chimeric antigen receptor (CAR) that specifically binds to CD19. In certain embodiments, the method comprises administering to a subject a dose of T cells, including CD4+ and CD8+ T cells, wherein the T cells comprise a chimeric antigen receptor (CAR) that specifically binds to CD19.

[0157] In some embodiments, the autoimmune disease is selected from the group consisting of Addison's disease, allergies, ankylosing spondylitis, asthma, atherosclerosis, autoimmune diseases of the ear, autoimmune diseases of the eye, autoimmune hepatitis, autoimmune parotitis, colitis, coronary heart disease, diabetes (including type 1 and / or type 2 diabetes), epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, recombinant human leukemia, ... In some embodiments, the systemic autoimmune disease includes, but is not limited to, immune response to a drug product, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, spondyloarthropathy, thyroiditis, transplant rejection, vasculitis, AIDS, atopic allergy, bronchial asthma, eczema, leprosy, schizophrenia, chronic fatigue syndrome, Alzheimer's disease, Parkinson's disease, myocardial infarction, stroke, autism, epilepsy, Arthus phenomenon, and anaphylaxis. In some embodiments, the systemic autoimmune disease includes, but is not limited to, systemic lupus erythematosus (SLE) and severe SLE, rheumatoid arthritis (RA), and systemic sclerosis. In some embodiments, the systemic autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), Sjogren's syndrome, progressive systemic sclerosis (i.e., scleroderma), idiopathic inflammatory myositis (IIM, including dermatomyositis, polymyositis, and necrotizing myositis), mixed connective tissue disorder (MCTD), relapsing-remitting multiple sclerosis, ANCA-associated vasculitis (AAV), Crohn's disease, myasthenia gravis, Behcet's disease, rheumatoid arthritis, primary progressive MS, IgA nephropathy, pemphigus vulgaris, myasthenia gravis, autoimmune hemolytic anemia, immune thrombocytopenia, IgG4-related disease, membranous nephropathy, cutaneous lupus erythematosus, sarcoidosis, light chain aminotransferase (LANA), and leukemia. These conditions may include inflammatory bowel disease, acute respiratory distress syndrome, atopic eczema, hereditary angioedema, hidradenitis suppurativa, inclusion body myositis, inflammatory bowel disease, mastocytosis, multifocal motor neuropathy, necrotizing myopathy, neuromyelitis optica spectrum disorder, mixed connective tissue disorder, POEMS syndrome, primary biliary cholangitis, psoriasis, rhesus hemolytic disease, Still's disease, type 1 diabetes, urticaria, capillary leak syndrome, cytokine release syndrome, erythema multiforme, pyoderma gangrenosum, X-linked agammaglobulinemia, antiphospholipid antibody syndrome, and chronic inflammatory demyelinating polyneuropathy (also called chronic inflammatory demyelinating polyneuropathy).

[0158] In some embodiments, the systemic autoimmune disease is SLE, for example, moderate SLE or severe refractory SLE, idiopathic inflammatory myopathy, systemic sclerosis, rheumatoid arthritis (RA), or multiple sclerosis. In some embodiments, the systemic autoimmune disease is SLE, for example, moderate SLE or severe refractory SLE, idiopathic inflammatory myopathy, systemic sclerosis, or multiple sclerosis. Some of the provided methods involve administering engineered cells or compositions containing engineered cells, for example, engineered T cells, to a subject with SLE, including severe refractory SLE. Methods and uses of the engineered cells (e.g., T cells) and / or compositions of the provided CD19-directed CARs are also provided, including methods for treating a subject with SLE, including severe refractory SLE, involving the administration of engineered cells and / or compositions thereof. In certain embodiments, the subject has severe refractory SLE. In some embodiments, a subject is selected for or identified as having severe refractory SLE, such as by the presence of certain characteristics or clinical signs indicating the presence of severe refractory SLE. Exemplary selection criteria are further described herein. In some embodiments, the provided methods and uses for CD19-directed CAR engineered cells (e.g., T cells) and / or compositions thereof include methods for treating a subject with severe refractory SLE who has failed at least two or more previous treatments. In certain embodiments, the method includes administering to a subject a dose of T cells, including CD4+ and CD8+ T cells, wherein the T cells comprise a chimeric antigen receptor (CAR) that specifically binds to CD19.

[0159] Also disclosed herein is a method of treating a systemic autoimmune disease, comprising administering to a subject having or suspected of having a severe or moderate systemic autoimmune disease a composition comprising engineered T cells expressing a CAR that targets CD19, wherein the composition is produced by a manufacturing process that yields an output composition exhibiting predetermined characteristics, wherein a plurality of output compositions are produced by repetition of the manufacturing process, optionally from a human biological sample that comprises a plurality of different individual subjects, and wherein the predetermined characteristics of an output composition of the plurality of output compositions are selected from the composition characteristics disclosed in Sections II-C and III, including, in any combination, the percentage of CD3+ cells, the ratio of CD4+ / CD8+ or CD4+CAR+ / CD8+CAR+ cells, the percentage of cells expressing apoptotic markers, the percentage of less differentiated cells, and iVCN and iVCN / VCN values.

[0160] In some embodiments, the methods and uses involve administering to a subject cells expressing a genetically engineered (recombinant) cell surface receptor in adoptive cell therapy, where the receptor is generally a chimeric receptor, e.g., a chimeric antigen receptor (CAR), that recognizes CD19 associated with and / or specific to the cell type from which it was derived. The cells are generally administered in a composition formulated for administration. In some embodiments, the cells are harvested from the subject prior to treatment with the intent of engineering the cells with a CD19-directed recombinant receptor (e.g., a CAR). In some embodiments, the cells are harvested by leukapheresis. In some embodiments, the cells have been harvested by leukapheresis. In some aspects, the cells are engineered by an ex vivo method that does not involve culturing the cells for expansion (hereinafter also referred to as a non-expansion process). Exemplary non-expansion processes for engineering the provided CAR-expressing therapeutic compositions are described in Section II-C.

[0161] In some embodiments, the subject has received one or more previous therapies for treating an autoimmune disease, for example, two or more previous therapies. In some embodiments, the subject has received one previous therapy for treating a systemic autoimmune disease. In some embodiments, the subject has received two previous therapies for treating a systemic autoimmune disease. In some embodiments, the subject has received three previous therapies for treating a systemic autoimmune disease.

[0162] In some embodiments, the systemic autoimmune disease is a refractory disease. In some embodiments, the refractory disease is characterized by the absence of a response to one or more previous treatments, for example, one or more standard treatments. In some embodiments, the refractory disease is characterized by the absence of a complete response to one or more previous treatments, for example, one or more standard treatments. In some embodiments, the subject is refractory to treatment with one or more previous treatments for treating the systemic autoimmune disease. In some embodiments, the subject is refractory to treatment with two or more previous treatments for treating the systemic autoimmune disease.

[0163] In some embodiments, the systemic autoimmune disease is a severe autoimmune disease. In some embodiments, the severe autoimmune disease is an autoimmune disease in which the subject has achieved a response to standard treatment, but the response is insufficient or partial. In some embodiments, the severe autoimmune disease is an autoimmune disease in which the subject's response can only be achieved by combining standard treatments in the patient.

[0164] In some embodiments, one or more previous therapies, e.g., two or more previous therapies, are standard therapies for treating autoimmune diseases. In some embodiments, the standard therapies are anti-inflammatory drugs, steroids, e.g., corticosteroids, analgesics (e.g., paracetamol or codeine), or immunosuppressants, or a combination thereof.

[0165] In some embodiments, the subject has not previously received CAR T cell therapy before administering the CD19-directed engineered CAR T cells according to the provided methods. In some embodiments, the subject has not received genetically modified T cell therapy. In some embodiments, the subject has not received a CD19-targeted therapy. Exemplary CD19-targeted therapies include, but are not limited to, anti-CD19 monoclonal antibodies or anti-CD19 bispecific antibodies. In some embodiments, the subject does not have hypersensitivity to fludarabine and / or cyclophosphamide.

[0166] In certain embodiments, prior to administration of the dose of CD19-directed engineered CAR T cells, the subject is administered or is undergoing lymphodepleting chemotherapy. Lymphodepletion can improve CAR T cell engraftment and activity by reducing homeostatic cytokines, CD4+CD25+ regulatory T cells, increasing SDF-1 in the bone marrow microenvironment, and by stimulating antigen-presenting cells (Grossman et al., Nat Rev Immunol. 2004; 4(5):387-395; Stachel et al., Pediatr Blood Cancer 2004; 43(6):644-50; Pinthus et al., J Clin Invest 2004; 114(12):1774-81; Turk et al., J Exp Med 2004; 200(6):771-82). In addition, LD chemotherapy can further reduce the risk and severity of cytokine release syndrome (CRS).

[0167] Thus, in some embodiments, the method includes administering a preconditioning agent, such as a lymphodepleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof, to the subject prior to administration of the engineered cells. For example, the subject can be administered the preconditioning agent at least 2 days, such as at least 3, 4, 5, 6, 7, 8, or 9 days, prior to administration of the engineered cells. In some embodiments, the subject is administered the preconditioning agent at most 9 days, such as at most 8, 7, 6, 5, 4, 3, or 2 days, prior to administration of the engineered cells.

[0168] In some embodiments, the subject is preconditioned with cyclophosphamide at a dose of 20 mg to 100 mg per kg of the subject's body weight, or about 20 mg to 100 mg per kg, e.g., 40 mg / kg to 80 mg / kg, or about 40 mg / kg to 80 mg / kg. In some embodiments, the subject is preconditioned with or administered 60 mg / kg or about 60 mg / kg of cyclophosphamide. In some embodiments, cyclophosphamide can be administered in a single dose or in multiple doses, such as daily, every other day, or every third day. In some embodiments, cyclophosphamide is administered once daily for one or two days. In some embodiments, when the lymphodepleting agent includes cyclophosphamide, the subject is administered cyclophosphamide at a dose of 1 m2 or more of the subject's body surface area, including the borders. 2 100 mg to 500 mg per m or about 100 mg to 500 mg per m , e.g., 200 mg / m 2 ~400mg / m 2 or approximately 200 mg / m 2 ~400mg / m 2 , or 250 mg / m 2 ~350mg / m 2 or approximately 250 mg / m 2 ~350mg / m 2 In some instances, the subject is administered a dose of about 100 mg / m 2In some cases, the subject is administered about 150 mg / m 2 In some cases, the subject is administered about 200 mg / m 2 In some cases, the subject is administered about 250 mg / m 2 In some cases, the subject is administered about 300 mg / m 2 In some embodiments, cyclophosphamide is administered in a single dose or in multiple doses, such as given daily, every other day, or every third day. In some embodiments, cyclophosphamide is administered daily, for example, for 1-5 days, for example, for 3-5 days. In some examples, the subject is administered 100 mg of cyclophosphamide per m of the subject's body surface area prior to initiation of cell therapy. 2 In some embodiments, the subject is administered approximately 300 mg of cyclophosphamide per m every day for three days. In some embodiments, the subject is administered a total of 300 mg / m prior to the initiation of cell therapy. 2 , 400 mg / m 2 , 500 mg / m 2 , 600 mg / m 2 , 700 mg / m 2 , 800 mg / m 2 , 900 mg / m 2 , 1000 mg / m 2 , 1200 mg / m 2 , 1500 mg / m 2 , 1800 mg / m 2 , 2000 mg / m 2 , 2500 mg / m 2 , 2700 mg / m 2 , 3000 mg / m 2 , 3300 mg / m 2 , 3600 mg / m 2 , 4000 mg / m 2 or 5000 mg / m 2 or approximately 300 mg / m 2 , 400 mg / m 2 , 500 mg / m 2 , 600 mg / m 2 , 700 mg / m 2 , 800 mg / m 2 , 900 mg / m2 , 1000 mg / m 2 , 1200 mg / m 2 , 1500 mg / m 2 , 1800 mg / m 2 , 2000 mg / m 2 , 2500 mg / m 2 , 2700 mg / m 2 , 3000 mg / m 2 , 3300 mg / m 2 , 3600 mg / m 2 , 4000 mg / m 2 or 5000 mg / m 2 of cyclophosphamide, or a range defined by any of the foregoing, is administered.

[0169] In some embodiments, when the lymphodepleting agent comprises fludarabine, the subject is administered fludarabine at a dose of up to, inclusive of 1 mg / m 2 or approximately 1 mg / m 2 ~100mg / m 2 or approximately 100 mg / m 2 , e.g., 10 mg / m 2 or approximately 10 mg / m 2 ~75mg / m 2 or approximately 75 mg / m 2 , 15 mg / m 2 or approximately 15 mg / m 2 ~50mg / m 2 or approximately 50 mg / m 2 , 20 mg / m 2 or approximately 20 mg / m 2 ~40mg / m 2 or approximately 40 mg / m 2 , 24 mg / m 2 or approximately 24 mg / m 2 ~35mg / m 2 or approximately 35 mg / m 2 In some instances, the subject is administered a dose of 10 mg / m 2 or approximately 10 mg / m 2 In some instances, subjects are administered 15 mg / m 2 or approximately 15 mg / m 2In some instances, the subject is administered 20 mg / m 2 or approximately 20 mg / m 2 In some instances, subjects are administered 25 mg / m 2 or approximately 25 mg / m 2 In some instances, the subject is administered 30 mg / m 2 or approximately 30 mg / m 2 In some embodiments, fludarabine is administered in a single dose, or in multiple doses, such as given daily, every other day, or every third day. In some embodiments, fludarabine is administered daily, for example, for 1 to 5 days, for example, for 3 to 5 days. In some examples, the subject is administered 100 mg of fludarabine per m of the subject's body surface area prior to initiation of cell therapy. 2 In some embodiments, the subject is administered 30 mg or about 30 mg of fludarabine per m 2 daily for three days. In some embodiments, the subject is administered a total of 10 mg / m 2 fludarabine per m 2 daily prior to the initiation of cell therapy. 2 , 20 mg / m 2 , 25 mg / m 2 , 30 mg / m 2 , 40 mg / m 2 , 50 mg / m 2 , 60 mg / m 2 , 70 mg / m 2 , 80 mg / m 2 , 90 mg / m 2 , 100 mg / m 2 , 120 mg / m 2 , 150 mg / m 2 , 180 mg / m 2 , 200 mg / m 2 , 250 mg / m 2 , 270 mg / m 2 , 300 mg / m 2 , 330 mg / m 2 , 360 mg / m 2 , 400 mg / m 2 or 500 mg / m 2 or approximately 10 mg / m 2 , 20 mg / m 2 , 25 mg / m 2 , 30 mg / m 2 , 40 mg / m2 , 50 mg / m 2 , 60 mg / m 2 , 70 mg / m 2 , 80 mg / m 2 , 90 mg / m 2 , 100 mg / m 2 , 120 mg / m 2 , 150 mg / m 2 , 180 mg / m 2 , 200 mg / m 2 , 250 mg / m 2 , 270 mg / m 2 , 300 mg / m 2 , 330 mg / m 2 , 360 mg / m 2 , 400 mg / m 2 or 500 mg / m 2 of cyclophosphamide, or a range defined by any of the foregoing, is administered.

[0170] In some embodiments, the lymphodepleting agent comprises a single agent, such as cyclophosphamide or fludarabine. In some embodiments, the subject is administered cyclophosphamide alone, without fludarabine or another lymphodepleting agent. In some embodiments, prior to administration, the subject is administered cyclophosphamide within 1 m of the subject's body surface area. 2 200-400 mg / m or approximately 200-400 mg / m, as appropriate, 300 mg / m 2 or approximately 300 mg / m 2 In some embodiments, the subject is receiving lymphodepleting therapy, including daily administration of cyclophosphamide at 200 mg / kg / day for 2-4 days. In some embodiments, the subject is administered fludarabine alone, e.g., without cyclophosphamide or other lymphodepleting agents. In some embodiments, prior to administration, the subject is administered fludarabine at a dose of 100 mg / kg / day for 2-4 days. 2 20-40 mg / m or approximately 20-40 mg / m as appropriate, 30 mg / m 2 or approximately 30 mg / m 2 She is receiving lymphodepleting therapy, which includes daily administration of fludarabine for 2 to 4 days.

[0171] In some embodiments, the lymphodepleting agent comprises a combination of drugs, for example, a combination of cyclophosphamide and fludarabine. Thus, the combination of drugs can include cyclophosphamide at any dose or administration schedule, such as those described above, and fludarabine at any dose or administration schedule, such as those described above. For example, in some embodiments, a subject is administered cyclophosphamide at or about 60 mg / kg (about 2 g / m 2 ) of cyclophosphamide, and 3 to 5 doses of 25 mg / m 2 of fludarabine is administered prior to the first or subsequent dose. In some embodiments, the subject is administered fludarabine (30 mg / m for 3 days) prior to administration of the cells. 2 / day) and cyclophosphamide (300 mg / m for 3 days 2 / day) (flu / cy) is administered intravenously in parallel. In some embodiments, the subject is administered one or more reduced, delayed, or eliminated doses of a lymphodepleting agent.

[0172] In some embodiments, the subject is premedicated, for example, to minimize the risk of infusion reactions. In some aspects, the premedication includes administering a pain reliever and / or an antihistamine. In some embodiments, the premedication includes administering acetaminophen and / or diphenhydramine, or another H1-antihistamine. In some embodiments, 30-60 minutes or about 30-60 minutes prior to treatment with the cell therapy, the patient is administered acetaminophen (e.g., 650 mg orally) and diphenhydramine (e.g., 25-50 mg IV or orally), or another H1-antihistamine.

[0173] In embodiments of any of the methods provided, the subject is a human subject.

[0174] A. Exemplary Diseases In some embodiments, the methods provided herein are directed to the treatment of autoimmune diseases caused by, associated with, and / or specific for cells expressing CD19, such as SLE, IIM, SSc, AAV, systemic sclerosis, highly active relapsing-remitting multiple sclerosis (MS), primary progressive MS, IgA nephropathy, pemphigus vulgaris, myasthenia gravis, demyelinating polyneuropathy, autoimmune hemolytic anemia, immune thrombocytopenia, IgG4-related disease, membranous nephropathy, primary Sjogren's syndrome, cutaneous lupus erythematosus, sarcoidosis, light chain amyloidosis, and the like.

[0014] It is used to treat rheumatoid arthritis, bullous pemphigoid, acute respiratory distress syndrome, atopic eczema, hereditary angioedema, hidradenitis suppurativa, inclusion body myositis, inflammatory bowel disease, mastocytosis, multifocal motor neuropathy, necrotizing myopathy, neuromyelitis optica spectrum disorder, mixed connective tissue disorder, POEMS syndrome, primary biliary cholangitis, psoriasis, rhesus hemolytic disease, Still's disease, type 1 diabetes, urticaria, capillary leak syndrome, cytokine release syndrome, erythema multiforme, pyoderma gangrenosum, antiphospholipid antibody syndrome, or X-linked agammaglobulinemia.

[0175] In some embodiments, the methods provided herein are used to treat SLE, IIM, AAV, systemic sclerosis, highly active relapsing-remitting multiple sclerosis (MS), primary progressive MS, IgA nephropathy, pemphigus vulgaris, or myasthenia gravis. In some embodiments, the methods provided herein are used to treat SLE. In some embodiments, the methods provided herein are used to treat IIM. In some embodiments, the methods provided herein are used to treat SSc. In some embodiments, the methods provided herein are used to treat MS.

[0176] In some embodiments, the methods provided herein are used to treat rheumatoid arthritis. In some embodiments, the systemic autoimmune disease is rheumatoid arthritis.

[0177] In some embodiments, the methods provided herein are used to treat myositis. In some embodiments, the systemic autoimmune disease is myositis.

[0178] In some embodiments, the methods provided herein are used to treat myasthenia gravis. In some embodiments, the systemic autoimmune disease is myasthenia gravis.

[0179] In some embodiments, the methods provided herein are used to treat bullous pemphigoid. In some embodiments, the systemic autoimmune disease is bullous pemphigoid.

[0180] In some embodiments, the methods provided herein are used to treat immune thrombocytopenia. In some embodiments, the systemic autoimmune disease is immune thrombocytopenia.

[0181] In some embodiments, the methods provided herein are used to treat autoimmune hemolytic anemia. In some embodiments, the systemic autoimmune disease is autoimmune hemolytic anemia.

[0182] In some embodiments, the methods provided herein are used to treat pemphigus vulgaris. In some embodiments, the systemic autoimmune disease is pemphigus vulgaris.

[0183] In some embodiments, the methods provided herein are used to treat demyelinating polyradiculoneuropathy. In some embodiments, the systemic autoimmune disease is demyelinating polyradiculoneuropathy.

[0184] In some embodiments, the methods provided herein are used to treat membranous nephropathy. In some embodiments, the systemic autoimmune disease is membranous nephropathy.

[0185] 1. Systemic lupus erythematosus (SLE) Systemic lupus erythematosus (SLE) is a systemic autoimmune disease caused by abnormal immune system activity, resulting in a variety of clinical manifestations. SLE is characterized by the production of autoantibodies against nuclear and cytoplasmic antigens, can affect several different organs, and is associated with numerous distinct clinical and immunological abnormalities characterized by a relapsing and remitting clinical course. [Yu H, Nagafuchi Y, Fujio K. Clinical and Immunological Biomarkers for Systemic Lupus Erythematosus. Biomolecules. 2021 Jun 22;11(7):928.] SLE manifests with numerous clinical manifestations, including renal, cutaneous, neuropsychiatric, and cardiovascular symptoms. The complexity, heterogeneity, and variability of lupus have historically led to a focus on treating symptoms rather than the disease itself. The basis for lupus disease heterogeneity includes genetics, pathogenic mechanisms (pathways, autoantibodies), demographics, ethnicity and race, and socioeconomic factors. This raises numerous challenges, including those related to prognosis, treatment optimization, efficacy, safety, and clinical trial design. (Bazzan M, Vaccarino A, Marletto F. Systemic lupus erythematosus and thrombosis. Thromb J. 2015 Apr 23;13:16. doi: 10.1186 / s12959-015-0043-3.)

[0186] In some embodiments, the systemic autoimmune disease is SLE, for example, moderate SLE or severe refractory SLE. Some of the provided methods involve administering engineered cells or compositions containing engineered cells, such as engineered T cells, to a subject with SLE, including severe refractory SLE. Methods and uses for the engineered cells (e.g., T cells) and / or compositions of the provided CD19-directed CARs are also provided, including methods for treating a subject with SLE, including severe refractory SLE, involving the administration of engineered cells and / or compositions thereof. In certain embodiments, the subject has severe refractory SLE. In some embodiments, the subject is selected for or identified as having severe refractory SLE, such as by the presence of certain characteristics or clinical signs indicating the presence of severe refractory SLE. Exemplary selection criteria are further described herein. In some embodiments, the methods and uses of the provided CD19-directed CAR-engineered cells (e.g., T cells) and / or compositions thereof include methods for treating a subject with severe SLE who has failed at least two or more previous treatments. In certain embodiments, the method includes administering to a subject a dose of T cells, including CD4+ and CD8+ T cells, wherein the T cells comprise a chimeric antigen receptor (CAR) that specifically binds to CD19.

[0187] Clinical features at the onset and during the progression of severe SLE include, but are not limited to, malar rash, arthritis, nephropathy, photosensitivity, thrombosis, sicca syndrome, serositis, nephropathy, neurologic complications, oral ulcers, thrombocytopenia, lymphadenopathy, discoid lesions, livedo reticularis, thrombosis, myositis, hemolytic anemia, pulmonary complications, skin lesions, and chorea. [Cervera R, et al. Systemic lupus erythematosus: clinical and immunologic patterns of disease expression in a cohort of 1,000 patients. The European Working Party on Systemic Lupus Erythematosus. Medicine (Baltimore). 1993 Mar;72(2):113-24. PMID: 8479324]. These disease manifestations result in a significant burden of illness, potentially leading to decreased physical function, unemployment, lower health-related quality of life (QoL), and a 10-year reduction in life expectancy. Increased hospitalizations and side effects of medications, including long-term oral corticosteroids (OCS or glucocorticoids and other immunosuppressive treatments), increase the disease burden in SLE.

[0188] In some cases, subjects develop lupus nephritis. Lupus nephritis (LN): Up to 60% of SLE patients develop LN, one of numerous proteinuric kidney diseases caused by renal inflammation caused by systemic lupus erythematosus (SLE). LN is a debilitating and costly disease that often leads to kidney failure, requiring dialysis or kidney transplantation and often resulting in death. In fact, patients with kidney failure have a more than 60-fold increased risk of premature death compared with typical SLE patients. The clinical sign of LN is leakage of blood protein into the urine, and the disease can be diagnosed by a number of factors, including the urinary protein / creatinine ratio (UPCR), with a UPCR greater than 0.5 mg / mg indicating an active condition. Additionally, certain blood markers can also be helpful in diagnosis, such as complement 3 (C3), complement 4 (C4), and anti-dsDNA antibodies.

[0189] Treatment of SLE is challenging due to the limited efficacy and poor tolerability of standard therapies. Because all of the currently used therapies for the treatment of SLE have well-known adverse effect profiles, there is a medical need to identify new targeted therapies, particularly agents that can reduce the need for corticosteroids and nonspecific cytotoxic agents.

[0190] Nearly 50 years after hydroxychloroquine was approved for use in discoid lupus and SLE, only one new treatment for SLE (belimumab) has been approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA). However, belimumab is not approved everywhere, and its adoption has been modest. Many drugs currently used to treat SLE, such as azathioprine, cyclophosphamide, and mycophenolate mofetil (MMF) / mycophenolic acid, are not approved for the disease. Furthermore, all of these drugs have well-documented safety issues and are not effective for all manifestations of lupus in all patients. Antimalarials (e.g., hydroxychloroquine) and corticosteroids can be used to control joint pain, arthritis, and rashes. Other treatments include nonsteroidal anti-inflammatory drugs (NSAIDs); analgesics for fever, joint pain, and arthritis; and topical sunscreens to minimize photosensitivity. For subjects with moderate or severe disease, tapering and completely weaning off OCS is often difficult, as it can cause long-term morbidity and contribute to premature cardiovascular mortality. Even low daily doses of 5-10 mg prednisone used chronically increase the risk of side effects such as cataracts, osteoporosis, and coronary artery disease.

[0191] Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used in patients with SLE for the symptomatic management of joint pain, mild arthritis, myalgia, serositis, and fever. NDAIDs do not have any immunosuppressive properties. NSAIDs can only be used short-term and are not suitable for patients with renal complications, hypertension, and pre-existing heart disease. NSAIDs can cause fluid retention, decreased kidney function, and interstitial nephritis.

[0192] Mycophenolate mofetil (MMF) is a specific inhibitor of inosine monophosphate dehydrogenase. MMF impairs de novo purine synthesis, an essential pathway in activated lymphocytes. Therefore, MMF inhibits both T and B lymphocyte proliferation and reduces antibody synthesis.

[0193] In some cases, rituximab is used to treat SLE, especially lupus nephritis.Rituximab is a chimeric anti-CD20 monoclonal antibody.Rituximab is an effective treatment for many autoimmune diseases, including rheumatoid arthritis and ANCA vasculitis.A small number of uncontrolled studies on lupus nephritis have shown that rituximab can potentially be effective in patients with lupus nephritis.

[0194] In some embodiments, inhibitors of type I interferon (IFN) are used to treat SLE. Type I interferon (IFN) is a cytokine that forms a crucial link between innate and adaptive immunity, and its involvement in SLE has been suggested by genetic susceptibility data and upregulation of interferon-induced gene expression in the majority of SLE patients. Sifalimumab is an anti-interferon-α monoclonal antibody. Sifalimumab has been shown to be effective and safe in some subjects, but the effects of treatment are often modest. Anifrolumab (MEDI-546): A monoclonal antibody that binds to IFNAR. Anifrolumab reduces disease activity compared to placebo in patients with moderate to severe SLE, but its efficacy has not met all primary endpoints.

[0195] Many subjects with SLE, including those with severe SLE, show an inadequate response or are refractory to existing treatments, such as treatments using any two or more of MMF, CYC, belimumab, rituximab, anifrolumab, azathioprine, mTX, csp, and voclosporin. Patients with severe, refractory SLE are often young adults who face lifelong treatments, frequent relapses, and cumulative organ dysfunction over time. Despite advances in SLE treatment, a significant proportion of patients with severe SLE do not respond and / or relapse, placing them at high risk of organ failure or death. There is a significant unmet need for SLE treatments with better efficacy and safety profiles than currently available treatments, particularly in subjects with severe, refractory SLE.

[0196] In some embodiments, a diagnosis of SLE can be made based on the criteria defined by the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) [Aringer et al. (2019) Arthritis Rheumatol. 71:1400-1412]. These criteria are based on the presence of a positive antinuclear antibody test and clinical features including discoid rash, oral ulcers, arthritis, serositis, renal damage, neuropathy, hematological disorders, and immune disorders. A mammal (e.g., a human) can be clinically classified as having SLE if he or she obtains a score of at least 10 points derived from weighted criteria. In some embodiments, a diagnosis of SLE can be made based on the presence of detectable SLE-associated antibodies in the subject's blood. In some embodiments, these antibodies include anti-dsDNA, anti-histone, anti-chromatin, and / or anti-Sm antibodies. In some embodiments, the subject has severe SLE characterized by at least one organ system categorized as BILAG A or at least two organ systems categorized as BILAG B. The term "BILAG" refers to the British Isles Lupus Assessment Group (BILAG) 2004, a disease index designed for patients with SLE based on intent-to-treat analysis (Isenberg et al., 2005). The term "organ system" used in connection with BILAG refers to the nine systems considered in the BILAG 2004 index: systemic, mucocutaneous, central nervous system, musculoskeletal, cardiovascular / respiratory, abdominal, renal, and hematological systems. The BILAG 2004 assessment consists of 101 questions (plus five additional items primarily required for calculating glomerular filtration rate). Each question is answered as follows: 0 = absent; 1 = improving; 2 = the same; 3 = worsening; and 4 = new. The index records disease activity over the past four weeks compared to the four weeks before. Based on the scoring for each of these questions, a predefined algorithm specific to each line results in a disease activity score for each line ranging from A to E: A=12, which is defined as severe disease requiring medium / large doses of corticosteroids (>20 mg prednisolone or equivalent), and / or initiation or increase of immunosuppressive medications, or high dose anticoagulation (INR>3) (Yee et al., Rheumatology, 2010). In some embodiments, Grade A represents very active disease requiring immunosuppressive medications and / or prednisone doses >20 mg / day or equivalent; B=8, which is defined as disease activity requiring moderate doses of immunosuppressants, e.g., <20 mg prednisolone, and / or certain medications, e.g., antimalarials, antiepileptics, antidepressants, and NSAIDs, or topical steroids. In some embodiments, Grade B represents moderate disease activity requiring lower doses of corticosteroids, topical steroids, topical immunosuppressants, antimalarials, or NSAIDs; C=1, which is defined as mild persistent disease activity requiring only symptomatic treatment, e.g., painkillers or NSAIDs. In some embodiments, grade C indicates mild stable disease; D=0, which is defined as the organ or system was once active but is no longer. In some embodiments, grade D indicates that there is no disease activity, but the system has been previously affected; E=0, which is defined as the organ or system has never been active. In some embodiments, a grade of E indicates no current or past disease activity.

[0197] In some embodiments, the subject has OCS-related organ damage. The OCS can include prednisone, prednisolone, and / or methylprednisolone. In some embodiments, the subject can be selected for SLE that does not respond to OCS treatment.

[0198] In some embodiments, the subject has a SLEDAI disease activity score of ≧10, which is an indicator of disease severity in SLE.

[0199] In some embodiments, severe disease is based on the presence of major organ involvement (renal, neurological, cardiovascular, or respiratory involvement) and the need for treatment with >7.5 mg / day of corticosteroids or immunosuppressants.

[0200] In some embodiments, the subject has previously received treatment with a glucocorticoid, an antimalarial agent, an immunosuppressant, an anti-CD20 antibody, an IFN inhibitor, or a soluble B-lymphocyte stimulatory factor (BLyS) inhibitor. In some embodiments, the immunosuppressant is azathioprine, cyclosporine (csp), cyclophosphamide (cyc), mizoribine, mycophenolate mofetil (MFF), mycophenolic acid, and / or methotrexate (mtx). In some embodiments, the glucocorticoid is an oral glucocorticoid, such as prednisone, prednisolone, and / or methylprednisolone. In some embodiments, the antimalarial agent is hydroxychloroquine. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, the IFN inhibitor is anifrolumab. In some embodiments, the BLyS inhibitor is belimumab.

[0201] In some embodiments, the subject is refractory to treatment with two or more previous treatments.In some embodiments, the two or more previous treatments (for example, two, three, four, five or more previous treatments) are selected from any two or more of the following: mycophenolic acid or its derivatives, cyclophosphamide (CYC), belimumab, rituximab, anifrolumab, azathioprine, methotrexate (mtx), cisplatin (CSP), obinutuzumab, cyclosporine, tacrolimus and / or voclosporin.In some embodiments, for the number of unsuccessful treatments, methotrexate and azathioprine are counted as 1. In some embodiments, the two or more previous treatments (e.g., two, three, four, five, or more previous treatments) are selected from any two or more of the following: mycophenolate mofetil (MFF), cyclophosphamide (cyc), belimumab, rituximab, anifrolumab, azathioprine, methotrexate, cyclosporine (csp), or voclosporin. In some embodiments, the subject has received two or more previous treatments (e.g., two, three, four, five, or more previous treatments) for lupus and has resulted in an inadequate response (e.g., as measured by SLE disease activity). In some embodiments, the subject has an inadequate response to two previous treatments. In some embodiments, the subject has an inadequate response to three previous treatments. In some embodiments, the subject has an inadequate response to four or more previous treatments. In some embodiments, the subject has failed to achieve clinical remission (e.g., three months after a given treatment) after being treated with any two or more previous treatments for lupus. In any embodiment, the subject is identified as having an inadequate response to the previous treatment prior to leukapheresis in connection with the manipulation of CD19-directed CAR T cell composition, or is selected if the subject has an inadequate response.An inadequate response to treatment is defined as a lack of response to an appropriate dose, an inadequate response, or a lack of sustained response.Intolerance is not considered an inadequate response.

[0202] In some embodiments, the subject does not have drug-induced SLE. In some embodiments, the subject does not have additional systemic autoimmune diseases, including but not limited to multiple sclerosis, psoriasis, and / or inflammatory bowel disease. In some embodiments, the subject does not have SLE overlap syndromes, including but not limited to rheumatoid arthritis, scleroderma, and / or mixed connective tissue disease. In some embodiments, the subject does not have clinically significant CNS pathology.

[0203] a. Response and Efficacy In some embodiments, the provided methods and uses involving administration of anti-CD19 CAR T cell therapy reduce SLE disease activity in a subject.

[0204] In some embodiments, the treatment is effective in reducing lupus disease activity. In some embodiments, lupus disease activity is measured by a disease activity score selected from the group consisting of British Isles Lupus Assessment Group 2004 (BILAG), SLE Disease Activity Index (SLEDAI-2K), SLEDAI-2K Responder Index 50 (SRI-50), composite SLE Responder Index (cSRI), minimal clinically important difference (MCID), patient-reported short form quality of life (SF-36), physical summary score (PCS) and / or mental summary score (MCS), and lupus-specific quality of life form (lupus-QOL), or a combination thereof.

[0205] In some embodiments, reducing SLE disease activity in a subject can include one or more of the following: a BILAG-based Composite Lupus Assessment (BICLA) response in the subject; a reduction in the subject's Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI) score compared to the subject's CLASI score before treatment; a reduction in the subject's tender and swollen joint count compared to the subject's tender and swollen joint count before treatment; the subject having a BILAG-2004 B score of up to 1 after treatment; the subject having a BILAG-2004 score of C or higher after treatment; the subject having an improvement in at least one patient-reported outcome (PRO) compared to before treatment; or a reduction in the subject's SLE flare rate compared to the subject's flare rate before treatment.

[0206] In some embodiments, a subject's BILAG score can be measured before and after administration of a CD19-targeted cell therapy. In some embodiments, patient-reported outcomes (PROs) are measured in a subject before and after administration of a CD19-targeted cell therapy. PROs can include the subject's Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F), Short Form 36 Health Survey version 2 (SF-36-v2), Mental Summary Score (MCS), and / or Physical Summary Score (PCS).

[0207] In some embodiments, as a result of treatment, a subject who had at least one organ system categorized as BILAG A or at least two organ systems categorized as BILAG B at baseline experiences a decrease in the score in any one organ system categorized as BILAG A or in at least two organ systems categorized as BILAG B by 1, without any other organ system progressing to BILAG A or B.

[0208] In some embodiments, reducing SLE disease activity in a subject comprises a BILAG-Based Composite Lupus Assessment (BICLA) response. In some embodiments, reducing SLE disease activity in a subject comprises a BICLA response by at least 4 weeks of treatment. In some embodiments, reducing SLE disease activity comprises a BICLA response by at least 8 weeks of treatment. In some embodiments, the BICLA response may be sustained in a subject for at least 52 weeks. In some embodiments, the BICLA response comprises a reduction in the subject's BILAG-2004 A and B domain scores to B / C / D and C / D, respectively.

[0209] In some embodiments, as a result of treatment, a subject who had at least one organ system categorized as BILAG A or at least two organ systems categorized as BILAG B at baseline has all organ systems categorized as either BILAG C or BILAG D / E after treatment.

[0210] In some embodiments, treatment results in a minimal clinically meaningful difference (MCID) of 1 for SRI-50.

[0211] In some embodiments, as a result of treatment, subjects who had at least one organ system categorized as BILAG A or at least two organ systems categorized as BILAG B at baseline have all organ systems categorized as either BILAG C or BILAG D / E after treatment, and no progression as measured by the SLEDAI-2K after treatment. In some embodiments, the SLE Disease Activity Index "SLEDAI-2K" (also referred to as "SLEDAI") is a validated tool developed as a comprehensive assessment of disease activity in SLE patients (Gladman et al., 2002). It represents the consensus of a group of experts in the field of lupus research. The SLEDAI-2K evaluates 24 descriptors (16 clinical signs and 8 laboratory values) across 9 organ systems. Descriptors are weighted differently based on their clinical significance using a dichotomous score (present / absent within the past 30 days). Descriptors must be attributable to active SLE or should not be scored. The SLEDAI-2K is intended to assess current lupus activity, not chronic disability. In some embodiments, exacerbation in the context of the SLEDAI-2K refers to worsening disease activity as measured by the SLEDAI-2K.

[0212] In some embodiments, disease activity is monitored by SRI-50. The "SRI-50" is an SLE disease activity index that includes the same 24 descriptors covering nine organ systems and generates a total score over the past 30 days, similar to the SLEDAI-2K, to indicate disease activity (Touma et al., 2012). Each SRI-50 descriptor has a definition for identifying a 50% or greater improvement and generates a corresponding descriptor score. Overall, the SRI-50 is an index developed to reflect partial and significant improvements in disease activity between visits.

[0213] In some embodiments, treatment results in an SRI (Systemic Lupus Erythematosus Responder Index) of >4, or SRI(4). A subject achieves SRI(4) if they meet all of the following criteria: a >4-point reduction in SLEDAI-2K from baseline; no new organ systems affected using BILAG-2004 as defined by one or more BILAG-2004 A or two or more BILAG-2004 B items compared to baseline; no worsening of the subject's lupus disease activity from baseline as defined by an increase of >0.30 points on a 3-point PGA VAS.

[0214] In some embodiments, SRI(X) (X=5, 6, 7, or 8) is defined by the proportion of subjects who meet the following criteria: a decrease from baseline of >X points in SLEDAI-2K; no new organ systems affected compared to baseline using BILAG-2004 as defined by one or more BILAG-2004 A or two or more BILAG-2004 B items; no worsening of the subject's lupus disease activity from baseline as defined by an increase of >0.30 points on a 3-point PGA VAS.

[0215] In some embodiments, disease activity is monitored by the "composite SLE responder index" (cSRI), an SLE disease index that incorporates two different systems, BILAG and SLEDAI-2K, and is defined as substantial response as measured by BILAG 2004 and no progression as measured by SLEDAI-2K.

[0216] In some embodiments, as a result of treatment, the subject has an improvement of 4 points or more on the SELENA-SLEDAI, the subject has no new organ systems categorized as BILAG A or more than one organ system categorized as BILAG B, and the subject has no increase of 0.3 points or more on the Physician's Global Assessment.

[0217] In some embodiments, disease activity is monitored using the Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI). CLASI is a tool used to measure disease severity and response to treatment. A 4-point or 20% reduction in the CLASI activity score is generally considered the cutoff for classifying a subject as a responder to treatment. In certain embodiments, treatment using a provided CD19-targeted cell therapy results in a reduction of the subject's CLASI score by at least 50% compared to the subject's baseline score. In some embodiments, CLASI is a validated index used to evaluate skin lesions in SLE, and is composed of two separate scores: the first score summarizes the inflammatory activity of the disease, and the second score is a measure of the damage caused by the disease. The activity score takes into account erythema, scaling / thickening, mucosal lesions, recent hair loss, and non-scarring alopecia. The damage score represents pigmentation abnormalities, scarring / atrophy / panniculitis, and scalp scarring. Subjects are asked if their dyspigmentation has persisted for more than 12 months; if so, the dyspigmentation score is doubled. Each of the above parameters is measured at 13 different anatomical locations, which are included because they are most commonly associated with cutaneous lupus erythematosus (CLE). The most severe lesion in each area is measured.

[0218] In some embodiments, as a result of treatment, the subject achieves a lupus low disease activity status (LLDAS). The LLDAS is a comparable, validated goal for SLE used to measure low disease activity. The LLDAS is defined by (1) no activity in major organ systems, SLE Disease Activity Index (SLEDAI)-2K ≤ 4, (2) no new lupus disease activity, (3) a SELENA-SLEDAI Physician Global Assessment (scale 0-3) ≤ 1, (4) a current daily dose of prednisolone (or equivalent) ≤ 7.5 mg, and (5) well-tolerated standard maintenance doses of immunosuppressants and approved biologics (Franklyn et al., 2015).

[0219] In some embodiments, as a result of treatment, subjects experience significant changes in SF-36 PCS and / or MCS compared to baseline. In some embodiments, the "Patient-Reported Short Form Quality of Life Assessment" (SF-36) is a widely validated general patient questionnaire that has been shown to be sensitive to changes in a variety of diseases, including hypertension and cardiovascular disease, diabetes, pulmonary disease, low back pain, rheumatoid arthritis (RA), and osteoarthritis [Ware JE, et al. (1992) Medical Care 30:473-483]. The SF-36 consists of 36 questions representing eight important health concepts, each of which is scored on a separate "domain" scale: physical functioning, role-physical, bodily pain, overall health, vitality, social functioning, role-emotional, and mental health (Ware et al. Medical Care, 1992). These eight scales are combined into two summary measures: physical (PCS) and mental (MCS) summary scores.

[0220] In some embodiments, as a result of treatment, subjects experience a significant change in the Health Assessment Questionnaire - Disability Index (HAQ-DI) compared to baseline. In some embodiments, the patient-reported quality of life assessment is a widely validated, general patient questionnaire that measures difficulty performing activities of daily living. Questions are rated on a scale of 0 to 3, with 0 indicating "no difficulty" and 3 indicating "unable to perform" (Allanore et al., 2020).

[0221] In some embodiments, by reducing the SLE disease activity of the subject, the number of tender and swollen joints is improved by at least 50% compared with the number of tender and swollen joints of the patient before treatment.In some embodiments, the number of swollen and tender joints is based on the left and right shoulder, elbow, wrist, metacarpophalangeal joint (MCP)1, MCP2, MCP3, MCP4, MCP5, distal interphalangeal (PIP)1, PIP2, PIP3, PIP4, PIP5 joints of the upper limbs, and the left and right knee of the lower limbs.For joint number assessment, active joints are defined as tender and swollen joints.

[0222] In some embodiments, reducing SLE disease activity in a subject includes preventing a flare in the subject. In some embodiments, a flare may be defined as a new BILAG-2004 A domain score of ≧1 or a new (worsening) BILAG-2004 B domain score of ≧2 compared to the subject's score one month ago.

[0223] In some embodiments, as a result of treatment, the subject experiences an increase in the time to first confirmed severe SLE flare or first confirmed major SLE flare.

[0224] In some embodiments, treatment results in an increase in the time to first confirmed severe SLE flare, where a severe SLE flare includes a subject with any new organ system categorized as BILAG A or any two new organ systems categorized as BILAG B.

[0225] In some embodiments, treatment results in an increase in the time to first confirmed major SLE flare, as defined by the Fortin definition of a major flare, including initiation or increase in immunosuppressant medication or high-dose corticosteroid therapy, hospitalization attributable to SLE, or death.

[0226] In some embodiments, the method of treatment involves reducing the oral corticosteroid (OCS) dose administered to the subject compared to the OCS dose administered to the subject before treatment. In some embodiments, reducing SLE disease activity in the subject is characterized by a reduction in the flare rate in the subject compared to the flare rate before treatment, and the method involves reducing the OCS dose administered to the subject compared to the OCS dose administered to the subject before treatment. In some embodiments, the OCS comprises prednisone, prednisolone, and / or methylprednisolone.

[0227] In some embodiments, as a result of treatment, the subject experiences a significant change in cumulative disability index as measured by the Systemic Lupus International Collaborating Clinics / American College of Rheumatology Disability Index (SLICC / ACR DI). In some embodiments, the Systemic Lupus Erythematosus International Collaborating Clinics / American College of Rheumatology (SLICC / ACR) is an index for cumulative organ damage (Dayal et al., Lupus 2002). SLE disability is defined as irreversible changes in an organ or system that have been present for at least 6 months.

[0228] In some embodiments, as a result of treatment, the subject experiences a significant change in daily dose of glucocorticoid.

[0229] In some embodiments, as a result of treatment, the subject has a significant improvement in lupus quality of life.

[0230] In some embodiments, as a result of treatment, the subject has a significant improvement in the global assessment of disease activity based on the minimal clinically meaningful difference (MCID). In some embodiments, MCID refers to a score obtained from the patient that indicates a change in clinical intervention that is meaningful to the patient.

[0231] In some embodiments, the method reduces SLE disease activity in the subject as characterized by reducing anti-dsDNA levels in the subject.

[0232] In some embodiments, subjects treated according to the provided methods are evaluated or monitored for a period of time after treatment to determine whether a complete or partial remission has occurred, hi some embodiments, subjects are evaluated or monitored to assess whether the remission achieved according to the measurements is maintained.

[0233] In some embodiments, remission is monitored using the Definition of Remission in Systemic Lupus Erythematosus (DORIS) (Modified: 2021 DORIS definition of remission in SLE: final recommendations from an international task force. Lupus Science & Medicine 2022;9:e000538corr1. doi: 10.1136 / lupus-2021-000538corr1). In some embodiments, remission is defined as a score of 0 on the Systemic Lupus Disease Activity Index (SLEDAI) and a rater's global assessment score of <0.5 (0-3). Subjects may be on stable antimalarials, immunosuppressants, biologics, and / or low-dose glucocorticoids (5 mg / day or less of prednisolone).

[0234] In some embodiments, the subject has lupus nephritis. In some embodiments, efficacy assessment can be based on urinary protein / creatinine ratio (UPCR), where a ratio of ≦0.5 mg / mg indicates a complete response; alternatively, or in addition, an eGFR of ≧60 mL / min / 1.73 m2, or no decline from baseline, and an eGFR of ≧20% are indicated. Other indicators of complete response include not requiring rescue medication, such as intravenous steroids or cyclophosphamide, or requiring ≦10 mg of prednisone for more than three consecutive days or a total of more than seven days. In some embodiments, complete remission (CR) is defined as a protein / creatinine ratio of ≦0.5 mg / mg and an eGFR of ≧60 mL / min / 1.73 m2, or no decline from baseline of ≧20%. Partial remission is defined as a 50% decrease in UPCR from baseline.

[0235] In some embodiments, treatment with the provided methods results in clinical remission of SLE in a subject, and the remission is maintained for more than 3 months. In some embodiments, treatment with the provided methods results in clinical remission of SLE in a subject, and the remission is maintained for more than 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, 3 years, 4 years, 5 years, or more. In some embodiments, treatment with the provided methods results in clinical remission of SLE in a subject, and the remission is maintained for more than 6 months. In some embodiments, treatment with the provided methods results in clinical remission of SLE in a subject, and the remission is maintained for more than 12 months. In some embodiments, treatment with the provided methods results in clinical remission of SLE in a subject, and the remission is maintained for more than 24 months. In some embodiments, treatment with the provided methods results in clinical remission of SLE in a subject, and the remission is maintained for more than 3 years. In some embodiments, treatment with the provided methods results in clinical remission of SLE in a subject, which is maintained in remission for greater than 4 years. In some embodiments, treatment with the provided methods results in clinical remission of SLE in a subject, which is maintained in remission for greater than 5 years.

[0236] In some embodiments, treatment with the provided methods results in long-term remission, defined as five consecutive years of no disease activity (SLE Disease Activity Index, SLEDAI=0) and no treatment (corticosteroids, antimalarials, or immunosuppressants).

[0237] In some cases, the pharmacokinetics of administered cells, such as adoptively transferred cells, are determined to assess the availability, e.g., bioavailability, of the administered cells. A method for determining the pharmacokinetics of adoptively transferred cells can include collecting peripheral blood from a subject to whom the engineered cells have been administered and determining the number or ratio of engineered cells in the peripheral blood. Approaches for selecting and / or isolating cells can include the use of chimeric antigen receptor (CAR)-specific antibodies (e.g., Brentjens et al., Sci. Transl. Med. 2013 Mar; 5(177): 177ra38), Protein L (Zheng et al., J. Transl. Med. 2012 Feb; 10:29), epitope tags, such as Strep-Tag sequences introduced directly into specific sites within the CAR, where binding reagents for Strep-Tag are used to directly assess the CAR (Liu et al. (2016) Nature Biotechnology, 34:430; International Patent Application Publication No. WO2015095895), and monoclonal antibodies that specifically bind to the CAR polypeptide (see International Patent Application Publication No. WO2014190273). Exogenous marker genes can be utilized in conjunction with therapies using engineered cells, in some cases to enable cell detection or selection, and in some cases to promote cell suicide. In some cases, a truncated epidermal growth factor receptor (EGFRt) can be co-expressed with a transgene of interest (CAR) in transduced cells (see, for example, U.S. Patent No. 8,802,374). EGFRt can contain an epitope recognized by the antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibodies or binding molecules, and can be used to identify or select cells engineered with an EGFRt construct and another recombinant receptor, such as a chimeric antigen receptor (CAR), and / or to eliminate or separate cells expressing the receptor. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. 2016 April; 34(4): 430-434.

[0238] In some embodiments, CARs in a biological sample obtained from a patient, e.g., blood + The number of T cells can be determined after a period of time following administration of the cell therapy, for example, to determine the pharmacokinetics of the cells. In some embodiments, detectable CAR T cells are detected in the blood of a subject or in the majority of subjects so treated by the method. + T cells, as appropriate, CAR + CD8 + T cells and / or CAR + CD4 + The number of T cells is greater than 1 cell per μL, greater than 5 cells per μL, or greater than 10 cells per μL.

[0239] 2. Idiopathic inflammatory myopathy (IIM) Idiopathic inflammatory myopathies (IIM) are a group of chronic autoimmune conditions that primarily affect proximal muscles. IIM includes dermatomyositis, polymyositis, and other disorders, such as immune-mediated necrotizing myopathy (IMNM), and many patients have antisynthetase syndrome (aSS). aSS is characterized by autoantibodies against aminoacyl-transferase (ATR) synthetases, which overlap with interstitial lung disease (ILD), myositis, and other conditions. IIM symptoms include skin lesions, muscle fatigue, and weakness, which significantly reduce patients' quality of life and put them at risk for various serious long-term complications. For example, 10–25% of IIM cases also have ILD, and 5% of cases are acute. 15–25% of patients with IIM have or will develop malignant lesions. One-third of patients with IIM will develop myocarditis, increasing their risk of congestive heart failure. The 10-year survival rate for IIM is 70% across various indications. Recently, only two medications have been approved for IIM, including IVIg and Acthar Gel, and no medications have been approved for aSS. There is strong evidence that B cells are involved, with IVIg approved for dermatomyositis and rituximab used as a treatment-withdrawal agent. There is evidence that B cells contribute to the pathogenesis, including complete resolution of aSS after anti-CD19 CAR-T cell therapy in a patient who was refractory to steroids, rituximab, tacrolimus, and cyclophosphamide.

[0240] In some embodiments, the systemic autoimmune disease is an idiopathic inflammatory myopathy (IIM), such as dermatomyositis, polymyositis, and / or immune-mediated necrotizing myopathy. In some embodiments, the patient has antisynthetase syndrome (aSS). Some provided methods involve administering engineered cells or compositions containing engineered cells, such as engineered T cells, to a subject with IIM, including dermatomyositis, polymyositis, and / or immune-mediated necrotizing myopathy. Methods and uses of the engineered cells (e.g., T cells) and / or compositions of the provided CD19-directed CARs are also provided, including methods for treating a subject with IIM, including dermatomyositis, polymyositis, and / or immune-mediated necrotizing myopathy, involving the administration of the engineered cells and / or compositions thereof. In certain embodiments, the subject has dermatomyositis, polymyositis, and / or immune-mediated necrotizing myopathy. In some embodiments, a subject is selected for or identified as having dermatomyositis, polymyositis, and / or immune-mediated necrotizing myopathy, such as by the presence of certain characteristics or clinical signs indicative of the presence of dermatomyositis, polymyositis, and / or immune-mediated necrotizing myopathy. In some embodiments, the provided methods and uses of CD19-directed CAR-engineered cells (e.g., T cells) and / or compositions thereof include methods for treating a subject with dermatomyositis, polymyositis, and / or immune-mediated necrotizing myopathy who has failed at least two or more previous treatments. In certain embodiments, the method includes administering to the subject a dose of T cells, including CD4+ and CD8+ T cells, wherein the T cells comprise a chimeric antigen receptor (CAR) that specifically binds to CD19.

[0241] In any of the embodiments herein, at or shortly before the time of administration of the composition comprising the engineered T cells, the subject has experienced a relapse after treatment with one or more prior therapies for IIM or has become refractory to the treatment.

[0242] In any of the embodiments herein, at or shortly before administration of the composition comprising the engineered T cells, the subject has experienced a relapse after remission following treatment with one or more prior therapies for the IIM or has become refractory to the treatment. In any of the embodiments herein, at or shortly before administration of the composition comprising the engineered T cells, the subject has experienced a relapse after treatment with one or more prior therapies for the IIM or has become refractory to the treatment. In any of the embodiments herein, the one or more prior therapies for the IIM do not include another dose of cells expressing a CAR.

[0243] In any of the embodiments herein, the one or more previous treatments for IIM may include corticosteroids, Octagam (IVIg), Acthar, or rituximab. In any of the embodiments herein, the one or more previous treatments for IIM include corticosteroids, Octagam, Acthar, or rituximab. In any of the embodiments herein, CD19-directed CAR engineered cells (e.g., T cells) and / or compositions thereof are used to treat patients with IIM that are refractory to previous treatments.

[0244] a. Response and Efficacy In some embodiments, the provided methods and uses involving administration of anti-CD19 CAR T cell therapy reduce IIM disease activity in a subject.

[0245] In some embodiments, the treatment is effective in reducing IIM disease activity, as measured by a disease activity score selected from the International Myositis Assessment and Clinical Studies Group (IMACS), the minimal clinically important difference (MCID), the Patient-Reported Short Form Quality of Life Assessment (SF-36) Physical Summary Score (PCS) and / or Mental Summary Score (MCS), or a combination thereof.

[0246] In some embodiments, reducing IIM disease activity in a subject may include one or more of the following: reducing the subject's IMACS score after treatment compared to the subject's IMACS score before treatment, alleviating the subject's skin lesions, muscle fatigue, and / or weakness compared to the subject's skin lesions, muscle fatigue, and / or weakness before treatment, or the subject having an improvement in at least one patient-reported outcome (PRO) compared to before treatment.

[0247] In some embodiments, a subject's IMACS score can be measured before and after administration of a CD19-targeted cell therapy. In some embodiments, patient-reported outcomes (PROs) are measured in a subject before and after administration of a CD19-targeted cell therapy. PROs can include the subject's Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F), 36-item Short Form Health Survey, version 2 (SF-36-v2), Mental Summary Score (MCS), and / or Physical Summary Score (PCS) scores.

[0248] In some embodiments, as a result of treatment, subjects experience a significant change in the Health Assessment Questionnaire-Disability Index (HAQ-DI) compared to baseline. In some embodiments, the patient-reported quality of life assessment is a widely validated, general patient questionnaire that measures difficulty performing activities of daily living. Questions are rated on a scale of 0 to 3, with 0 indicating "no difficulty" and 3 indicating "unable to perform" (Allanore et al., 2020).

[0249] In some embodiments, as a result of treatment, the subject has a significant improvement in the global assessment of disease activity based on the minimal clinically meaningful difference (MCID). In some embodiments, MCID refers to a score obtained from the patient that indicates a change in clinical intervention that is meaningful to the patient.

[0250] In some embodiments, treatment with the provided methods results in clinical remission of IIM in a subject, and the remission is maintained for more than 3 months. In some embodiments, treatment with the provided methods results in clinical remission of IIM in a subject, and the remission is maintained for more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24 months, 3, 4, 5 years, or more. In some embodiments, treatment with the provided methods results in clinical remission of IIM in a subject, and the remission is maintained for more than 6 months. In some embodiments, treatment with the provided methods results in clinical remission of IIM in a subject, and the remission is maintained for more than 12 months. In some embodiments, treatment with the provided methods results in clinical remission of IIM in a subject, and the remission is maintained for more than 24 months. In some embodiments, treatment with the provided methods results in clinical remission of IIM in a subject, and the remission is maintained for more than 3 years. In some embodiments, treatment with the provided methods results in clinical remission of IIM in the subject, and the remission is maintained for greater than 4 years. In some embodiments, treatment with the provided methods results in clinical remission of IIM in the subject, and the remission is maintained for greater than 5 years.

[0251] In some embodiments, treatment with the provided methods results in long-term remission, which is defined as five consecutive years of no disease activity and no treatment (corticosteroids, IVIg, rituximab, or immunosuppressants).

[0252] In some embodiments, treatment results in reduced muscle weakness or a reduction in the progression of muscle weakness. In some embodiments, treatment results in improved muscle strength. In some embodiments, treatment results in reduced muscle weakness or a reduction in the progression of muscle weakness in the upper limbs. In some embodiments, treatment results in reduced muscle weakness or a reduction in the progression of muscle weakness in the lower limbs. In some embodiments, treatment results in reduced muscle weakness or a reduction in the progression of muscle weakness in the neck flexors. In some embodiments, treatment results in reduced muscle weakness or a reduction in the progression of muscle weakness in proximal muscles.

[0253] In some embodiments, treatment results in a reduction in skin lesions. In some embodiments, treatment results in a reduction in heliotrope rash symptoms. In some embodiments, treatment results in a reduction in Gottron papules. In some embodiments, treatment results in a reduction in Gottron signs.

[0254] In some embodiments, treatment results in a reduction in dysphagia or esophageal motility disorders. In some embodiments, treatment results in an improvement in swallowing or esophageal motility function.

[0255] In some embodiments, treatment reduces the presence of anti-Jo-a (anti-histidyl-tRNA synthetase) autoantibodies. In some embodiments, treatment leads to the absence of detectable anti-Jo-a (anti-histidyl-tRNA synthetase) autoantibodies. In some embodiments, treatment leads to a decrease in serum levels of creatine kinase, lactate dehydrogenase, aspartate aminotransferase, and / or alanine aminotransferase.

[0256] In some embodiments, the treatment reduces endomysial infiltration of mononuclear cells, which surround but do not invade muscle fibers. In some embodiments, the treatment reduces perimysial and / or perivascular infiltration of mononuclear cells. In some embodiments, the treatment reduces perifacial atrophy. In some embodiments, the treatment reduces rimming vacuoles present in muscle biopsy samples.

[0257] In some cases, the pharmacokinetics of administered cells, such as adoptively transferred cells, are determined to assess the availability, e.g., bioavailability, of the administered cells. A method for determining the pharmacokinetics of adoptively transferred cells can include collecting peripheral blood from a subject to whom the engineered cells have been administered and determining the number or ratio of engineered cells in the peripheral blood. Approaches for selecting and / or isolating cells can include the use of chimeric antigen receptor (CAR)-specific antibodies (e.g., Brentjens et al., Sci. Transl. Med. 2013 Mar; 5(177): 177ra38), Protein L (Zheng et al., J. Transl. Med. 2012 Feb; 10:29), epitope tags, such as Strep-Tag sequences introduced directly into specific sites within the CAR, where binding reagents for Strep-Tag are used to directly assess the CAR (Liu et al. (2016) Nature Biotechnology, 34:430; International Patent Application Publication No. WO2015095895), and monoclonal antibodies that specifically bind to the CAR polypeptide (see International Patent Application Publication No. WO2014190273). Exogenous marker genes can be utilized in conjunction with therapies using engineered cells, in some cases to enable cell detection or selection, and in some cases to promote cell suicide. In some cases, a truncated epidermal growth factor receptor (EGFRt) can be co-expressed with a transgene of interest (CAR) in transduced cells (see, for example, U.S. Patent No. 8,802,374). EGFRt can contain an epitope recognized by the antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibodies or binding molecules, and can be used to identify or select cells engineered with an EGFRt construct and another recombinant receptor, such as a chimeric antigen receptor (CAR), and / or to eliminate or separate cells expressing the receptor. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. 2016 April; 34(4): 430-434.

[0258] In some embodiments, CARs in a biological sample obtained from a patient, e.g., blood + The number of T cells can be determined after a period of time following administration of the cell therapy, for example, to determine the pharmacokinetics of the cells. In some embodiments, detectable CAR T cells are detected in the blood of a subject or in the majority of subjects so treated by the method. + T cells, as appropriate, CAR + CD8 + T cells and / or CAR + CD4 + The number of T cells is greater than 1 cell per μL, greater than 5 cells per μL, or greater than 10 cells per μL.

[0259] 3. Systemic sclerosis (SSc) Systemic sclerosis (SSc) is an autoimmune disease that primarily affects the skin and can cause organ system involvement. It is characterized by fibrosis affecting the skin and internal organs, and there are three major types of the disease. The first, limited SSc, accounts for approximately 60% of cases and is limited to cutaneous involvement with some vascular and pulmonary involvement. The second, diffuse SSc, is the most severe, accounting for approximately 35% of cases. Diffuse SSc is characterized by widespread cutaneous involvement and is associated with more severe multiorgan involvement, including interstitial lung disease (ILD) and renal failure. The third type, sine SSc, is the rarest, accounting for approximately 5% of cases. Sine SSc does not involve skin involvement and is associated with varying levels of organ involvement. SSc disease progression can cause fibrosis in the heart, lungs, kidneys, and other organs. Patients' quality of life is severely impaired, with a 10-year survival rate of approximately 72%. An estimated 32,000 patients in the United States have diffuse SSc, and approximately half develop ILD. B cells are thought to play a role in the development of SSc, and off-label use of rituximab has shown some efficacy.

[0260] In some embodiments, the systemic autoimmune disease is SSc, for example, limited SSc, diffuse SSc, or sine SSc. Some of the provided methods involve administering engineered cells or compositions containing engineered cells, such as engineered T cells, to a subject with SSc, including limited SSc, diffuse SSc, or sine SSc. Also provided are methods and uses for the engineered cells (e.g., T cells) and / or compositions of the provided CD19-directed CARs, including methods for treating a subject with SSc, including limited SSc, diffuse SSc, or sine SSc, involving the administration of engineered cells and / or compositions thereof. In certain embodiments, the subject has limited SSc. In some embodiments, a subject is selected for or identified as having limited SSc, diffuse SSc, or sine SSc, such as by the presence of certain characteristics or clinical signs indicative of the presence of limited SSc, diffuse SSc, or sine SSc. In some embodiments, the provided methods and uses of CD19-directed CAR-engineered cells (e.g., T cells) and / or compositions thereof include methods for treating a subject with SSc who has failed at least two or more previous therapies. In certain embodiments, the method includes administering to the subject a dose of T cells, including CD4+ and CD8+ T cells, wherein the T cells comprise a chimeric antigen receptor (CAR) that specifically binds to CD19.

[0261] In any of the embodiments herein, at the time of or immediately prior to administration of the composition comprising the engineered T cells, the subject has experienced a relapse after treatment with one or more prior therapies for SSc followed by remission or has become refractory to the treatment.

[0262] In any of the embodiments herein, at or shortly before administration of the composition comprising the engineered T cells, the subject has experienced a relapse after remission following treatment with one or more prior therapies for SSc or has become refractory to the treatment. In any of the embodiments herein, at or shortly before administration of the composition comprising the engineered T cells, the subject has experienced a relapse after treatment with one or more prior therapies for SSc or has become refractory to the treatment. In any of the embodiments herein, the one or more prior therapies for SSc did not include another dose of cells expressing a CAR.

[0263] In any of the embodiments herein, the one or more previous treatments for SSc may include mycophenolate and / or methotrexate if the subject does not have ILD. In any of the embodiments herein, the one or more previous treatments for SSc may include mycophenolate, cyclophosphamide, and / or tricizumab (Actemra) if the subject has ILD. In any of the embodiments herein, the one or more previous treatments for SSc may include administration of mycophenolate, methotrexate, cyclophosphamide, and / or tricizumab (Actemra), followed by administration of B cell depletion therapy, such as rituximab and / or a VEGFR inhibitor, such as nintedanib. In any of the embodiments herein, CD19-directed CAR engineered cells (e.g., T cells) and / or compositions thereof are used to treat patients with SSc who are refractory to previous treatments.

[0264] a. Response and Efficacy In some embodiments, the provided methods and uses involving administration of anti-CD19 CAR T cell therapy reduce SSc disease activity in a subject.

[0265] In some embodiments, the treatment is effective in reducing SSc disease activity, as measured by a disease activity score selected from the modified Rodnan skin score, forced vital capacity, the European Scleroderma Study Group (EScSG) index, the minimal clinically significant difference (MCID), the Patient-Reported Short Form Quality of Life Assessment (SF-36) physical summary score (PCS) and / or mental summary score (MCS), or a combination thereof.

[0266] In some embodiments, reducing SSc disease activity in a subject can include one or more of the following: reducing the subject's EScSG index score after treatment compared to the subject's EScSG index score before treatment; alleviating the subject's skin effects, ILD, and / or pulmonary arterial hypertension compared to the subject's skin effects, ILD, and / or pulmonary arterial hypertension before treatment; or the subject having an improvement in at least one patient-reported outcome (PRO) compared to before treatment.

[0267] In some embodiments, a subject's EScSG index score can be measured before and after administration of a CD19-targeted cell therapy. In some embodiments, patient-reported outcomes (PROs) are measured in the subject before and after administration of a CD19-targeted cell therapy. PROs can include the subject's Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F), 36-item Short Form Health Survey, version 2 (SF-36-v2), Mental Summary Score (MCS), and / or Physical Summary Score (PCS).

[0268] In some embodiments, as a result of treatment, subjects experience a significant change in the Health Assessment Questionnaire-Disability Index (HAQ-DI) compared to baseline. In some embodiments, the patient-reported quality of life assessment is a widely validated, general patient questionnaire that measures difficulty performing activities of daily living. Questions are rated on a scale of 0 to 3, with 0 indicating "no difficulty" and 3 indicating "unable to perform" (Allanore et al., 2020).

[0269] In some embodiments, as a result of treatment, the subject has a significant improvement in the global assessment of disease activity based on the minimal clinically meaningful difference (MCID). In some embodiments, MCID refers to a score obtained from the patient that indicates a change in clinical intervention that is meaningful to the patient.

[0270] In some embodiments, the treatment results in a decrease in the modified Rodnan skin score. In some embodiments, the treatment results in a decrease in skin thickness. In some embodiments, the treatment results in a decrease in skin thickness on the fingers, hands, forearms, upper arms, face, anterior chest, abdomen, thighs, legs, and / or feet. In some embodiments, the treatment reduces the score on the EScSG index.

[0271] In some embodiments, treatment with the provided methods results in clinical remission of SSc in a subject, with the remission maintained for more than 3 months. In some embodiments, treatment with the provided methods results in clinical remission of SSc in a subject, with the remission maintained for more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24 months, 3, 4, 5, or more years. In some embodiments, treatment with the provided methods results in clinical remission of SSc in a subject, with the remission maintained for more than 6 months. In some embodiments, treatment with the provided methods results in clinical remission of SSc in a subject, with the remission maintained for more than 12 months. In some embodiments, treatment with the provided methods results in clinical remission of SSc in a subject, with the remission maintained for more than 24 months. In some embodiments, treatment with the provided methods results in clinical remission of SSc in a subject, with the remission maintained for more than 3 years. In some embodiments, treatment with the provided methods results in clinical remission of SSc in a subject, which is maintained in remission for greater than 4 years. In some embodiments, treatment with the provided methods results in clinical remission of SSc in a subject, which is maintained in remission for greater than 5 years.

[0272] In some embodiments, treatment with provided methods results in long-term remission, which is defined as five consecutive years of no disease activity and no treatment (corticosteroids, methotrexate, mycophenolate, cyclophosphamide, tocilizumab, IVIg, rituximab, nintedanib, or immunosuppressants).

[0273] In some cases, the pharmacokinetics of administered cells, such as adoptively transferred cells, are determined to assess the availability, e.g., bioavailability, of the administered cells. A method for determining the pharmacokinetics of adoptively transferred cells can include collecting peripheral blood from a subject to whom the engineered cells have been administered and determining the number or ratio of engineered cells in the peripheral blood. Approaches for selecting and / or isolating cells can include the use of chimeric antigen receptor (CAR)-specific antibodies (e.g., Brentjens et al., Sci. Transl. Med. 2013 Mar; 5(177): 177ra38), Protein L (Zheng et al., J. Transl. Med. 2012 Feb; 10:29), epitope tags, such as Strep-Tag sequences introduced directly into specific sites within the CAR, where binding reagents for Strep-Tag are used to directly assess the CAR (Liu et al. (2016) Nature Biotechnology, 34:430; International Patent Application Publication No. WO2015095895), and monoclonal antibodies that specifically bind to the CAR polypeptide (see International Patent Application Publication No. WO2014190273). Exogenous marker genes can be utilized in conjunction with therapies using engineered cells, in some cases to enable cell detection or selection, and in some cases to promote cell suicide. In some cases, a truncated epidermal growth factor receptor (EGFRt) can be co-expressed with a transgene of interest (CAR) in transduced cells (see, for example, U.S. Patent No. 8,802,374). EGFRt can contain an epitope recognized by the antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibodies or binding molecules, and can be used to identify or select cells engineered with an EGFRt construct and another recombinant receptor, such as a chimeric antigen receptor (CAR), and / or to eliminate or separate cells expressing the receptor. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. 2016 April; 34(4): 430-434.

[0274] In some embodiments, CARs in a biological sample obtained from a patient, e.g., blood + The number of T cells can be determined after a period of time following administration of the cell therapy, for example, to determine the pharmacokinetics of the cells. In some embodiments, detectable CAR T cells are detected in the blood of a subject or in the majority of subjects so treated by the method. + T cells, as appropriate, CAR + CD8 + T cells and / or CAR + CD4 + The number of T cells is greater than 1 cell per μL, greater than 5 cells per μL, or greater than 10 cells per μL.

[0275] 4.Multiple Sclerosis (MS) Multiple sclerosis (MS) has two major subtypes: relapsing MS and progressive MS. Relapsing MS is associated with an immune-dependent mechanism of disease characterized by cycles of relapse and remission, while progressive MS is associated with an immune-independent mechanism of disease and characterized by steadily worsening symptoms. Early symptoms of MS include fatigue, weakness, and muscle spasms, which can progress to advanced or severe disease characterized by vision and bladder problems along with cognitive changes and physical disability. Approximately 33% of patients require the use of a wheelchair within 20 years of diagnosis. Patients with MS have an approximately 80% increased risk of death. B cells are thought to play a key role in the pathogenesis of MS, as evidenced by the role of anti-CD20 mAbs used in treatment.

[0276] In some embodiments, the systemic autoimmune disease is MS, e.g., relapsing MS (RMS) or progressive MS (PMS). In some embodiments, the systemic autoimmune disease is highly active RMS. In some embodiments, the MS is clinically isolated syndrome (CIS), relapsing-remitting MS (RRMS), active secondary progressive MS (aSPMS), non-active secondary progressive MS (naSPMS), inactive secondary progressive MS (iSPMS), or primary progressive MS (PPMS). In some embodiments, the MS is aSPMS. In some embodiments, the systemic autoimmune disease is aSPMS. Among the methods provided are treatment methods involving administering engineered cells or compositions containing engineered cells, e.g., engineered T cells, to a subject with MS, including CIS, RRMS, aSPMS, naSPMS, iSPMS, or PPMS. Methods and uses of the engineered cells (e.g., T cells) of the provided CD19-directed CARs and / or compositions thereof are also provided, including methods for treating subjects with MS, including CIS, RRMS, aSPMS, naSPMS, iSPMS, or PPMS, involving the administration of the engineered cells and / or compositions thereof. In certain embodiments, the subject has CIS, RRMS, aSPMS, naSPMS, iSPMS, or PPMS. In certain embodiments, the subject has RRMS, aSPMS, iSPMS, or PPMS. In some embodiments, the subject is selected for or identified as having RRMS, aSPMS, iSPMS, or PPMS, such as by the presence of certain characteristics or clinical signs indicating the presence of RRMS, aSPMS, iSPMS, or PPMS. In some embodiments, the methods and uses of the engineered cells (e.g., T cells) of the provided CD19-directed CARs and / or compositions thereof include methods for treating subjects with MS who have failed at least two or more previous treatments.In certain embodiments, the method includes administering to a subject a dose of T cells comprising CD4+ and CD8+ T cells, wherein the T cells comprise a chimeric antigen receptor (CAR) that specifically binds to CD19.

[0277] In any of the embodiments herein, at the time of or immediately prior to administration of the composition comprising the engineered T cells, the subject has experienced a relapse after remission following treatment with one or more prior therapies for MS or has become refractory to the treatment.

[0278] In any of the embodiments herein, at or shortly before administration of the composition comprising the engineered T cells, the subject has experienced a relapse after remission following treatment with one or more prior therapies for MS, or has become refractory to the treatment. In any of the embodiments herein, at or shortly before administration of the composition comprising the engineered T cells, the subject has experienced a relapse after treatment with one or more prior therapies for MS, or has become refractory to the treatment. In any of the embodiments herein, the one or more prior therapies for MS do not include another dose of cells expressing a CAR. In any of the embodiments herein, the one or more prior therapies include two prior disease-modifying therapies (DMTs), one of the prior therapies corresponding to an anti-CD20 antibody.

[0279] In any of the embodiments herein, the one or more previous therapies for MS may include glucocorticoids, plasma exchange, IVIg, adrenocorticotropic hormone (ACTH), fingolimod, siponimod, ozanimod, natalizumab, teriflunomide, ocrelizumab, ofatumumab, alemtuzumab, dimethyl fumarate. In any of the embodiments herein, CD19-directed CAR engineered cells (e.g., T cells) and / or compositions thereof are used to treat patients with MS that is refractory to previous therapies.

[0280] In any of the embodiments herein, at the time of or immediately prior to administration of a composition comprising the engineered T cells, the subject is unable to complete a standard dexterity test. In some embodiments, the subject is unable to complete the 9-Hole Peg Test (9-HPT) for each hand in <240 seconds, or the subject is unable to perform the 25-foot Timed Walk Test (T25FWT) in <150 seconds.

[0281] The 9-Hole Peg Test described herein involves performing the following task: a seated subject holds nine dowels (approximately 7 mm in diameter and 32 mm in length) in one hand and, with the other hand, places them randomly, one at a time, into a board with nine holes. Timing begins when the first peg is placed in a hole and ends when the last peg is placed. The examiner holds the board steady on the tabletop during the test. The test is administered with the dominant hand. If the patient drops a peg, the examiner stops the timer and the patient begins the test again.

[0282] The "25-Foot Timed Walk" or "T25FWT" described herein is a quantitative mobility and leg function performance test based on a timed 25-walk. Patients are directed to one end of a clearly marked 25-foot course and instructed to walk 25 feet as quickly but safely as possible. Time is calculated from the start of the instruction to depart and ends when the patient reaches the 25-foot mark. The patient is immediately tasked with walking the same distance again. Patients may use assistive devices when performing this task. The score on the T25FWT is the average of two completed trials.

[0283] In some embodiments, subjects do not have MS lesions or symptoms that may increase their risk of neurotoxicity, including, but not limited to, tumor-like lesions (greater than or equal to 3 cm within the five years prior to screening. In some embodiments, subjects do not experience a decreased level of consciousness and / or have no active, clinically significant concomitant central nervous system pathology other than MS that may confound the ability to interpret study results or complicate the identification or assessment of neurotoxicity.

[0284] a. Response and Efficacy In some embodiments, the provided methods and uses involving administration of anti-CD19 CAR T-cell therapy reduce MS disease activity in a subject.

[0285] In some embodiments, the treatment is effective in reducing MS disease activity, as measured by a disease activity score selected from the Expanded Disability Status Scale (EDSS), disease stage, Multiple Sclerosis Functional Composite (MSFC), minimal clinically important change (MCID), Patient-Reported Short Form Quality of Life (SF-36) physical summary score (PCS) and / or mental summary score (MCS), or a combination thereof.

[0286] In some embodiments, reducing MS disease activity in a subject can include one or more of the following: reducing the subject's EDSS index score after treatment compared to the subject's EDSS index score before treatment; improving the subject's energy, pain, fatigue, muscle strength, walking distance, mental health, or vision impairment compared to the subject's energy, pain, fatigue, muscle strength, walking distance, mental health, or vision impairment before treatment; or the subject having an improvement in at least one patient-reported outcome (PRO) compared to before treatment.

[0287] In some embodiments, the subject's EDSS index score can be measured before and after administration of CD19-targeted cell therapy. In some embodiments, patient-reported outcomes (PROs) are measured in the subject before and after administration of CD19-targeted cell therapy. PROs can include the subject's Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F), 36-item Short Form Health Survey, version 2 (SF-36-v2), Mental Summary Score (MCS), and / or SF-36, Physical Summary Score (PCS) scores.

[0288] In some embodiments, as a result of treatment, subjects experience a significant change in the Health Assessment Questionnaire-Disability Index (HAQ-DI) compared to baseline. In some embodiments, the patient-reported quality of life assessment is a widely validated, general patient questionnaire that measures difficulty performing activities of daily living. Questions are rated on a scale of 0 to 3, with 0 indicating "no difficulty" and 3 indicating "unable to perform" (Allanore et al., 2020).

[0289] In some embodiments, treatment results in a reduction in the subject's EDSS score. In some embodiments, treatment results in a reduction in the subject's disease stage score. In some embodiments, treatment results in a reduction in the subject's MSFC score.

[0290] In some embodiments, as a result of treatment, the subject has a significant improvement in global assessment of disease activity based on minimal clinically meaningful difference (MCID). In some embodiments, MCID refers to a score obtained from the patient that indicates a change in clinical intervention that is meaningful to the patient. In some embodiments, the subject's walking speed increases after treatment. In some embodiments, the treatment results in an increase in the subject's dexterity, for example, arm or hand dexterity. In some embodiments, the treatment results in improved cognitive function, such as numerical calculation or as measured by a speeded auditory serial addition test. In some embodiments, the treatment results in an increase in the subject's energy. In some embodiments, the treatment results in a reduction in the subject's pain. In some embodiments, the treatment results in a reduction in visual impairment. In some embodiments, the treatment results in improved bladder control or bowel management.

[0291] In some embodiments, treatment with the provided methods results in a subject achieving clinical remission of MS, with the remission being maintained for more than 3 months. In some embodiments, treatment with the provided methods results in a subject achieving clinical remission of MS, with the remission being maintained for more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24 months, 3, 4, 5, or more years. In some embodiments, treatment with the provided methods results in a subject achieving clinical remission of MS, with the remission being maintained for more than 6 months. In some embodiments, treatment with the provided methods results in a subject achieving clinical remission of MS, with the remission being maintained for more than 12 months. In some embodiments, treatment with the provided methods results in a subject achieving clinical remission of MS, with the remission being maintained for more than 24 months. In some embodiments, treatment with the provided methods results in a subject achieving clinical remission of MS, with the remission being maintained for more than 3 years. In some embodiments, treatment with the provided methods results in a subject achieving clinical remission of MS that is maintained for greater than 4 years, hi some embodiments, treatment with the provided methods results in a subject achieving clinical remission of MS that is maintained for greater than 5 years.

[0292] In some embodiments, treatment with provided methods results in long-term remission, which is defined as five consecutive years without disease activity and without treatment (fingolimod, siponimod, ozanimod, natalizumab, dimethyl fumarate, teriflunomide, ocrelizumab, ofatumumab, alemtuzumab, anti-CD20 antibodies, or immunosuppressants).

[0293] In some cases, the pharmacokinetics of administered cells, such as adoptively transferred cells, are determined to assess the availability, e.g., bioavailability, of the administered cells. A method for determining the pharmacokinetics of adoptively transferred cells can include collecting peripheral blood from a subject to whom the engineered cells have been administered and determining the number or ratio of engineered cells in the peripheral blood. Approaches for selecting and / or isolating cells can include the use of chimeric antigen receptor (CAR)-specific antibodies (e.g., Brentjens et al., Sci. Transl. Med. 2013 Mar; 5(177): 177ra38), Protein L (Zheng et al., J. Transl. Med. 2012 Feb; 10:29), epitope tags, such as Strep-Tag sequences introduced directly into specific sites within the CAR, where binding reagents for Strep-Tag are used to directly assess the CAR (Liu et al. (2016) Nature Biotechnology, 34:430; International Patent Application Publication No. WO2015095895), and monoclonal antibodies that specifically bind to the CAR polypeptide (see International Patent Application Publication No. WO2014190273). Exogenous marker genes can be utilized in conjunction with therapies using engineered cells, in some cases to enable cell detection or selection, and in some cases to promote cell suicide. In some cases, a truncated epidermal growth factor receptor (EGFRt) can be co-expressed with a transgene of interest (CAR) in transduced cells (see, for example, U.S. Patent No. 8,802,374). EGFRt can contain an epitope recognized by the antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibodies or binding molecules, and can be used to identify or select cells engineered with an EGFRt construct and another recombinant receptor, such as a chimeric antigen receptor (CAR), and / or to eliminate or separate cells expressing the receptor. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. 2016 April; 34(4): 430-434.

[0294] In some embodiments, CARs in a biological sample obtained from a patient, e.g., blood + The number of T cells can be determined after a period of time following administration of the cell therapy, for example, to determine the pharmacokinetics of the cells. In some embodiments, detectable CAR T cells are detected in the blood of a subject or in the majority of subjects so treated by the method. + T cells, as appropriate, CAR + CD8 + T cells and / or CAR + CD4 + The number of T cells is greater than 1 cell per μL, greater than 5 cells per μL, or greater than 10 cells per μL.

[0295] 5. Rheumatoid arthritis (RA) In some embodiments, the systemic autoimmune disease is rheumatoid arthritis (RA). Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that affects 1% of the population. Disease progression is characterized by destructive inflammation of the joints, which can lead to progressive disability and reduced survival. The synovial membrane of RA is infiltrated by activated immune cells, most abundantly macrophages and T cells, resulting in the chronic production of proinflammatory cytokines and matrix metalloproteinases, which causes inflammation and cartilage and bone degradation (Choy EH and Panayi GS,, N Engl Jmed. 2001; 344:907-916).

[0296] The patient to be treated may have RA as determined according to the 1987 ACR criteria. The patient may test positive for rheumatoid factor (RF) and / or anti-cyclic citrullinated peptide (CCP) IgG antibodies before treatment. A positive RF and anti-CCP antibody test confirms the diagnosis of RA. The patient may have had RA for at least 5 years or at least 7 years, for example, 5 to 10 years.

[0297] In some embodiments, the methods and uses of the provided CD19-directed CAR-engineered cells (e.g., T cells) and / or compositions thereof include methods for treating a subject with MS who has failed at least two or more previous treatments. In certain embodiments, the method includes administering to a subject a dose of T cells, including CD4+ and CD8+ T cells, wherein the T cells comprise a chimeric antigen receptor (CAR) that specifically binds to CD19.

[0298] In any of the embodiments herein, at the time of or immediately prior to administration of the composition comprising the engineered T cells, the subject has experienced a relapse after remission following treatment with one or more prior therapies for RA or has become refractory to the treatment.

[0299] In any of the embodiments herein, at or shortly before administration of the composition comprising the engineered T cells, the subject has experienced a relapse after remission following treatment with one or more prior therapies for RA or has become refractory to the treatment. In any of the embodiments herein, at or shortly before administration of the composition comprising the engineered T cells, the subject has experienced a relapse after treatment with one or more prior therapies for RA or has become refractory to the treatment. In any of the embodiments herein, the one or more prior therapies for RA do not include another dose of cells expressing a CAR.

[0300] In some embodiments, the provided methods and uses involving administration of anti-CD19 CAR T-cell therapy reduce RA disease activity in a subject. In some embodiments, the reduction in RA disease activity is evident when there is a clinical benefit to the subject after administration of the anti-CD19 CAR T-cell therapy.

[0301] One measure of how well-controlled RA is is the Disease Activity Score (DAS) (Fransen & van Riel Clin Exp Rheumatol 23:S93-S99 2005). The DAS is calculated by a healthcare professional based on various validated measures of disease activity, including the physical symptoms of RA. A decrease in the DAS represents a decrease in disease severity. A DAS of less than 2.6 indicates disease remission. A DAS of 2.6 to 3.2 indicates low disease activity. A DAS higher than 3.2 indicates increased disease activity, and at this level, patients may be evaluated to determine whether a change in treatment is warranted. A DAS higher than 5.1 indicates severe disease activity. Variations in calculating the DAS may include assessing different numbers of joints in patients and monitoring different blood constituents. The DAS28 is a disease activity score in which 28 joints in the body are assessed to determine the number of tender and swollen joints (Prevoo et al. Arthritis Rheum 38:44-48 1995). When the DAS28 calculation includes a measurement of C-reactive protein (CRP) instead of erythrocyte sedimentation rate (ESR), it is called DAS28-CRP (Smolen et al. Rheumatology 42:244-257 2003; Wells G, et al. Annals of the Rheumatic Diseases 68: 954-960 2009). CRP is considered a more direct measure of inflammation than ESR and is more sensitive to short-term changes (Kushner, Arthritis Rheum 34: 1065-68 1991). CRP production has been associated with radiological advances in RA [van Leeuwen MA, et al. Br J Rheumatol 32(suppl 3):9-13 1993] and is considered at least as valid as ESR for measuring RA disease activity (Mallya RK, et al. J Rheumatol 9:224-8 1982; Wolfe F. J Rheumatol 24: 1477-85 1997).

[0302] The American College of Rheumatology (ACR) proposed a set of criteria for classifying RA. The commonly used criteria are the ACR 1987 revised criteria (Arnett et al. Arthritis Rheum. 31:315-324 1988). A diagnosis of RA according to the ACR criteria requires that patients meet a minimum number of listed criteria, such as tender or swollen joint counts, stiffness, pain, radiological indications, and serum rheumatoid factor measurements. The ACR 20, ACR 50, and ACR 70 are commonly used evaluation scales to express the effectiveness of RA treatment, especially in clinical trials. The ACR 20 represents a 20% improvement in the measured ACR criteria. Similarly, the ACR 50 represents a 50% improvement in the measured ACR criteria, and the ACR 70 represents a 70% improvement in the measured ACR criteria. An individual patient-reported measure of functional disability in RA patients is the Health Assessment Questionnaire-Disability Index (HAQ-DI). The HAQ-DI score represents the patient's reported physical function in terms of their ability to perform daily tasks, including the level of difficulty they experience when performing these activities. By recording the patient's ability to perform daily activities, the HAQ-DI score can be used as a measure of the patient's quality of life.

[0303] Clinical benefit can include remission of RA. Typically, remission is defined by a DAS28-CRP of less than 2.6.

[0304] Clinical benefit can be at least a 20%, at least a 50%, or at least a 70% improvement in treatment efficacy as determined by the 1987 ACR criteria, i.e., clinical benefit can be achieving an ACR20, an ACR 50, or an ACR 70, respectively.

[0305] One form of clinical benefit that is of particular value to RA patients is an improvement in their ability to perform daily activities. The methods of the present disclosure may include an improvement in patient self-reported functional impairment as measured by a health assessment questionnaire known as HAQ-DI. Methods that include providing clinical benefits to RA patients, including improving the physical function of RA patients as determined by HAQ-DI, as well as compositions and kits for use in such methods, are all aspects of the present disclosure. The clinical benefit may include improving the physical function of RA patients as determined by HAQ-DI. In certain embodiments, a statistically significant improvement in HAQ-DI is achieved within 12, 10, 8, or 6 weeks, or within 4 weeks, or within 2 weeks of starting treatment according to the present disclosure. The improvement may be an improvement of at least 0.25 in HAQ-DI, i.e., a decrease of 0.25 or more in the patient's HAQ-DI score. In certain embodiments, the improvement is an improvement of at least 0.30, 0.40, or 0.45 in the HAQ-DI score. Improvement is generally measured relative to the patient's baseline mean HAQ-DI score prior to treatment with an inhibitor in accordance with the present disclosure.

[0306] Patients can be monitored during and / or after a course of treatment with an anti-CD19 CAR T cell therapy to assess the level of clinical benefit, for example, by measuring DAS28-CRP and / or determining clinical benefit according to ACR criteria and / or measuring HAQ-DI. Methods can include determining that clinical benefit is achieved, e.g., a specified reduction in DAS28-CRP and / or achievement of an ACR 20, ACR50, or ACR70 is met, and / or the HAQ-DI score improves, as discussed elsewhere herein.

[0307] B. Medication In some embodiments, a dose of engineered cells is administered to a subject according to the provided method and / or by using the provided article of manufacture or composition.In some embodiments, the size or timing of the dose is determined according to the specific disease or condition of the subject.In some cases, the size or timing of the dose for a specific disease can be empirically determined in consideration of the provided description.

[0308] In some of any of the provided embodiments, the dose of T cells, such as engineered T cells expressing a recombinant receptor, is enriched for, or comprises a cell composition or population enriched for, CD3+ T cells, CD4+ T cells, CD8+ T cells, or CD4+ T cells and CD8+ T cells. In some of any such embodiments, more than or about 70%, 75%, 80%, 85%, 90%, 95%, or 98% of the cells in the dose of T cells are CD3+ T cells, CD4+ T cells, CD8+ T cells, or CD4+ T cells and CD8+ T cells. In some of any such embodiments, greater than or about 70%, 75%, 80%, 85%, 90%, 95%, or 98% of the cells in the dose of T cells are CD3+ T cells. In some of any of the provided embodiments, the dose of T cells includes both CD4+ and CD8+ cells. In some of any such embodiments, greater than or about 70%, 75%, 80%, 85%, 90%, 95%, or 98% of the cells in the dose of T cells are CD4+ and CD8+ T cells.

[0309] In some embodiments, the dose of cells is 0.1 x 10 per kilogram of subject body weight. 5 or approximately 0.1 x 10 5 of CD19-directed CAR engineered cells (cells / kg) ~2 x 10 6 Or about 2 x 106 cells / kg, e.g., 0.1 x 10 5 or approximately 0.1 x 10 5 cells / kg~0.5×10 5 Or about 0.5 x 10 5 cells / kg, 0.5×10 5 Or about 0.5 x 10 5 cells / kg~1×10 5 Or about 1 x 10 5 cells / kg, 1×10 5 Or about 1 x 10 5 cells / kg~1.5×10 5 Or about 1.5 x 10 5 cells / kg, 1.5×10 5 Or about 1.5 x 10 5 cells / kg~2×10 5 Or about 2 x 10 5 cells / kg, 2×10 5 Or about 2 x 10 5 cells / kg~2.5×10 5 Or about 2.5 x 10 5 cells / kg, 2.5×10 5 Or about 2.5 x 10 5 cells / kg~3×10 5 Or about 3 x 10 5 cells / kg, 3×10 5 Or about 3 x 10 5 cells / kg~3.5×10 5 Or about 3.5 x 10 5 cells / kg, 3.5×10 5 Or about 3.5 x 10 5 cells / kg~4×10 5 Or about 4 x 10 5 cells / kg, 4×10 5 Or about 4 x 10 5 cells / kg~4.5×10 5 or about 4.5 x 10 5 cells / kg, 4.5×10 5 or about 4.5 x 10 5 cells / kg~5×10 5 Or about 5 x 10 5 cells / kg, 5×10 5 Or about 5 x 105 cells / kg~5.5×10 5 Or about 5.5 x 10 5 cells / kg, 5.5×10 5 Or about 5.5 x 10 5 cells / kg~6×10 5 Or about 6 x 10 5 cells / kg, 6×10 5 Or about 6 x 10 5 cells / kg~6.5×10 5 or about 6.5 x 10 5 cells / kg, 6.5×10 5 or about 6.5 x 10 5 cells / kg~7×10 5 Or about 7 x 10 5 cells / kg, 7×10 5 Or about 7 x 10 5 cells / kg~7.5×10 5 Or about 7.5 x 10 5 cells / kg, 7.5×10 5 Or about 7.5 x 10 5 cells / kg~8×10 5 Or about 8 x 10 5 cells / kg, or 8 x 10 5 Or about 8 x 10 5 cells / kg~10×10 5 Or about 10 x 10 5 In some embodiments, the dose of cells comprises 2 x 10 cells / kg of the subject's body weight. 5 of CD19-directed CAR engineered cells (cells / kg) or less, e.g., 3 x 10 5 cells / kg or approximately 3 x 10 5 Cells / kg or less, 4×10 5 cells / kg or approximately 4 x 10 5 Cells / kg or less, 5×10 5 cells / kg or approximately 5 x 10 5 Cells / kg or less, 6×10 5 cells / kg or approximately 6 x 10 5 Cells / kg or less, 7×10 5 cells / kg or approximately 7 x 10 5 Cells / kg or less, 8×10 5cells / kg or approximately 8 x 10 5 Cells / kg or less, 9×10 5 cells / kg or approximately 9 x 10 5 Cells / kg or less, 1×10 6 cells / kg or approximately 1 x 10 6 cells / kg or less, or 2 x 10 6 cells / kg or approximately 2 x 10 6 In some embodiments, the dose of cells is at least 0.1 x 10 cells / kg or less. 5 Or at least about 0.1 × 10 5 of, or 0.1 x 10 5 or approximately 0.1 x 10 5 of CD19-directed CAR engineered cells (cells / kg), e.g., at least 0.2 x 10 5 Or at least about 0.2 × 10 5 or 0.2 × 10 5 or approximately 0.2 x 10 5 cells / kg, at least 0.3 × 10 5 Or at least about 0.3 × 10 5 or 0.3 × 10 5 or approximately 0.3 x 10 5 cells / kg, at least 0.4 × 10 5 Or at least about 0.4 × 10 5 or 0.4 × 10 5 or approximately 0.4 x 10 5 cells / kg, at least 0.5 × 10 5 Or at least about 0.5 × 10 5 or 0.5 × 10 5 Or about 0.5 x 10 5 cells / kg, at least 0.6 × 10 5 Or at least about 0.6 × 10 5 or 0.6 × 10 5 or approximately 0.6 x 10 5 cells / kg, at least 0.7 × 10 5 Or at least about 0.7 × 10 5 or 0.7 × 10 5 or about 0.7 x 10 5cells / kg, at least 0.8 × 10 5 Or at least about 0.8 × 10 5 or 0.8 × 10 5 or about 0.8 x 10 5 cells / kg, at least 0.9 × 10 5 Or at least about 0.9 × 10 5 or 0.9 × 10 5 or approximately 0.9 x 10 5 cells / kg, at least 0.1 x 10 6 Or at least about 0.1 × 10 6 or 0.1 × 10 6 or approximately 0.1 x 10 6 cells / kg, or at least 0.2 × 10 6 Or at least about 0.2 × 10 6 or 0.2 × 10 6 or approximately 0.2 x 10 6 In some embodiments, the number of cells is the number of viable cells, e.g., viable T cells, e.g., viable CD3+ cells, that express a CD19-directed CAR.

[0310] In certain embodiments, the individual populations of cells, or subtypes of cells, range from at or about 100,000 to at or about 100 billion cells, and / or that amount of cells per kilogram of subject body weight, e.g., at or about 100,000 to at or about 50 billion cells (e.g., at or about 5 million cells, at or about 25 million cells, at or about 50 million cells, at or about 1 billion cells, at or about 5 billion cells, at or about 20 billion cells, at or about 30 billion cells, at or about 30 billion cells, at or about 40 billion cells, at or about 5 ... cells, 40 billion or about 40 billion cells, or a range defined by any two of the foregoing values), 1 million or about 1 million to 50 billion or about 50 billion cells (e.g., 5 million or about 5 million cells, 25 million or about 25 million cells, 500 million or about 500 million cells, 1 billion or about 1 billion cells, 5 billion or about 5 billion cells, 20 billion or about 20 billion cells, 30 billion or about 30 billion cells, 40 billion or about 40 billion cells, or a range defined by any two of the foregoing values), e.g., 10 million or about 10 million to 100 billion or about 100 billion cells (e.g., 20 million or about 20 million cells, 30 million or about 30 million cells, 40 million or about 40 million cells, 60 million or about 60 million cells, 70 million or about 70 million cells, 80 million or about 80 million cells, 90 million or about 90 million cells, 10 billion or about 10 billion cells, 25 billion or about 25 billion cells, 50 billion or about 50 billion cells, 75 billion or about 75 billion cells, 90 billion or about 90 billion cells, or a range defined by any two of the foregoing values), and In some cases, 100 million or about 100 million to 50 billion or about 50 billion cells (e.g., 120 million or about 120 million cells, 250 million or about 250 million cells, 350 million or about 350 million cells, 650 million or about 650 million cells, 800 million or about 800 million cells, 900 million or about 900 million cells, 3 billion or about 3 billion cells, 30 billion or about 30 billion cells, 45 billion or about 45 billion cells), or any value therebetween and / or per kilogram of the subject's body weight, are administered to the subject.Dosage may vary depending on the characteristics of the disease or disorder and / or the patient and / or other treatments. In some embodiments, such values ​​refer to the number of recombinant receptor-expressing cells, while in other embodiments, they refer to the number of T cells or total cells in the administered composition. In some embodiments, the number of cells refers to the number of such cells that are viable cells.

[0311] In some embodiments, the dose of cells is a constant dose of cells or a fixed dose of cells, and thus the dose of cells is not tied to or based on the subject's body surface area or weight, hi some embodiments, administering a higher number of cytotoxic cells based on the subject's weight may contribute to an increased risk of toxicity, such as neurotoxicity, in the subject.

[0312] In some embodiments, the dose of engineered cells is a total of 1 x 10 expressing a CD19-directed CAR. 5 or about 1 x 10 5 ~1×10 8 or about 1 x 10 8 A total of 1 x 10 T cells expressing CD19-directed CAR 5 or about 1 x 10 5 ~1.0×10 7 or approximately 1.0 x 10 7 A total of 1 x 10 T cells expressing CD19-directed CAR 5 or about 1 x 10 5 ~1.0×10 6 or approximately 1.0 x 10 6 A total of 1 x 10 T cells expressing CD19-directed CAR 6 or about 1 x 10 6 ~1.0×10 8 or approximately 1.0 x 10 8 A total of 1 x 10 T cells expressing CD19-directed CAR 6 or about 1 x 10 6 ~1.0×10 7 or approximately 1.0 x 10 7 A total of 5 x 10 T cells expressing CD19-directed CAR 6 or about 5 x 10 6 ~1.0×10 8or approximately 1.0 x 10 8 A total of 5 x 10 T cells expressing CD19-directed CAR 6 or about 5 x 10 6 ~1.0×10 7 or approximately 1.0 x 10 7 T cells, a total of 10 x 10 expressing CD19-directed CAR 6 or about 10 x 10 6 ~1.0×10 8 or approximately 1.0 x 10 8 In some embodiments, the number of cells is the number of such cells that are viable cells, such as viable T cells.

[0313] In some embodiments, the dose of engineered cells is a total of 1 x 10 expressing a CD19-directed CAR. 5 or about 1 x 10 5 ~1×10 8 or about 1 x 10 8 A total of 1 x 10 viable T cells expressing CD19-directed CAR 5 or about 1 x 10 5 ~1.0×10 7 or approximately 1.0 x 10 7 A total of 1 x 10 viable T cells expressing CD19-directed CAR 5 or about 1 x 10 5 ~1.0×10 6 or approximately 1.0 x 10 6 A total of 1 x 10 viable T cells expressing CD19-directed CAR 6 or about 1 x 10 6 ~1.0×10 8 or approximately 1.0 x 10 8 A total of 1 x 10 viable T cells expressing CD19-directed CAR 6 or about 1 x 10 6 ~1.0×10 7 or approximately 1.0 x 10 7 A total of 5 x 10 viable T cells expressing CD19-directed CAR 6 or about 5 x 10 6 ~1.0×10 8 or approximately 1.0 x 10 8 A total of 5 x 10 viable T cells expressing CD19-directed CAR6 or about 5 x 10 6 ~1.0×10 7 or approximately 1.0 x 10 7 A total of 10 x 10 viable T cells expressing CD19-directed CAR 6 or about 10 x 10 6 ~1.0×10 8 or approximately 1.0 x 10 8 containing viable T cells.

[0314] In some embodiments, the dose of cells is relatively low. In some embodiments, the anti-CD19 CAR T cell compositions for use in the provided embodiments comprise cells with a less differentiated phenotype, with the majority of these cells having a naive-like or central memory cell phenotype. Furthermore, in provided embodiments, the composition comprises a population of T cells in which more than 25% of the T cells (e.g., CD3+ T cells) express a CAR, e.g., more than 30%, 35%, 40%, 45%, or 50% of the T cells (e.g., CD3+ T cells) express a CAR. In some embodiments, the composition comprises a population of T cells in which more than 50% of the T cells (e.g., CD3+ T cells) express a CAR, e.g., more than 60%, more than 70%, or more than 80% of the T cells express a CAR. Without wishing to be bound by theory, compositions having the characteristics as provided herein ensure that the cells exhibit higher potency and greater persistence in a subject, while minimizing or reducing the potential toxicity of CAR-expressing T cells. In some embodiments, the dose of anti-CD19 CAR T cells exhibits greater potency, persistence, and / or less toxicity than cells of alternative compositions comprising a higher percentage of more differentiated cells (e.g., having a higher proportion of effector T cells). In some embodiments, the dose of anti-CD19 CAR T cells exhibits greater potency, persistence, and / or less toxicity than cells of alternative compositions comprising a lower percentage of cells expressing the CAR. In some embodiments, the dose of engineered cells is greater than or equal to a total of 10 x 10 expressing a CD19-directed CAR. 7 The amount administered may be less than 1000 T cells.

[0315] In some embodiments, the dose of engineered cells is a total of 9 x 10 expressing a CD19-directed CAR. 7 In some embodiments, the dose of engineered cells may be administered in an amount that is less than 8 x 10 total T cells expressing a CD19-directed CAR. 7 In some embodiments, the dose of engineered cells may be administered in an amount that is less than 7.5 x 10 total viable T cells expressing a CD19-directed CAR. 7 In some embodiments, the dose of engineered cells may be administered in an amount that is less than 7.0 x 10 total viable T cells expressing a CD19-directed CAR. 7 In some embodiments, the dose of engineered cells may be administered in an amount that is less than 6.0 x 10 total viable T cells expressing a CD19-directed CAR. 7 In some embodiments, the dose of engineered cells may be administered in an amount that is less than 1 x 10 total viable T cells expressing a CD19-directed CAR. 6 Or about 1 x 10 6 ~50×10 6 Or about 50 x 10 6 A total of 1 x 10 viable T cells expressing CD19-directed CAR 6 Or about 1 x 10 6 ~40×10 6 Or about 40 x 10 6 A total of 1 x 10 viable T cells expressing CD19-directed CAR 6 Or about 1 x 10 6 ~30×10 6 Or about 30 x 10 6 A total of 1 x 10 viable T cells expressing CD19-directed CAR 6 Or about 1 x 10 6 ~20×10 6 Or about 20 x 10 6 A total of 1 x 10 viable T cells expressing CD19-directed CAR 6 Or about 1 x 10 6 ~10×10 6 Or about 10 x 10 6A total of 1 x 10 viable T cells expressing CD19-directed CAR 6 Or about 1 x 10 6 ~5×10 6 Or about 5 x 10 6 A total of 1 x 10 viable T cells expressing CD19-directed CAR 6 Or about 1 x 10 6 ~2.5×10 6 Or about 2.5 x 10 6 A total of 2.5 x 10 viable T cells expressing CD19-directed CAR 6 Or about 2.5 x 10 6 ~50×10 6 Or about 50 x 10 6 A total of 2.5 x 10 viable T cells expressing CD19-directed CAR 6 Or about 2.5 x 10 6 ~40×10 6 Or about 40 x 10 6 A total of 2.5 x 10 viable T cells expressing CD19-directed CAR 6 Or about 2.5 x 10 6 ~30×10 6 Or about 30 x 10 6 A total of 2.5 x 10 viable T cells expressing CD19-directed CAR 6 Or about 2.5 x 10 6 ~20×10 6 Or about 20 x 10 6 A total of 2.5 x 10 viable T cells expressing CD19-directed CAR 6 Or about 2.5 x 10 6 ~10×10 6 Or about 10 x 10 6 A total of 2.5 x 10 viable T cells expressing CD19-directed CAR 6 Or about 2.5 x 10 6 ~5×10 6 Or about 5 x 10 6 A total of 2.5 x 10 viable T cells expressing CD19-directed CAR 6 Or about 2.5 x 10 6 ~50×10 6 Or about 50 x 10 6 A total of 5 x 10 viable T cells expressing CD19-directed CAR6 Or about 5 x 10 6 ~50×10 6 Or about 50 x 10 6 A total of 5 x 10 viable T cells expressing CD19-directed CAR 6 Or about 5 x 10 6 ~40×10 6 Or about 40 x 10 6 A total of 5 x 10 viable T cells expressing CD19-directed CAR 6 Or about 5 x 10 6 ~30×10 6 Or about 30 x 10 6 A total of 5 x 10 viable T cells expressing CD19-directed CAR 6 Or about 5 x 10 6 ~20×10 6 Or about 20 x 10 6 A total of 5 x 10 viable T cells expressing CD19-directed CAR 6 Or about 5 x 10 6 ~10×10 6 Or about 10 x 10 6 A total of 10 x 10 viable T cells expressing CD19-directed CAR 6 Or about 10 x 10 6 ~50×10 6 Or about 50 x 10 6 A total of 10 x 10 viable T cells expressing CD19-directed CAR 6 Or about 10 x 10 6 ~40×10 6 Or about 40 x 10 6 A total of 10 x 10 viable T cells expressing CD19-directed CAR 6 Or about 10 x 10 6 ~30×10 6 Or about 30 x 10 6 A total of 10 x 10 viable T cells expressing CD19-directed CAR 6 Or about 10 x 10 6 ~20×10 6 Or about 20 x 10 6 A total of 20 x 10 viable T cells expressing CD19-directed CAR 6 Or about 20 x 10 6 ~50×106 Or about 50 x 10 6 A total of 20 x 10 viable T cells expressing CD19-directed CAR 6 Or about 20 x 10 6 ~40×10 6 Or about 40 x 10 6 A total of 20 x 10 viable T cells expressing CD19-directed CAR 6 Or about 20 x 10 6 ~30×10 6 Or about 30 x 10 6 A total of 30 x 10 viable T cells expressing CD19-directed CAR 6 Or about 30 x 10 6 ~50×10 6 Or about 50 x 10 6 A total of 30 x 10 viable T cells expressing CD19-directed CAR 6 Or about 30 x 10 6 ~40×10 6 Or about 40 x 10 6 of viable T cells, or a total of 40 x 10 expressing CD19-directed CAR 6 Or about 40 x 10 6 ~50×10 6 Or about 50 x 10 6 are viable T cells.

[0316] In some embodiments, the dose of engineered cells is a total of 0.1 x 10 expressing a CD19-directed CAR. 6 or approximately 0.1 x 10 6 A total of 0.2 x 10 T cells expressing CD19-directed CAR 6 or approximately 0.2 x 10 6 A total of 0.25 x 10 viable T cells expressing CD19-directed CAR 6 or approximately 0.25 x 10 6 A total of 0.5 x 10 viable T cells expressing CD19-directed CAR 6 or approximately 0.5 x 10 6 A total of 0.75 x 10 viable T cells expressing CD19-directed CAR 6 or approximately 0.75 x 10 6 T cells, a total of 2 x 10 expressing CD19-directed CAR6 or about 2 x 10 6 A total of 3 x 10 viable T cells expressing CD19-directed CAR 6 or about 3 x 10 6 A total of 4 x 10 viable T cells expressing CD19-directed CAR 6 or about 4 x 10 6 A total of 6 x 10 viable T cells expressing CD19-directed CAR 6 or about 6 x 10 6 A total of 7 x 10 viable T cells expressing CD19-directed CAR 6 or about 7 x 10 6 A total of 8 x 10 viable T cells expressing CD19-directed CAR 6 or about 8 x 10 6 A total of 9 x 10 viable T cells expressing CD19-directed CAR 6 or about 9 x 10 6 A total of 10 x 10 viable T cells expressing CD19-directed CAR 6 or about 10 x 10 6 A total of 11 x 10 viable T cells expressing CD19-directed CAR 6 or about 11 x 10 6 A total of 12 x 10 viable T cells expressing CD19-directed CAR 6 or about 12 x 10 6 A total of 13 x 10 viable T cells expressing CD19-directed CAR 6 or about 13 x 10 6 A total of 14 x 10 viable T cells expressing CD19-directed CAR 6 or about 14 x 10 6 A total of 15 x 10 viable T cells expressing CD19-directed CAR 6 or about 15 x 10 6 A total of 16 x 10 viable T cells expressing CD19-directed CAR 6 or approximately 16 x 10 6 A total of 17 x 10 viable T cells expressing CD19-directed CAR 6 or approximately 17 x 10 6 A total of 18 x 10 viable T cells expressing CD19-directed CAR 6 or about 18 x 10 6A total of 19 x 10 viable T cells expressing CD19-directed CAR 6 or approximately 19 x 10 6 A total of 25 x 10 viable T cells expressing CD19-directed CAR 6 or about 25 x 10 6 A total of 35 x 10 viable T cells expressing CD19-directed CAR 6 or about 35 x 10 6 A total of 45 x 10 viable T cells expressing CD19-directed CAR 6 or about 45 x 10 6 A total of 60 x 10 viable T cells expressing CD19-directed CAR 6 or about 60 x 10 6 A total of 70 x 10 viable T cells expressing CD19-directed CAR 6 or about 70 x 10 6 A total of 75 x 10 T cells expressing CD19-directed CAR 6 or about 75 x 10 6 A total of 80 x 10 T cells expressing CD19-directed CAR 6 or about 80 x 10 6 A total of 90 x 10 viable T cells expressing CD19-directed CAR 6 or about 90 x 10 6 , 100×10 6 from surviving T cells.

[0317] In some embodiments, the dose of engineered cells is a total of 5 x 10 expressing a CD19-directed CAR. 6 or about 5 x 10 6 ~50×10 6 or about 50 x 10 6 In some embodiments, the dose of engineered cells is a total of 10 x 10 viable T cells expressing a CD19-directed CAR. 6 or about 10 x 10 6 ~50×10 6 or about 50 x 10 6 are viable T cells.

[0318] In some embodiments, the dose of engineered cells is about 5 x 10 total cells expressing a CD19-directed CAR.6 In some embodiments, the number of viable T cells expressing a CD19-directed CAR is about 5 x 10 6 A single dose of T cells is administered to the subject.

[0319] In some embodiments, the dose of engineered cells comprises a total of about 10 x 10 cells expressing a CD19-directed CAR. 6 In some embodiments, the number of viable T cells expressing a CD19-directed CAR is about 10 x 10 6 A single dose of T cells is administered to the subject.

[0320] In some embodiments, the dose of engineered cells is about a total of about 15 x 10 expressing a CD19-directed CAR. 6 In some embodiments, the number of viable T cells expressing a CD19-directed CAR is about 15 x 10 6 A single dose of T cells is administered to the subject.

[0321] In some embodiments, the dose of engineered cells is about 20 x 10 total cells expressing a CD19-directed CAR. 6 In some embodiments, the number of viable T cells expressing a CD19-directed CAR is about 20 x 10 6 A single dose of T cells is administered to the subject.

[0322] In some embodiments, the dose of engineered cells comprises a total of about 25 x 10 cells expressing a CD19-directed CAR. 6 In some embodiments, the number of viable T cells expressing a CD19-directed CAR is about 25 x 10 6 A single dose of T cells is administered to the subject.

[0323] In some embodiments, the dose of engineered cells is about 30 x 10 total cells expressing a CD19-directed CAR. 6 In some embodiments, the number of viable T cells expressing a CD19-directed CAR is about 30 x 10 6 A single dose of T cells is administered to the subject.

[0324] In some embodiments, the dose of engineered cells is about 40 x 10 total cells expressing a CD19-directed CAR. 6 In some embodiments, the number of viable T cells expressing a CD19-directed CAR is about 40 x 10 6 A single dose of T cells is administered to the subject.

[0325] In some embodiments, the dose of engineered cells is about 50 x 10 total cells expressing a CD19-directed CAR. 6 In some embodiments, the number of viable T cells expressing a CD19-directed CAR is about 50 x 10 6 A single dose of T cells is administered to the subject.

[0326] In some embodiments, the number is CD3 + , CD8 + or total CD4+ and CD8+ counts, in some cases recombinant receptor expression (e.g., CAR + In some embodiments, the number of cells is the number of such cells that are viable cells.

[0327] In some embodiments, the dose of T cells is CD4 + T cells, CD8 + T cells, or CD4 + and CD8 + Contains T cells.

[0328] In some embodiments, the dose of T cells is CD4 + T cells, CD8 + T cells, or CD4 + and CD8 + Contains T cells.

[0329] In some embodiments, the dose of cells, e.g., recombinant receptor-expressing T cells, is administered to the subject as a single dose, or only once within a period of 2 weeks, 1 month, 3 months, 6 months, 1 year or more.

[0330] With respect to adoptive cell therapy, administration of a given "dose" encompasses administration of a given amount or number of cells as a single composition and / or a single, uninterrupted administration, such as, for example, a single injection or continuous infusion, as well as administration of a given amount or number of cells as divided doses or as multiple compositions provided in multiple individual compositions or infusions over a specified period of time, for example, over a period of three days or less. Thus, in some situations, a dose is a single or continuous administration of a specified number of cells given or initiated at a single time point. However, in some situations, a dose is administered in multiple injections or infusions over a period of three days or less, for example, by multiple infusions over a period of three or two days, once daily, or over a one-day period.

[0331] In certain embodiments, the number and / or concentration of cells refers to the number of recombinant receptor (e.g., CAR)-expressing cells. In other embodiments, the number and / or concentration of cells refers to the number or concentration of administered T cells.

[0332] In some embodiments, the subject receives multiple doses of cells, for example, two or more doses or multiple consecutive doses. In some embodiments, two doses are administered to the subject. In some embodiments, the subject receives consecutive doses, for example, the second dose is administered approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days after the first dose. In some embodiments, multiple consecutive doses are administered after the first dose, and thus, the additional dose is administered after the administration of the consecutive dose. In some aspects, the number of cells administered to the subject in the additional dose is the same or similar to that of the first dose and / or the consecutive dose. In some embodiments, the additional dose is greater than the previous dose.

[0333] In some embodiments, the size of the dose is determined based on one or more criteria, such as the subject's response to previous treatments, and / or the likelihood or incidence of the subject developing a toxic outcome, e.g., CRS, macrophage activation syndrome, neurotoxicity, and / or the host immune response to the administered cells and / or recombinant receptor.

[0334] In some embodiments, the time between the administration of the first dose and the administration of the subsequent dose is about 9 to about 35 days, about 14 to about 28 days, or 15 to 27 days. In some embodiments, the administration of the subsequent dose is more than about 14 days after the administration of the first dose and less than about 28 days after the administration of the first dose. In some embodiments, the time between the first dose and the subsequent dose is about 21 days. In some embodiments, an additional dose, e.g., a subsequent dose, is administered after the administration of the subsequent dose. In some embodiments, an additional subsequent dose is administered at least about 14 days after the administration of the previous dose and less than about 28 days after the administration of the previous dose. In some embodiments, an additional dose is administered less than about 14 days after the previous dose, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 days after the previous dose. In some embodiments, no dose is administered less than about 14 days after the previous dose and / or no dose is administered more than about 28 days after the previous dose.

[0335] In some embodiments, the dose of cells is generally large enough to be effective in reducing disease burden.

[0336] In certain embodiments, the number and / or concentration of cells refers to the number of recombinant receptor (e.g., CAR)-expressing cells. In other embodiments, the number and / or concentration of cells refers to the number or concentration of total cells, T cells, or peripheral blood mononuclear cells (PBMCs) administered.

[0337] In some embodiments, the method also includes administering one or more additional doses of cells expressing a chimeric antigen receptor (CAR) and / or lymphocyte depletion therapy, and / or one or more steps of the method are repeated. In some embodiments, the one or more additional doses are the same as the initial dose. In some embodiments, the one or more additional doses are different from the initial dose, for example, higher than the initial dose, for example, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times or more than the initial dose, or about 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times or more than the initial dose, or lower than the initial dose, for example, higher, for example, half, third, quarter, fifth, sixth, seventh, eighth, ninth, or tenth or less than the initial dose. In some embodiments, the administration of one or more additional doses is determined based on the subject's response to the initial treatment or any previous treatments, and / or the likelihood or incidence of the subject developing a toxic outcome, e.g., CRS, macrophage activation syndrome, neurotoxicity, and / or the host immune response to the administered cells and / or recombinant receptor.

[0338] C.Toxicity In some embodiments, provided methods are designed to include or include features that result in a lower rate and / or less severe occurrence of treatment-emergent or serious adverse events (AEs), particularly noteworthy AEs, laboratory abnormalities, or dose-limiting toxicities (DLTs). Adverse events are defined by the Common Terminology Criteria for Adverse Events (CTCAE). AEs range from 1 to 5, with 1 being mild and 5 being death. In some embodiments, provided methods are designed to include or include features that result in a lower rate and / or less severe occurrence of DLTs. In some embodiments, DLTs are characterized by hematologic, non-hematologic, and / or organ-specific adverse events or side effects compared to baseline.

[0339] In some embodiments, the methods provided include, for example, alternative cell therapies, e.g., alternative CARs. + The administration of the T cell composition and / or a different administration of cells, e.g., a defined ratio, is designed to include or includes a feature that results in a lower rate and / or a lower degree of toxicity, toxic outcomes or symptoms, toxicity-promoting profiles, factors, or characteristics, such as symptoms or outcomes associated with or indicative of cytokine release syndrome (CRS) or neurotoxicity (NT), compared to administration of the T cell composition and / or a different administration of cells, e.g., a administration of cells not administered at a defined ratio. Cytokine release syndrome (CRS) and neurotoxicity can be graded according to the American Society for Transplantation and Cellular Therapy (ASTCT) consensus grading system (see, e.g., Lee et al. Biol Blood Marrow Transplant. 2019 Apr;25(4):625-38).

[0340] In some embodiments, the engineered T cells (e.g., CCR7 + CD45RA + , CD27 + CCR7 + , or CD62L - CCR7 +While less differentiated T cells (higher proportions of engineered T cells with naive-like or central memory phenotypes, such as phenotypes selected from) are expected to be more active than more differentiated cells, research indicates that the safety of cell therapy can be managed. In some embodiments, robust efficacy and increased safety are obtained by providing lower doses of the composition, for example, compared to cell compositions produced by processes in which the cells are more differentiated, e.g., processes including cell expansion. In some embodiments, it is recognized that even higher doses of cells of the provided anti-CD19 CAR compositions can be administered while maintaining a lower degree of toxicity, such as severe cytokine release syndrome (CRS) or severe neurotoxicity. Thus, the provided methods, in some embodiments, provide alternative cell therapies, for example, alternative CARs having more differentiated engineered T cells than those administered herein. + compared to methods involving administration of a T cell composition, a higher dose of engineered T cells (e.g., 50×10 6 CAR-expressing T cells, for example, 100 x 10 6 or about 100 x 10 6 This involves the administration of CAR-expressing T cells.

[0341] In some embodiments, the provided methods do not result in a high rate or likelihood of toxic or toxic outcomes, such as neurotoxicity (NT) or cytokine release syndrome (CRS), or reduce the rate or likelihood of toxic or toxic outcomes, for example, compared to certain other cell therapies. In some embodiments, the methods do not result in or increase the risk of severe NT (sNT), severe CRS (sCRS), macrophage activation syndrome, fever of at least 38 degrees Celsius or at least about 38 degrees Celsius for 3 or more days, and plasma CRP levels of at least 20 mg / dL or at least about 20 mg / dL. In some embodiments, more than 30%, 35%, 40%, 50%, 60%, or more of subjects treated according to the provided methods do not experience any grade of CRS or any grade of neurotoxicity. In some embodiments, more than 50% of the treated subjects (e.g., at least 60%, at least 70%, at least 80%, at least 90% or more of the treated subjects) do not exhibit cytokine release syndrome (CRS) greater than Grade 2 and / or cytotoxicity greater than Grade 2. In some embodiments, at least 50% of the subjects (e.g., at least 60%, at least 70%, at least 80%, at least 90% or more of the treated subjects) treated according to the method do not exhibit a severe toxic outcome (e.g., severe CRS or severe neurotoxicity), e.g., do not exhibit neurotoxicity of Grade 3 or greater and / or do not exhibit severe CRS, or do not exhibit within a certain period of time following treatment, e.g., within 1 week, 2 weeks, or 1 month of administration of the cells. In some embodiments, parameters evaluated to determine certain toxicities include, but are not limited to, adverse events (AEs), dose-limiting toxicities (DLTs), CRS, and NT.

[0342] The administration of adoptive T cell therapy, such as treatment with T cells expressing chimeric antigen receptors, can induce toxic effects or outcomes, such as cytokine release syndrome and neurotoxicity. In some cases, such effects or outcomes are accompanied by high levels of circulating cytokines, which may underlie the observed toxicity.

[0343] In some embodiments, toxic outcomes are, are associated with, or indicate cytokine release syndrome (CRS) or severe CRS (sCRS).CRS, for example, sCRS, can occur in some cases after adoptive T cell therapy and other biological products are administered to subjects.See Davila et al., Sci Transl Med 6, 224ra25 (2014); Brentjens et al., Sci. Transl. Med. 5, 177ra38 (2013); Grupp et al., N. Engl. J. Med. 368, 1509-1518 (2013); and Kochenderfer et al., Blood 119, 2709-2720 (2012); Xu et al., Cancer Letters 343 (2014) 172-78.

[0344] Typically, CRS is caused by an excessive systemic immune response mediated by, for example, T cells, B cells, NK cells, monocytes, and / or macrophages. Such cells can release large amounts of inflammatory mediators, such as cytokines and chemokines. Cytokines can trigger acute inflammatory responses and / or induce organ endothelial damage, which can lead to microvascular leakage, heart failure, or death. Severe, life-threatening CRS can lead to pulmonary infiltration and injury, renal failure, or disseminated intravascular coagulation. Other severe, life-threatening toxicities can include cardiac toxicity, respiratory distress, neurological toxicity, and / or liver failure. In some embodiments, fever, particularly high fevers (≥38.5°C or ≥101.3°F), is associated with CRS or its risk. In some cases, characteristics or symptoms of CRS mimic those of an infection. In some embodiments, infection is also suspected in subjects with CRS symptoms, and culture monitoring and empirical antibiotic therapy may be administered. Other conditions associated with CRS can include cardiac dysfunction, adult respiratory distress syndrome, renal and / or hepatic failure, coagulopathy, disseminated intravascular coagulation, and capillary leak syndrome.

[0345] CRS can be treated with anti-inflammatory therapy, such as anti-IL-6 therapy, such as anti-IL-6 antibodies, such as tocilizumab, or antibiotics or other drugs as described. Outcomes, signs, and symptoms of CRS are known and include those described herein. In some embodiments, if a particular dosing regimen or administration affects or does not affect a given CRS-related outcome, sign, or symptom, the particular outcome, sign, and symptom and / or its amount or extent may be specified.

[0346] In the setting of administering CAR-expressing cells, CRS typically occurs 6 to 20 days after infusion of the CAR-expressing cells. See Xu et al., Cancer Letters 343 (2014) 172-78. In some cases, CRS occurs less than 6 days after CAR T cell infusion or more than 20 days after CAR T cell infusion. The incidence and timing of CRS may be related to baseline cytokine levels at the time of infusion. Generally, CRS causes elevated serum levels of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and / or interleukin (IL)-2. Other cytokines that can be rapidly induced during CRS are IL-1β, IL-6, IL-8, and IL-10.

[0347] Exemplary outcomes associated with CRS include fever, rigors, chills, hypotension, dyspnea, acute respiratory distress syndrome (ARDS), encephalopathy, elevated ALT / AST, renal failure, cardiac damage, hypoxia, neurological disorders, and death. Neurological complications include delirium, seizure-like activity, confusion, difficulty finding words, aphasia, and / or obtundation. Other outcomes associated with CRS include fatigue, nausea, headache, seizures, tachycardia, myalgia, rash, acute vascular leak syndrome, liver dysfunction, and renal failure. In some embodiments, CRS is associated with an increase in one or more factors, such as serum ferritin, d-dimer, aminotransferase, lactate dehydrogenase, and triglycerides, or is associated with hypofibrinogenemia or hepatosplenomegaly. Other exemplary signs or symptoms associated with CRS include hemodynamic instability, febrile neutropenia, elevated serum C-reactive protein (CRP), changes in coagulation parameters (e.g., international normalized ratio (INR), prothrombin time (PTI) and / or fibrinogen), changes in cardiac and other organ function and / or absolute neutrophil count (ANC).

[0348] In some embodiments, the CRS-related outcome includes one or more of the following: persistent fever, e.g., a fever above a designated temperature, e.g., at or about 38 degrees Celsius, for two or more days, e.g., three or more days, e.g., four or more days, or at least three consecutive days; fever above 38 or about 38 degrees Celsius; elevation of cytokines, e.g., a maximum fold change of at least two cytokines (e.g., at least two from the group consisting of interferon gamma (IFNγ), GM-CSF, IL-6, IL-10, Flt-3L, fractalkine, and IL-5, and / or tumor necrosis factor alpha (TNFα)) compared to pre-treatment levels, e.g., a maximum fold change of at least 250 or about 250 in at least one of such cytokines; and / or at least one clinical sign of toxicity, e.g., hypotension (e.g., administration of at least one intravenous vasoactive hypoxia (e.g., plasma oxygen (PO2) levels below 90% or about 90%); and / or one or more neurological disorders (including mental status changes, obtundation, and seizures). In some embodiments, neurotoxicity (NT) can be observed in parallel with CRS.

[0349] Exemplary CRS-related outcomes include elevated serum levels or high serum levels of one or more factors, including cytokines and chemokines and other factors associated with CRS.Exemplary outcomes also include increased synthesis or secretion of one or more of these factors.Such synthesis or secretion can be by T cells or cells that interact with T cells, such as innate immune cells or B cells.

[0350] In some embodiments, CRS-related serum factors or CRS-related outcomes include inflammatory cytokines and / or chemokines, including interferon gamma (IFN-γ), IL-7, IL-12, sIL-2Ra, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein (MIP)-1, tumor necrosis factor alpha (TNFα), IL-6, and IL-10, IL-1β, IL-8, IL-2, MIP-1, Flt-3L, fractalkine, and / or IL-5. In some embodiments, the factors or outcomes include C-reactive protein (CRP). In addition to being an early and easily measurable risk factor for CRS, CRP is also a marker of cellular expansion. In some embodiments, subjects who are measured to have high levels of CRP, such as ≥ 15 mg / dL, have CRS. In some embodiments, subjects who are measured to have high levels of CRP do not have CRS. In some embodiments, measuring CRS includes measuring CRP and another factor indicative of CRS.

[0351] In some embodiments, one or more inflammatory cytokines or chemokines are monitored before, during, or after CAR treatment. In some embodiments, the one or more cytokines or chemokines include IFN-γ, TNF-α, IL-2, IL-1β, IL-6, IL-7, IL-8, IL-10, IL-12, sIL-2Rα, granulocyte-macrophage colony-stimulating factor (GM-CSF), or macrophage inflammatory protein (MIP). In some embodiments, IFN-γ, TNF-α, and IL-6 are monitored.

[0352] CRS criteria that appear to correlate with the onset of CRS have been developed to predict which patients are likely at risk for developing sCRS (see Davilla et al. Science translational medicine. 2014;6(224):224ra25). Factors include fever, hypoxia, hypotension, neurological changes, and elevated serum levels of inflammatory cytokines, such as the set of seven cytokines (IFNγ, IL-5, IL-6, IL-10, Flt-3L, fractalkine, and GM-CSF). Other guidelines for the diagnosis and management of CRS are known (see, e.g., Lee et al., Blood. 2014;124(2):188-95; Lee et al., Biol Blood Marrow Transplant 2019;25(4):625-38). In some embodiments, the criteria reflecting CRS grade are detailed in Table 1 below.

[0353] [Table 1]

[0354] In some embodiments, the criteria reflecting the CRS grade are those detailed in Table 2 below.

[0355] [Table 2]

[0356] In some embodiments, high-dose vasopressor therapy includes those set forth in Table 3 below.

[0357] [Table 3]

[0358] In some embodiments, the toxic outcome is severe CRS. In some embodiments, the toxic outcome is the absence of severe CRS (e.g., moderate or mild CRS). In some embodiments, a subject is deemed to develop "severe CRS" ("sCRS") in response to or secondary to administration of a dose of a cell therapy or cells thereof if, after administration, the subject exhibits: (1) a fever of at least 38 degrees Celsius for at least three days; (2) cytokine elevations, including any of the following: (a) an increase of at least 75% for at least two of the following seven cytokine groups: interferon gamma (IFNγ), GM-CSF, IL-6, IL-10, Flt-3L, fractalkine, and IL-5, compared to levels immediately after administration; and / or (b) a maximum fold change of at least 250 for at least one of the following seven cytokine groups compared to levels immediately after administration: interferon gamma (IFNγ), GM-CSF, IL-6, IL-10, Flt-3L, fractalkine, and IL-5; and (c) at least one clinical sign of toxicity, such as hypotension (requiring at least one intravenous vasopressor) or hypoxia (PO2<90%) or one or more neurological disorders (including mental status changes, obtundation, and / or seizures). In some embodiments, severe CRS includes CRS of Grade 3 or higher, such as those shown in Tables 1 and 2.

[0359] In some embodiments, toxicity outcomes, e.g., levels of CRS-related outcomes, e.g., serum levels of indicators of CRS, are measured by ELISA. In some embodiments, fever and / or C-reactive protein (CRP) levels can be measured. In some embodiments, subjects with fever and a CRP of ≥ 15 mg / dL can be considered at high risk for developing severe CRS. In some embodiments, CRS-related serum factors or CRS-related outcomes include elevated levels and / or concentrations of inflammatory cytokines and / or chemokines, including Flt-3L, fractalkine, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 beta (IL-1β), IL-2, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, interferon gamma (IFN-γ), macrophage inflammatory protein (MIP)-1, MIP-1, sIL-2Rα, or tumor necrosis factor alpha (TNFα). In some embodiments, the factor or outcome includes C-reactive protein (CRP). In addition to being an early and easily measurable risk factor for CRS, CRP is also a marker of cellular expansion. In some embodiments, subjects who are measured to have high levels of CRP, such as ≧15 mg / dL, have CRS. In some embodiments, subjects who are measured to have high levels of CRP do not have CRS. In some embodiments, measuring CRS includes measuring CRP and another factor indicative of CRS.

[0360] In some embodiments, outcomes associated with severe CRS or CRS of grade 3 or higher, e.g., grade 4 or higher, include one or more of the following: persistent fever, e.g., a fever at a designated temperature, e.g., above 38 or about 38 degrees Celsius, for two or more days, e.g., three or more days, e.g., four or more days, or at least three consecutive days; fever above 38 or about 38 degrees Celsius; elevated cytokines, e.g., at least two cytokines [e.g., interferon gamma (IFNγ), GM-CSF, IL-6, IL-10, Flt-3L, fractalkine, and IL-5, and / or and tumor necrosis factor alpha (TNFα) compared to pre-treatment levels, e.g., a maximum fold change of at least 75 or about 75, or a maximum fold change of at least one of such cytokines of at least 250 or about 250; and / or at least one clinical sign of toxicity, e.g., hypotension (e.g., as measured by at least one intravenous vasopressor); hypoxia (e.g., a plasma oxygen (PO2) level of less than 90% or about 90%); and / or one or more neurological disorders (including mental status changes, obtundation, and seizures). In some embodiments, severe CRS includes CRS requiring management or care in an intensive care unit (ICU).

[0361] In some embodiments, CRS, e.g., severe CRS, includes the combination of (1) persistent fever (fever of at least 38 degrees Celsius for at least three days) and (2) a serum CRP level of at least 20 mg / dL or at least about 20 mg / dL. In some embodiments, CRS includes hypotension requiring the use of two or more vasopressors, or respiratory failure requiring mechanical ventilation. In some embodiments, the dosage of the vasopressor is increased during a second or subsequent administration.

[0362] In some embodiments, severe CRS or Grade 3 CRS includes increased alanine aminotransferase, increased aspartate aminotransferase, chills, febrile neutropenia, headache, left ventricular dysfunction, encephalopathy, hydrocephalus, and / or tremors.

[0363] Methods for measuring or detecting various outcomes may be specified.

[0364] In some embodiments, the toxic outcome is neurotoxicity or is associated with neurotoxicity. In some embodiments, symptoms associated with clinical risk of neurotoxicity include confusion, delirium, aphasia, expressive aphasia, obtundation, myoclonus, lethargy, altered mental status, convulsions, seizure-like activity, seizures (confirmed by electroencephalography (EEG), if appropriate), elevated levels of beta amyloid (Aβ), elevated levels of glutamate, and elevated levels of oxygen radicals. In some embodiments, neurotoxicity is graded based on severity (e.g., using a 1-5 scale) (see, e.g., Guido Cavaletti & Paola Marmiroli Nature Reviews Neurology 6, 657-666 (December 2010); National Cancer Institute-Common Toxicity Criteria version 4.03 (NCI-CTCAE v4.03)).

[0365] In some instances, neurological symptoms may be the initial symptoms of sCRS. In some embodiments, neurological symptoms appear to begin 5-7 days after cell therapy infusion. In some embodiments, the duration of neurological changes may range from 3-19 days. In some cases, resolution of neurological changes occurs after other symptoms of sCRS have resolved. In some embodiments, the time and extent of resolution of neurological changes is not hastened by treatment with anti-IL-6 and / or steroids.

[0366] In some embodiments, a subject is considered to develop "severe neurotoxicity" in response to or secondary to administration of a dose of a cell therapy or cells thereof if, after administration, the subject exhibits any of the following symptoms that limit self-care (e.g., bathing, dressing and undressing, eating, using the toilet, taking medication): 1) symptoms of peripheral motor neuropathy, including inflammation or degeneration of peripheral motor nerves; 2) symptoms of peripheral sensory neuropathy, including inflammation or degeneration of peripheral sensory nerves, paresthesia, e.g., distortion of perception resulting in abnormal and unpleasant sensations, neuralgia, e.g., intense pain sensations along a nerve or nerve group, and / or paresthesia, e.g., dysfunction of sensory neurons resulting in abnormal skin sensations such as tingling, numbness, pressure, cold, and warmth in the absence of stimulation. In some embodiments, severe neurotoxicity includes neurotoxicity that is Grade 3 or higher, such as those shown in Table 4.

[0367] [Table 4]

[0368] In some embodiments, the method alleviates symptoms associated with CRS or neurotoxicity compared to other methods. In some aspects, the provided method alleviates / reduces symptoms, outcomes, or factors associated with CRS, including symptoms, outcomes, or factors associated with severe CRS or CRS of grade 3 or higher compared to other methods. For example, a subject treated according to the method may be free of detectable symptoms, outcomes, or factors of CRS, such as any of those described, e.g., shown in Table 1 and Table 2, e.g., severe CRS or CRS of grade 3 or higher, and / or may exhibit reduced symptoms, reduced outcomes, or reduced factors. In some embodiments, subjects treated according to the present methods may exhibit reduced symptoms of neurotoxicity, such as weakness or numbness in the limbs, loss of memory, vision, and / or intelligence, uncontrollable, obsessive and / or compulsive behavior, delusions, headaches, cognitive and behavioral problems (including motor control, cognitive decline, and autonomic nervous system dysfunction), and sexual dysfunction, compared to subjects treated by other methods. In some embodiments, subjects treated according to the present methods may exhibit reduced symptoms associated with peripheral motor neuropathy, peripheral sensory neuropathy, paresthesia, neuralgia, or paresthesia.

[0369] In some embodiments, the method reduces outcomes associated with neurotoxicity, including nervous system and / or brain damage, e.g., neuronal death. In some aspects, the method reduces levels of factors associated with neurotoxicity, e.g., beta amyloid (Aβ), glutamate, and oxygen radicals.

[0370] In some embodiments, the toxic outcome is a dose-limiting toxicity (DLT). In some embodiments, the toxic outcome is a dose-limiting toxicity. In some embodiments, the toxic outcome is the absence of dose-limiting toxicity. In some embodiments, a dose-limiting toxicity (DLT) is defined as any Grade 3 or higher toxicity, as assessed by any known or published guideline for assessing specific toxicity, including any of those described above and the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) Version 4.0.

[0371] In some embodiments, the low rate, risk, or likelihood of developing toxicity, e.g., CRS or neurotoxicity, or severe CRS or neurotoxicity, e.g., Grade 3 or higher CRS or neurotoxicity, observed with administering a dose of T cells according to the provided methods and / or using the provided articles of manufacture or compositions allows for the administration of cell therapy in an outpatient setting. In some embodiments, the administration of cell therapy, e.g., T cells (e.g., CAR T cells), according to the provided methods and / or using the provided articles of manufacture or compositions, allows for the administration of cell therapy in an outpatient setting. + Administration of the dose of T cells is done on an outpatient basis or does not require hospital admission, for example, an overnight hospital stay.

[0372] In some embodiments, cell therapy, such as T cell (e.g., CAR) therapy, is administered according to the provided methods and / or using the provided articles of manufacture or compositions, including to subjects treated outpatient. + A subject receiving a dose of a therapeutic agent (e.g., T cells) is not administered any intervention to treat toxicity prior to or in conjunction with administration of the cell dose unless or until the subject exhibits signs or symptoms of toxicity, e.g., neurotoxicity or CRS. Exemplary agents for treating, delaying, attenuating, or ameliorating toxicity are described in Section IC.

[0373] In some embodiments, including in subjects treated outpatient, cell therapy, e.g., T cells (e.g., CAR + If a subject administered a dose of T cells develops a fever, the subject is administered a fever-reducing treatment or is instructed to receive or administer a fever-reducing treatment. In some embodiments, fever in a subject is characterized by the subject's body temperature being at or above (or measured at) a particular threshold temperature or level. In some aspects, the threshold temperature is associated with at least a mild fever, at least a moderate fever, and / or at least a severe fever. In some embodiments, the threshold temperature is a particular temperature or range. For example, the threshold temperature may be 38, 39, 40, 41 or 42 degrees Celsius, or about 38, 39, 40, 41 or 42 degrees Celsius, or at least about 38, 39, 40, 41 or 42 degrees Celsius, and / or may be in the range of 38 or about 38 degrees Celsius to 39 or about 39 degrees Celsius, in the range of 39 or about 39 degrees Celsius to 40 or about 40 degrees Celsius, in the range of 40 or about 40 degrees Celsius to 41 or about 41 degrees Celsius, or in the range of 41 or about 41 degrees Celsius to 42 or about 42 degrees Celsius.

[0374] In some embodiments, the treatment designed to reduce fever comprises treatment with antipyretic drugs.Antipyretic drugs include any drug that reduces fever, such as compounds, compositions or components, for example, any number of drugs known to have antipyretic effects, such as NSAIDs (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), salicylates (e.g., aspirin, choline salicylate, magnesium salicylate, and sodium salicylate), paracetamol, acetaminophen, metamizole, nabumetone, phenaxone, antipyrine, and antipyretic drugs.In some embodiments, the antipyretic drug is acetaminophen.In some embodiments, acetaminophen can be administered orally or intravenously at a dose of 12.5 mg / kg every 4 hours at most.In some embodiments, the antipyretic drug is ibuprofen or aspirin.

[0375] In some embodiments, if fever is persistent fever, the subject will be administered alternative treatment for treating toxicity.For subjects who are treated in outpatient clinics, if the subject has persistent fever and / or is determined to have persistent fever or will have persistent fever, the subject will be instructed to return to the hospital.In some embodiments, if the subject has a fever of or above the appropriate threshold temperature, and after designated treatment, for example, treatment designed to reduce fever, for example, treatment with antipyretic drugs such as NSAIDs or salicylates, for example, ibuprofen, acetaminophen or aspirin, the subject's fever or body temperature does not decrease, or does not decrease by a specified amount or more than a specified amount (for example, more than 1°C, generally not by about 0.5°C, 0.4°C, 0.3°C or 0.2°C, or not by more than about 0.5°C, 0.4°C, 0.3°C or 0.2°C). For example, a subject is considered to have a persistent fever if he or she exhibits, or is determined to exhibit, a fever of at least 38 or 39 degrees Celsius, or at least about 38 or 39 degrees Celsius, which does not decrease by at or about 0.5°C, 0.4°C, 0.3°C, or 0.2°C, or by more than 0.5°C, 0.4°C, 0.3°C, or 0.2°C, or by more than or about 0.5°C, 0.4°C, 0.3°C, or 0.2°C, or by 1%, 2%, 3%, 4%, or 5%, or by about 1%, 2%, 3%, 4%, or 5%, over 6, 8, 12, or 24 hours, even after treatment with an antipyretic such as acetaminophen. In some embodiments, the dosage of the antipyretic is a dosage normally effective to reduce fever, or a particular type of fever, e.g., fever associated with a bacterial or viral infection, e.g., a localized or systemic infection, in such a subject.

[0376] In some embodiments, a subject has, and / or is determined to have, or is considered to have, a persistent fever if he or she exhibits a fever at or above the appropriate threshold temperature, and if the subject's fever or body temperature does not rise or fall by about 1° C. or by more than about 1° C., generally by about 0.5° C., 0.4° C., 0.3° C., or 0.2° C., or by more than about 0.5° C., 0.4° C., 0.3° C., or 0.2° C. Such absence of rise or fall above or below a certain amount is generally measured over a predetermined period of time (e.g., over 24 hours, 12 hours, 8 hours, 6 hours, 3 hours, or 1 hour, which may be measured from the first sign of fever or from the first temperature above the indicated threshold). For example, in some embodiments, a subject is considered or determined to exhibit a persistent fever if he or she exhibits a fever of at least 38 or 39 degrees Celsius, or at least about 38 or 39 degrees Celsius, that does not fluctuate in temperature by more than 0.5°C, 0.4°C, 0.3°C, or 0.2°C, or by more than about 0.5°C, 0.4°C, 0.3°C, or 0.2°C, over 6, 8, 12, or 24 hours.

[0377] In some embodiments, the fever is a persistent fever; in some aspects, the subject is not diagnosed with a persistent fever when the subject is determined to have a persistent fever, e.g., from such a determination or from a first treatment that may induce toxicity, e.g., a cell therapy, e.g., T cells, e.g., CAR + Treatment may occur within 1, 2, 3, 4, 5, 6 hours or less of the first such determination after the dose of T cells.

[0378] In some embodiments, one or more interventions or agents for treating toxicity, such as toxicity-targeting therapies, are administered when or shortly after a subject is determined or confirmed (e.g., first determined or confirmed) to have persistent fever, e.g., as measured according to any of the above-described embodiments. In some embodiments, the toxicity-targeting therapies are administered within a certain time period of such confirmation or determination, e.g., within 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, or 8 hours of the confirmation or determination.

[0379] In some embodiments, the provided methods do not result in a high rate or likelihood of toxicity or toxic outcomes, such as immune effector cell-associated neurotoxicity syndrome (ICANS), or reduce the rate or likelihood of toxicity or toxic outcomes, compared to certain other cell therapies. In some embodiments, the methods do not result in or increase the risk of ICANS.

[0380] Exemplary ICANS-related outcomes include the ICANS grading scheme developed by the CAR T-Cell Therapy-Associated TOXicity (CARTOX) consensus group, which consists of a 10-point grading classification (CARTOX-10) that combines the essential components of the Mini Mental State Assessment to assess the grade of encephalopathy by fluctuations in concentration, speech, handwriting, and orientation (see, e.g., Neelapu et al. Nat Rev Clin Oncol., 2018, 15:47-62).

[0381] In some embodiments, outcomes related to a method for classifying the severity of ICANS by using an immune effector cell encephalopathy (ICE) score, in some embodiments, ICE scoring includes assessing receptive aphasia.

[0382] II. Cellular Therapy and Cell Engineering In some embodiments, the cell therapy (e.g., T cell therapy) methods disclosed herein involve administering engineered cells expressing a recombinant receptor (e.g., a CAR) designed to recognize and / or specifically bind to an antigen associated with a disease or condition, such as severe and refractory SLE. In certain embodiments, the antigen bound or recognized by the recombinant receptor (e.g., a CAR) is CD19. In some embodiments, binding to the antigen results in a response, e.g., an immune response, to such antigen. In some embodiments, the cells contain or are engineered to contain a recombinant receptor, e.g., a chimeric antigen receptor (CAR). Recombinant receptors, e.g., CARs, generally comprise an extracellular antigen (or ligand) binding domain specific for the antigen linked to one or more intracellular signaling components, in some embodiments via a linker and / or transmembrane domain. In some embodiments, the engineered cells are provided in pharmaceutical compositions and formulations suitable for administration to a subject, e.g., for adoptive cell therapy. Therapeutic methods for administering the cells and compositions to a subject, e.g., a patient, are also provided.

[0383] In some embodiments, the cells contain one or more nucleic acids introduced by genetic engineering, thereby expressing recombinant or genetically engineered products of such nucleic acids. In some embodiments, gene transfer is accomplished by first stimulating the cells, e.g., by combining them with a stimulatory agent that induces a response such as proliferation, survival, and / or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells and expansion in culture to numbers effective for clinical application.

[0384] A. Chimeric Antigen Receptors (e.g., CD19-targeted CARs) In some embodiments of the provided methods and uses, the chimeric receptor, e.g., a chimeric antigen receptor, contains one or more domains that combine a ligand binding domain (e.g., an antibody or antibody fragment) that provides specificity for a desired antigen (e.g., CD19) with an intracellular signaling domain. In some embodiments, the intracellular signaling domain is a stimulatory or activating intracellular domain portion, e.g., a T cell stimulatory or activation domain, that provides a primary activation or activation signal. In some embodiments, the intracellular signaling domain contains, or further contains, a costimulatory signaling domain to facilitate effector function. In some embodiments, the chimeric receptor, when engineered into an immune cell, can modulate T cell activation, and in some cases, modulate T cell differentiation or homeostasis, thereby resulting in engineered cells with improved in vivo longevity, survival, and / or persistence, such as for use in adoptive cell therapy methods.

[0385] Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells are described in, for example, International Patent Application Publication Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. 2002131960, 2013287748, 20130149337, U.S. those described in Patent Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353 and 8,479,118, European Patent Application No. EP2537416, and / or Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) pLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some embodiments, the antigen receptor includes a CAR such as that described in U.S. Patent No. 7,446,190 and that described in International Patent Application Publication No. WO / 2014055668A1.Examples of CARs include those disclosed in any of the publications mentioned above, e.g., WO2014031687, U.S. Patent No. 8,339,645, U.S. Patent No. 7,446,179, U.S. Patent Application Publication No. 2013 / 0149337, U.S. Patent No. 7,446,190, U.S. Patent No. 8,389,282, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, U.S. Patent No. 8,339,645, U.S. Patent No. 7,446,179, U.S. Patent Application Publication No. 2013 / 0149337, U.S. Patent No. 7,446,190, and U.S. Patent No. 8,389,282.

[0386] Chimeric receptors, e.g., CARs, generally comprise an extracellular antigen-binding domain, e.g., a portion of an antibody molecule, generally the variable heavy (V) domain of an antibody, e.g., an scFv antibody fragment. H ) chain region and / or variable light (V L ) chain regions. In some embodiments, the antibody or antigen-binding portion thereof is expressed on a cell as part of a recombinant receptor, e.g., a chimeric receptor (e.g., a CAR), that binds, e.g., specifically binds, an antigen (e.g., CD19).

[0387] In some embodiments, the antigen targeted by the receptor is a polypeptide. In certain embodiments, the antigen target is CD19. In some embodiments, the antigen is selectively expressed on B cells, which is used to treat autoimmune or inflammatory conditions such as lupus. In some embodiments, the CAR typically comprises one or more antibodies or antigen-binding fragments or portions thereof that target CD19 in its extracellular portion.

[0388] Chimeric receptors, e.g., CARs, generally comprise an extracellular antigen-binding domain that is the antigen-binding portion of an antibody molecule. In some embodiments, the antigen-binding domain is a portion of an antibody molecule, generally the variable heavy (V) domain of an antibody, e.g., an scFv antibody fragment. H ) chain region and / or variable light (V L In some embodiments, a CAR is an antigen-binding portion of an antibody molecule, such as the variable heavy (V) chain region of a monoclonal antibody (mAb). H ) chain and variable light (V L In some embodiments, the antigen-binding domain comprises a single-domain antibody (sdAb), e.g., a sdFv, a nanobody, a V H H and V NAR In some embodiments, the antigen-binding fragment comprises antibody variable regions joined by a flexible linker.

[0389] In some embodiments, an antibody or antigen-binding fragment (e.g., scFv or V H The antibody or antigen-binding fragment (e.g., scFv) specifically recognizes an antigen, such as CD19. In some embodiments, the antibody or antigen-binding fragment is derived from or is a variant of an antibody or antigen-binding fragment that specifically binds to CD19. In some embodiments, the antigen is CD19. In some embodiments, the antibody or antigen-binding fragment (e.g., scFv) contains a variable heavy chain and a variable light chain with six CDRs, CDRH1-3 and CDRL1-3, that confer binding to CD19.

[0390] The terms "complementarity-determining region" and "CDR," synonymous with "hypervariable region" or "HVR," are known to refer, in some cases, to noncontiguous sequences of amino acids in an antibody variable region that confer antigen specificity and / or binding affinity. Generally, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region, and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. It is also known that "framework region" and "FR" refer, in some cases, to the non-CDR portions of the heavy and light chain variable regions. Generally, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region, and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region.

[0391] The precise amino acid sequence boundaries of a given CDR or FR can be determined using the methods described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745." ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 Jan;27(1):55-77 ("IMGT" numbering scheme); Honegger A and Plueckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun 8;309(3):657-70 ("Aho" numbering scheme); and Martin et al., "Modeling antibody hypervariable loops: a combined algorithm," PNAS, 1989, 86(23):9268-9272 ("AbM" numbering scheme).

[0392] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. Both the Kabat and Chothia numbering schemes are based on the most common antibody region sequence lengths, with insertions accommodated by insertion letters, e.g., "30a," and deletions occurring in some antibodies. The two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM scheme, used by Oxford Molecular's AbM antibody modeling software, is a compromise between the Kabat and Chothia definitions.

[0393] Table 5 below lists exemplary positions of the boundaries of CDR-L1, CDR-L2, CDR-L3, and CDR-H1, CDR-H2, and CDR-H3 as defined by the Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between the CDRs; for example, FR-L1 is located before CDR-L1, FR-L2 is located between CDR-L1 and CDR-L2, FR-L3 is located between CDR-L2 and CDR-L3, etc. Note that the Kabat numbering scheme shown places insertions at H35A and H35B, so the ends of the Chothia CDR-H1 loop, when numbered using the Kabat numbering convention shown, vary between H32 and H34 depending on the length of the loop.

[0394] [Table 5]

[0395] Thus, unless otherwise specified, the "CDRs" or "complementarity determining regions" or individual designated CDRs (e.g., CDR-H1, CDR-H2, CDR-H3) of a given antibody or region thereof, e.g., the variable region thereof, will be understood to encompass a (or particular) complementarity determining region as defined by any of the schemes described above or other known schemes. For example, a particular CDR (e.g., CDR-H3) may be a CDR of a given V H or V L When a variable region amino acid sequence is described as containing the amino acid sequence of a corresponding CDR in the variable region, it is understood that such CDR has the sequence of the corresponding CDR (e.g., CDR-H3) in the variable region, defined according to either the scheme described above or other known schemes. In some embodiments, a specific CDR sequence is specified. Although exemplary CDR sequences of the provided antibodies are described using various numbering schemes, it is understood that the provided antibodies can include CDRs described according to any of the other numbering schemes described above or other numbering schemes known to those skilled in the art.

[0396] Similarly, unless otherwise specified, the FRs or individual designated FRs (e.g., FR-H1, FR-H2, FR-H3, FR-H4) of a given antibody or region thereof, e.g., its variable region, are understood to encompass a (or specific) framework region defined by any known scheme. In some cases, schemes are specified for the identification of specific CDRs, FRs, or CDRs, such as CDRs as defined by the Kabat, Chothia, AbM, or Contact method or other known schemes. In other cases, the specific amino acid sequences of the CDRs or FRs are provided.

[0397] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy and light chains of a native antibody (V H and V L) generally have a similar structure, with each domain containing four conserved framework regions (FRs) and three CDRs. [See, e.g., Kindt et al., Kuby Immunology, 6t h ed., W.H. Freeman and Co., page 91 (2007). H and V L The domain may be sufficient to confer antigen binding specificity. Furthermore, an antibody that binds to a particular antigen can be isolated by isolating the V from the antibody that binds to that antigen. H or V L Complementary V domains L or V H Domains can be isolated by screening libraries of each (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0398] Among the antibodies provided are antibody fragments. An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; variable heavy chains (V); H ) domains, single chain antibody molecules, e.g., scFv and single domain V H and multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single-chain antibody fragment, e.g., an scFv, comprising a variable heavy chain region and / or a variable light chain region.

[0399] A single domain antibody (sdAb) is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody. In some embodiments, the CAR comprises an antibody heavy chain domain that specifically binds to an antigen, such as an antigen on a B cell, such as CD19. Exemplary single domain antibodies include sdFv, nanobodies, V H H or V NAR Examples include:

[0400] Antibody fragments can be produced by various techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells. In some embodiments, antibodies are recombinantly produced fragments, e.g., fragments containing non-naturally occurring configurations, e.g., those having two or more antibody regions or chains linked by synthetic linkers, e.g., peptide linkers. In some embodiments, antibody fragments are fragments that are not produced by enzymatic digestion of naturally occurring intact antibodies. In some embodiments, the antibody fragment is an scFv.

[0401] A "humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from non-human FRs. A humanized antibody may also contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody refers to a variant of a non-human antibody that retains the specificity and affinity of the parent non-human antibody but has typically been humanized to reduce immunogenicity to humans. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0402] In some embodiments, the scFv is a V derived from an antibody or antigen fragment specific for CD19. H and V LContains: In some embodiments, the extracellular binding domain of the CD19 CAR is selected from the group consisting of, for example, SJ25C1 [Bejcek et al., Cancer Res. 55:2346-2351 (1995)], HD37 [Pezutto et al., J. Immunol. 138(9):2793-2799 (1987)], 4G7 [Meeker et al., Hybridoma 3:305-320 (1984)], B43 [Bejcek (1995)], BLY3 [Bejcek (1995)], B4 [Freedman et al., 70:418-427 (1987)], B4 HB12b [Kansas & Tedder, J. Immunol. 147:4094-4102 (1991); Yazawa et al., Proc. Natl. Acad. Sci. USA 102:15178-15183 (2005); Herbst et al., J. Pharmacol. Exp. Ther. 335:213-222 (2010)], BU12 [Callard et al., J. Immunology, 148(10): 2983-2987 (1992)], and CLB-CD19 [De Rie Cell. Immunol. 118:368-381(1989)]. In any of these embodiments, the extracellular binding domain of the CD19 CAR is derived from an antibody specific for CD19, including the V H , V L And / or may comprise or consist of one or more CDRs.In some embodiments, the antibody or antibody fragment that binds to CD19 is a mouse-derived antibody, such as FMC63 and SJ25C1.In some embodiments, the antibody or antibody fragment is a human antibody, for example, as described in US Patent Application Publication No. 2016 / 0152723.

[0403] In some embodiments, the antigen binding domain is a V derived from FMC63, which in some aspects may be an scFv. H and / or V LFMC63 generally refers to a murine monoclonal IgG1 antibody raised against Nalm-1 and -16 cells expressing CD19 of human origin [Ling, NR, et al. (1987). Leucocyte typing III. 302]. In some embodiments, the FMC63 antibody comprises CDR-H1 and CDR-H2 set forth in SEQ ID NOs: 38 and 39, respectively, and CDR-H3 set forth in SEQ ID NO: 40 or 54, and CDR-L1 set forth in SEQ ID NO: 35, and CDR-L2 set forth in SEQ ID NO: 36 or 55, and CDR-L3 set forth in SEQ ID NO: 37 or 56. In some embodiments, the FMC63 antibody comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 41. H ), and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 L ) is included.

[0404] In some embodiments, the scFv comprises a variable light chain containing the CDR-L1 sequence of SEQ ID NO: 35, the CDR-L2 sequence of SEQ ID NO: 36, and the CDR-L3 sequence of SEQ ID NO: 37, and / or a variable heavy chain containing the CDR-H1 sequence of SEQ ID NO: 38, the CDR-H2 sequence of SEQ ID NO: 39, and the CDR-H3 sequence of SEQ ID NO: 40, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the scFv comprises the variable heavy chain region of FMC63 set forth in SEQ ID NO: 41 and the variable light chain region of FMC63 set forth in SEQ ID NO: 42, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0405] In some embodiments, the FMC63 antibody comprises CDR-H1 and CDR-H2 sequences set forth in SEQ ID NOs: 38 and 39, respectively, and CDR-H3 sequences set forth in SEQ ID NO: 40 or 54, and CDR-L1 sequences set forth in SEQ ID NO: 35, and CDR-L2 sequences set forth in SEQ ID NO: 36 or 55, and CDR-L3 sequences set forth in SEQ ID NO: 37 or 56. In some embodiments, the FMC63 antibody comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 41. H ), and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 L In some embodiments, the scFv comprises a variable light chain containing the CDR-L1 sequence of SEQ ID NO: 35, the CDR-L2 sequence of SEQ ID NO: 36, and the CDR-L3 sequence of SEQ ID NO: 37, and / or a variable heavy chain containing the CDR-H1 sequence of SEQ ID NO: 38, the CDR-H2 sequence of SEQ ID NO: 39, and the CDR-H3 sequence of SEQ ID NO: 40, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the scFv comprises the variable heavy chain region of FMC63 set forth in SEQ ID NO: 41 and the variable light chain region of FMC63 set forth in SEQ ID NO: 42, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0406] In some embodiments, the variable heavy chain and the variable light chain are connected by a linker. In some embodiments, the linker is set forth in SEQ ID NO: 24. In some embodiments, the scFv comprises, in order, V H , linker, and V L In some embodiments, the scFv comprises, in order, V L , linker, and V HIn some embodiments, the scFv is encoded by the sequence of nucleotides set forth in SEQ ID NO: 25, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 25. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 43, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 43.

[0407] In some embodiments, the antigen binding domain is a VFv derived from SJ25C1, which in some aspects may be an scFv. H and / or V L SJ25C1 is a murine monoclonal IgG1 antibody raised against Nalm-1 and -16 cells expressing CD19 of human origin [Ling, NR, et al. (1987). Leucocyte typing III. 302]. In some embodiments, the SJ25C1 antibody comprises CDR-H1, CDR-H2, and CDR-H3 set forth in SEQ ID NOs: 47 to 49, respectively, and CDR-L1, CDR-L2, and CDR-L3 set forth in SEQ ID NOs: 44 to 46, respectively. In some embodiments, the SJ25C1 antibody comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 50. H ), and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 51 LIn some embodiments, the svFv comprises a variable light chain containing the CDR-L1 sequence of SEQ ID NO: 44, the CDR-L2 sequence of SEQ ID NO: 45, and the CDR-L3 sequence of SEQ ID NO: 46, and / or a variable heavy chain containing the CDR-H1 sequence of SEQ ID NO: 47, the CDR-H2 sequence of SEQ ID NO: 48, and the CDR-H3 sequence of SEQ ID NO: 49, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the scFv comprises the variable heavy chain region of SJ25C1 set forth in SEQ ID NO:50 and the variable light chain region of SJ25C1 set forth in SEQ ID NO:51, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the variable heavy chain and variable light chain are connected by a linker. In some embodiments, the linker is set forth in SEQ ID NO:52. In some embodiments, the scFv comprises, in order, V H , linker, and V L In some embodiments, the scFv comprises, in order, V L , linker, and V H In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO:53, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:53.

[0408] In some embodiments, the linker is set forth in SEQ ID NO: 23. In some embodiments, the linker has the formula -PGGG-(SGGGG)5-P-, where P is proline, G is glycine, and S is serine, and the linker is set forth in SEQ ID NO: 22.

[0409] In some embodiments, the anti-CD19 CAR comprises an antigen binding domain described in PCT Publication No. WO2015187528. In some embodiments, the anti-CD19 CAR is a CAR described in PCT Publication No. WO2015187528.

[0410] In some embodiments, anti-CD19 CAR comprises an antigen binding domain that is a single-chain antibody derived from a fully human antibody.In some embodiments, the single-chain antibody is an scFv.Exemplary fully human anti-CD19 antibodies are described in PCT Publication No. WO2016033570, PCT Publication No. WO2020233589, US Patent Application Publication No. 2010 / 0104509 and US Patent Application Publication No. 20220220200.

[0411] Exemplary antigen receptors, e.g., CARs, also include the CARs of the FDA-approved products BREYANZI® (lisocabtagene maraleucel), TECARTUS™ (brexucabtagene autoleucel), KYMRIAH™ (tisagenlecleucel), and YESCARTA™ (axicabtagene ciloleucel). In some of any of the provided embodiments, the CAR is a CAR of BREYANZI® (lisocabtagene maraleucel), TECARTUS™ (brexucabtagene autoleucel), KYMRIAH™ (tisagenlecleucel), or YESCARTA™ (axicabtagene ciloleucel). In some of any of the provided embodiments, the CAR is a CAR of BREYANZI® (lisocabtagene maraleucel, see: Sehgal et al., 2020, Journal of Clinical Oncology 38:15_suppl, 8040; Teoh et al., 2019, Blood 134(Supplement_1):593; and Abramson et al., 2020, The Lancet 396(10254):839-852). In some of any of the provided embodiments, the CAR is a CAR of TECARTUS™ (brexocabtagene maraleucel, see: Mian and Hill, 2021, Expert Opin Biol Ther; 21(4):435-441; and Wang et al., 2021, Blood 138(Supplement 1):744).In some of any of the provided embodiments, the CAR is a CAR of KYMRIAH™ (tisagenlecleucel, see: Bishop et al., 2022, N Engl J Med 386:629:639; Schuster et al., 2019, N Engl J Med 380:45-56; Halford et al., 2021, Ann Pharmacother 55(4):466-479; Mueller et al., 2021, Blood Adv. 5(23):4980-4991; and Fowler et al., 2022, Nature Medicine 28:325-332). In some of any of the provided embodiments, the CAR is YESCARTA™ (axicabtagene siloleucel, see: Neelapu et al., 2017, N Engl J Med 377(26):2531-2544; Jacobson et al., 2021, The Lancet 23(1):P91-103; and Locke et al., 2022, N Engl J Med 386:640-654).

[0412] In some embodiments, the recombinant receptor, e.g., a chimeric antigen receptor, comprises one or more antigen-binding domains, e.g., antibodies or fragments thereof, and one or more intracellular signaling regions or domains (synonymously referred to as cytoplasmic signaling domains or regions). In some embodiments, the recombinant receptor, e.g., a CAR, further comprises a spacer and / or a transmembrane domain or portion. In some embodiments, the spacer and / or transmembrane domain can link the extracellular portion containing the antigen-binding domain with the intracellular signaling region or domain.

[0413] In some embodiments, the recombinant receptor, e.g., CAR, further comprises a spacer, which may include a hinge domain. In some embodiments, the spacer is a CD8α hinge domain, e.g., a human CD8α hinge domain. In some embodiments, the CD8α hinge domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 93. In some embodiments, the hinge domain comprises a CD28 hinge domain, e.g., a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 94. In some embodiments, the CD28 hinge domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 95. In some embodiments, the hinge domain has an amino acid sequence having at least 80% sequence identity, e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, to any of the foregoing.

[0414] In some embodiments, the spacer comprises at least a portion of an immunoglobulin constant region or a mutant or modified version thereof, such as a hinge region, e.g., an IgG4 hinge region, and / or a C H 1 / C Land / or Fc region. In some embodiments, the recombinant receptor further comprises a spacer and / or hinge region. In some embodiments, the constant region or portion is a constant region or portion of a human IgG, such as IgG4 or IgG1. In some aspects, the portion of the constant region functions as a spacer region between the antigen recognition component, e.g., scFv, and the transmembrane domain. The spacer can be of a length that results in increased cellular responsiveness after antigen binding compared to the absence of the spacer. In some examples, the spacer is 12 or about 12 amino acids in length, or is 12 amino acids or less in length. Exemplary spacers include spacers having at least about 10-229 amino acids, about 10-200 amino acids, about 10-175 amino acids, about 10-150 amino acids, about 10-125 amino acids, about 10-100 amino acids, about 10-75 amino acids, about 10-50 amino acids, about 10-40 amino acids, about 10-30 amino acids, about 10-20 amino acids, or about 10-15 amino acids, including any integer between the endpoints of any of these recited ranges. In some embodiments, the spacer region has no more than about 12 amino acids, no more than about 119 amino acids, or no more than about 229 amino acids. Exemplary spacers include an IgG4 hinge alone, an IgG4 hinge linked to the CH2 and CH3 domains, or an IgG4 hinge linked to the CH3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, Hudecek et al. (2015) Cancer Immunol Res. 3(2): 125-135, or International Patent Application Publication No. WO2014031687.

[0415] In some embodiments, the spacer, e.g., the hinge-only spacer shown in SEQ ID NO: 1 and encoded by the sequence shown in SEQ ID NO: 2, contains only the hinge region of an IgG, e.g., only the hinge of an IgG4 or IgG1.H 2 and / or C H In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to the C3 domain. H 2 and C H In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, e.g., as set forth in SEQ ID NO: 4, linked to the 3 domains. H an Ig hinge linked to only three domains, e.g., an IgG4 hinge, such as that set forth in SEQ ID NO: 3. In some embodiments, the spacer is or includes a glycine-serine rich sequence or other flexible linker, e.g., a known flexible linker. In some embodiments, the constant region or portion is an IgD constant region or portion. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 5. In some embodiments, the spacer has a sequence of amino acids that exhibits at least, or at least about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 1, 3, 4, and 5.

[0416] In some embodiments, the spacer is (a) a polypeptide spacer comprising or consisting of all or a portion of an immunoglobulin hinge or a modified version thereof, or comprising about 15 amino acids or less and not including the CD28 or CD8 extracellular region; (b) a polypeptide spacer comprising or consisting of all or a portion of an immunoglobulin hinge, optionally an IgG4 hinge, or a modified version thereof, and / or comprising about 15 amino acids or less and not including the CD28 or CD8 extracellular region; or (c) a polypeptide spacer that is 12 or about 12 amino acids in length and / or not including all or a portion of an immunoglobulin hinge, optionally an IgG4 hinge, or a modified version thereof. or (d) a polypeptide spacer comprising or consisting of the sequence of amino acids set forth in SEQ ID NO: 1, 3-5, 27-34 or 24, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto; or (e) a polypeptide spacer comprising or consisting of the formula X1PPX2P set forth in SEQ ID NO: 26, where X1 is glycine, cysteine ​​or arginine and X2 is cysteine ​​or threonine.

[0417] In some embodiments, an antigen receptor comprises an intracellular domain directly or indirectly linked to an extracellular domain. In some embodiments, a chimeric antigen receptor comprises a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises an ITAM. For example, in some aspects, an antigen recognition domain (e.g., an extracellular domain) is generally linked to one or more intracellular signaling components, e.g., a signaling component that mimics activation by an antigen receptor complex, such as a TCR complex in the case of a CAR, and / or signals through another cell surface receptor. In some embodiments, a chimeric antigen receptor comprises a transmembrane domain linked or fused between the extracellular domain (e.g., an scFv) and the intracellular signaling domain. Thus, in some embodiments, an antigen binding entity (e.g., an antibody) is linked to one or more transmembrane and intracellular signaling domains.

[0418] In one embodiment, a transmembrane domain that is naturally associated with one of the domains in a receptor, such as a CAR, is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to prevent the domain from binding to the transmembrane domain of the same or a different surface membrane protein, in order to minimize interaction with other members of the receptor complex.

[0419] In some embodiments, the transmembrane domain is derived from either natural or synthetic sources. If the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. The transmembrane region includes (i.e., includes at least) the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 (4-1BB), or CD154. Alternatively, the transmembrane domain in some embodiments is synthetic. In some aspects, synthetic transmembrane domains primarily comprise hydrophobic residues, e.g., leucine and valine. In some aspects, triplets of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain. In some embodiments, the linkage is via a linker, spacer, and / or transmembrane domain. In some embodiments, the transmembrane domain comprises the transmembrane portion of CD28 or a variant thereof. The extracellular domain and the transmembrane domain may be directly or indirectly linked. In some embodiments, the extracellular domain and the transmembrane domain are linked by a spacer, such as any of those described herein.

[0420] In some embodiments, the transmembrane domain is a transmembrane domain of human CD28 or a variant thereof, e.g., the 27 amino acid transmembrane domain of human CD28 (Accession No. P10747.1). In some embodiments, the transmembrane domain is a transmembrane domain comprising the sequence of amino acids set forth in SEQ ID NO:8, or a sequence of amino acids that exhibits at least, or at least about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8. In some embodiments, the transmembrane domain-containing portion of the recombinant receptor comprises the sequence of amino acids set forth in SEQ ID NO:9, or a sequence of amino acids having at least, or at least about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0421] In some embodiments, the transmembrane domain is a transmembrane domain of human CD8α. In some embodiments, the transmembrane domain is a transmembrane domain comprising the sequence of amino acids set forth in SEQ ID NO:96, or a sequence of amino acids that exhibits at least, or at least about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:96.

[0422] In some embodiments, the recombinant receptor, e.g., a CAR, comprises at least one intracellular signaling component, e.g., an intracellular signaling region or domain. T cell activation is described in some embodiments as mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation by the TCR (primary cytoplasmic signaling sequences) and those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). In some embodiments, a CAR comprises one or both of such signaling components. Some intracellular signaling sequences mimic or approximate signals from natural antigen receptors, signals from such receptors in combination with costimulatory receptors, and / or signals from costimulatory receptors alone. In some embodiments, a short oligo- or polypeptide linker, e.g., a linker 2-10 amino acids in length, e.g., one containing glycine and serine, e.g., a glycine-serine doublet, is present between the transmembrane domain and the cytoplasmic signaling domain of the CAR, forming a link between these domains.

[0423] In some embodiments, upon ligation of the CAR, the cytoplasmic domain or intracellular signaling region of the CAR activates at least one of the normal effector functions or responses of immune cells, e.g., T cells, engineered to express the CAR. For example, in some situations, the CAR induces T cell function, e.g., cytolytic activity or T-helper activity, e.g., secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling region of an antigen receptor component or costimulatory molecule is used in place of an intact immune activation chain, e.g., if it transmits an effector function signal. In some embodiments, the intracellular signaling region, e.g., the intracellular signaling region comprising the intracellular domain, comprises the cytoplasmic sequence of a T cell receptor (TCR), and in some aspects also comprises the cytoplasmic sequence of a co-receptor that, in a natural context, acts in concert with such receptor to initiate signal transduction after antigen receptor engagement, and / or any derivative or variant of such molecule, and / or any synthetic sequence having the same function. In some embodiments, the intracellular signaling region, e.g., the intracellular domain, comprises the cytoplasmic sequence of a region or domain involved in providing a costimulatory signal.

[0424] In some embodiments, CAR comprises a primary cytoplasmic signaling sequence that regulates the primary activation of TCR complex.The primary cytoplasmic signaling sequence that acts to stimulate can contain a signaling motif known as immunoreceptor tyrosine-based activation motif or ITAM.Examples of ITAM that contain primary cytoplasmic signaling sequence include those derived from CD3 zeta chain, FcR gamma, CD3 gamma, CD3 delta and CD3 epsilon.In some embodiments, the cytoplasmic signaling molecule in CAR contains a cytoplasmic signaling domain, a part thereof, or a sequence derived from CD3 zeta.

[0425] In some embodiments, the receptor comprises an intracellular component of the TCR complex, e.g., a TCR CD3 chain, e.g., CD3 zeta chain, which mediates T cell activation and cytotoxicity. Thus, in some embodiments, the antigen-binding portion is linked to one or more cell signaling modules. In some embodiments, the cell signaling module comprises a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. In some embodiments, the receptor further comprises a portion of one or more additional molecules, e.g., Fc receptor gamma, CD8 alpha, CD8 beta, CD4, CD25, or CD16. For example, in some embodiments, a CAR or other chimeric receptor comprises a chimeric molecule of CD3-zeta (CD3-ζ) or Fc receptor gamma with CD8 alpha, CD8 beta, CD4, CD25, or CD16.

[0426] In some embodiments, the intracellular (or cytoplasmic) signaling region comprises a human CD3 chain, optionally a CD3 zeta stimulatory signaling domain or a functional variant thereof, such as the 112 AA cytoplasmic domain of isoform 3 of human CD3ζ (Accession No. P20963.2), or a CD3 zeta signaling domain as described in U.S. Pat. No. 7,446,190 or U.S. Pat. No. 8,911,993. In some embodiments, the intracellular signaling region comprises the sequence of amino acids set forth in SEQ ID NO: 13, 14, or 15, or a sequence of amino acids that exhibits at least, or at least about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13, 14, or 15. In some embodiments, the CD3 zeta signaling domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the CD3 zeta signaling domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the CD3 zeta signaling domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15.

[0427] For natural TCR, full activation generally requires not only TCR signal transduction but also costimulatory signal.Therefore, in some embodiments, CAR also comprises the component for generating secondary or costimulatory signal to promote full activation.In other embodiments, CAR does not comprise the component for generating costimulatory signal.In some embodiments, additional CAR is expressed in the same cell to provide the component for generating secondary or costimulatory signal.

[0428] In some embodiments, the chimeric antigen receptor contains the intracellular domain of a T cell costimulatory molecule. In some embodiments, the CAR comprises the signaling domain and / or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, OX40 (CD134), CD27, DAP10, DAP12, ICOS, and / or other costimulatory receptors. In some embodiments, the CAR comprises the costimulatory region or domain of CD28 or 4-1BB, for example, human CD28 or human 4-1BB.

[0429] In some embodiments, the intracellular signaling region or domain comprises the intracellular costimulatory signaling domain of human CD28 or a functional variant or portion thereof, e.g., the 41 amino acid domain thereof, and / or such a domain having an LL to GG substitution at positions 186-187 of the native CD28 protein. In some embodiments, the intracellular signaling domain may comprise the sequence of amino acids set forth in SEQ ID NO: 10 or 11, or a sequence of amino acids that exhibits at least, or at least about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10 or 11. In some embodiments, the intracellular region comprises the intracellular costimulatory signaling domain of 4-1BB or a functional variant or portion thereof, e.g., the 42 amino acid cytoplasmic domain of human 4-1BB (e.g., Accession No. Q07011.1), or a functional variant or portion thereof, e.g., the sequence of amino acids set forth in SEQ ID NO: 12, or a sequence of amino acids exhibiting at least, or at least about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12.

[0430] In some embodiments, the same CAR comprises both a primary (or activating) cytoplasmic signaling region and a costimulatory signaling component.

[0431] In some embodiments, the activation domain is contained in one CAR, but the costimulatory component is provided by another CAR that recognizes a different antigen. In some embodiments, the CAR comprises an activating or stimulatory CAR and a costimulatory CAR, both expressed on the same cell (see WO2014 / 055668). In some aspects, the cell comprises one or more stimulatory or activating CARs and / or costimulatory CARs. In some embodiments, the cell further comprises an inhibitory CAR [iCAR, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013)], e.g., a CAR that recognizes an antigen other than that associated with and / or specific for the disease or condition, such that binding of the inhibitory CAR to its ligand reduces or inhibits the activation signal delivered by the disease-targeting CAR, e.g., reducing off-target effects.

[0432] In some embodiments, the two receptors induce activation signals and inhibitory signals to cells, respectively; thus, the ligation of one of the receptors to the antigen activates the cell or induces a response, while the ligation of the second inhibitory receptor to the antigen induces a signal that suppresses or weakens the response.An example is the combination of activating CAR and inhibitory CAR (iCAR).This strategy is used, for example, in situations where activating CAR binds to an antigen that is expressed during disease or condition but is also expressed on normal cells, and inhibitory receptor binds to a separate antigen that is expressed on normal cells but not on disease or condition cells, to reduce the possibility of off-target effects.

[0433] In some embodiments, the chimeric receptor is or comprises an inhibitory CAR (e.g., iCAR), which comprises an intracellular component that dampens or suppresses an immune response in a cell, e.g., an ITAM-promoted response and / or a costimulatory-promoted response. Exemplary of such intracellular signaling components are those found on immune checkpoint molecules, including PD-1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptor, and EP2 / 4 adenosine receptors, including A2AR. In some embodiments, the engineered cell comprises an inhibitory CAR that comprises such an inhibitory molecule or a signaling domain derived therefrom, such that the inhibitory CAR serves to dampen a cellular response, e.g., a response induced by an activation and / or costimulatory CAR.

[0434] In some cases, CARs are referred to as first-, second-, and / or third-generation CARs. In some embodiments, first-generation CARs provide only a CD3 chain-inducing signal upon antigen binding; in some embodiments, second-generation CARs provide such a signal and a costimulatory signal, for example, include an intracellular signaling domain from a costimulatory receptor such as CD28 or CD137; in some embodiments, third-generation CARs in some embodiments include multiple costimulatory domains from different costimulatory receptors.

[0435] In some embodiments, the CAR comprises one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., a primary activation domain, in the cytoplasmic portion. Exemplary CARs include the intracellular components of CD3-zeta, CD28, and 4-1BB.

[0436] In some embodiments, the antigen receptor further comprises a marker, and / or cells expressing a CAR or other antigen receptor further comprise a surrogate marker, e.g., a cell surface marker, which can be used to confirm transduction and manipulation of the cells to express the receptor. In some embodiments, the marker comprises all or a portion (e.g., a truncated form) of CD34, NGFR, or epidermal growth factor receptor, e.g., a truncated version of such a cell surface receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a linker sequence, e.g., a cleavable linker sequence, e.g., a polynucleotide encoding T2A. For example, the marker, and optionally the linker sequence, can be any of those disclosed in published patent application WO2014031687. For example, the marker can be a truncated EGFR (tEGFR), optionally linked to a linker sequence such as a T2A cleavable linker sequence.

[0437] Exemplary polypeptides of truncated EGFR (e.g., tEGFR) include the sequence of amino acids set forth in SEQ ID NO: 7 or 16, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7 or 16. Exemplary T2A linker sequences include the sequence of amino acids set forth in SEQ ID NO: 6 or 17, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 6 or 17.

[0438] In some embodiments, the marker is a molecule that is not naturally found on or at the surface of T cells, e.g., a cell surface protein, or portion thereof. In some embodiments, the molecule is a non-self molecule, e.g., a non-self protein, i.e., one that is not recognized as "self" by the immune system of the host into which the cells are adoptively transferred.

[0439] In some embodiments, the marker does not serve a therapeutic function and / or produce no effect other than being used as a marker for genetic manipulation, e.g., to select successfully manipulated cells. In other embodiments, the marker may be a therapeutic molecule or a molecule that otherwise exerts some desired effect, e.g., a ligand that the cell encounters in vivo, e.g., a costimulatory or immune checkpoint molecule that enhances and / or attenuates the response of the cell upon adoptive transfer and encounter with the ligand.

[0440] In some embodiments, the chimeric antigen receptor comprises an extracellular portion containing an antibody or fragment described herein. In some aspects, the chimeric antigen receptor comprises an extracellular portion containing an antibody or fragment described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment is an scFv or single domain V H The chimeric antigen receptor comprises an antibody, wherein the intracellular domain contains an ITAM. In some embodiments, the intracellular signaling domain comprises the signaling domain of the zeta chain of the CD3-zeta (CD3ζ) chain. In some embodiments, the CD3-zeta chain is a human CD3-zeta chain. In some embodiments, the intracellular signaling region further comprises a CD28 and CD137 (4-1BB, TNFRSF9) costimulatory domain linked to the CD3 zeta intracellular domain. In some embodiments, the CD28 is human CD28. In some embodiments, the 4-1BB is human 4-1BB. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain disposed between the extracellular domain and the intracellular signaling domain. In some embodiments, the transmembrane domain comprises the transmembrane portion of CD28. The extracellular domain and the transmembrane domain may be directly or indirectly linked. In some embodiments, the extracellular domain and the transmembrane domain are linked by a spacer, such as any described herein.

[0441] In some embodiments, the CAR comprises an antibody, e.g., an antibody fragment; a transmembrane domain that is or contains the transmembrane portion of CD28 or a functional variant thereof; and an intracellular signaling domain that contains the signaling portion of CD28 or a functional variant thereof and the signaling portion of CD3-zeta or a functional variant thereof. For example, in some embodiments, the CAR comprises an antibody, e.g., an antibody fragment, including an scFv, specific for CD19, e.g., any of those described above; a spacer, e.g., a spacer containing a portion of an immunoglobulin molecule, e.g., a hinge region and / or one or more constant regions of a heavy chain molecule, e.g., an Ig-hinge-containing spacer; and a transmembrane domain containing all or a portion of a transmembrane domain derived from CD28, an intracellular signaling domain derived from CD28, and a CD3-zeta signaling domain.

[0442] In some embodiments, the CAR comprises an antibody, e.g., an antibody fragment; a transmembrane domain that is or contains the transmembrane portion of CD28 or a functional variant thereof; and an intracellular signaling domain that contains the signaling portion of 4-1BB or a functional variant thereof and the signaling portion of CD3-zeta or a functional variant thereof. In some such embodiments, the receptor further comprises a spacer, e.g., a hinge-only spacer, containing a portion of an Ig molecule, such as a human Ig molecule, e.g., an Ig hinge, such as an IgG4 hinge. In some embodiments, the CAR comprises an antibody or fragment, e.g., an scFv, specific for CD19, e.g., any of the above; a spacer, e.g., any of the Ig-hinge-containing spacers; a transmembrane domain derived from CD28; an intracellular signaling domain derived from 4-1BB; and a signaling domain derived from CD3-zeta.

[0443] In certain embodiments, the CAR is a CD19-directed CAR containing an scFv antigen-binding domain from FMC63; an immunoglobulin hinge spacer; a transmembrane domain; and an intracellular signaling domain containing a costimulatory signaling region that is the signaling domain of 4-1BB and the signaling domain of the CD3-zeta (CD3ζ) chain. In some embodiments, the scFv has the sequence set forth in SEQ ID NO: 43. In some embodiments, the scFv has a VL having CDRs with the amino acid sequence RASQDISKYLN (SEQ ID NO: 35), the amino acid sequence of SRLHSGV (SEQ ID NO: 36), and the amino acid sequence of GNTLPYTFG (SEQ ID NO: 37); and a VH having CDRs with the amino acid sequence DYGVS (SEQ ID NO: 38), the amino acid sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39), and YAMDYWG (SEQ ID NO: 40). In some embodiments, the transmembrane domain has the sequence set forth in SEQ ID NO: 8. In some embodiments, the transmembrane domain has a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 8. In some embodiments, the 4-1BB costimulatory signaling domain has a sequence set forth in SEQ ID NO: 12. In some embodiments, the 4-1BB costimulatory signaling domain has a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12. In some embodiments, the CD3-zeta domain has the sequence set forth in SEQ ID NO: 13. In some embodiments, the CD3 zeta domain has a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the CAR contains a hinge-containing immunoglobulin spacer between the scFv and the transmembrane domain. In some embodiments, the spacer is set forth in SEQ ID NO: 1.

[0444] In particular embodiments of any of the provided methods, the CAR contains, in order from N-terminus to C-terminus, an extracellular antigen-binding domain that is an scFv set forth in SEQ ID NO: 43, a spacer set forth in SEQ ID NO: 1, a transmembrane domain set forth in SEQ ID NO: 8, a 4-1BB costimulatory signaling domain set forth in SEQ ID NO: 12, and a signaling domain of the CD3-zeta (CD3ζ) chain set forth in SEQ ID NO: 13.

[0445] In some embodiments, the CAR has a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 91. In some embodiments, the CAR comprises the sequence set forth in SEQ ID NO: 91. In some embodiments, the CAR is set forth in SEQ ID NO: 91. In some embodiments, the CAR is a CD19 CAR such as is present in lysocavtagenemaraleucel.

[0446] In some embodiments, the CAR is encoded by a sequence of nucleotides having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 92. In some embodiments, the CAR is encoded by a sequence of nucleotides set forth in SEQ ID NO:92.

[0447] In some embodiments, the CAR contains, in order from N-terminus to C-terminus, an extracellular antigen-binding domain that is an scFv comprising the variable heavy chain region of FMC63 set forth in SEQ ID NO: 41 and the variable light chain region of FMC63 set forth in SEQ ID NO: 42, such as the scFv set forth in SEQ ID NO: 43; a CD8α hinge domain of SEQ ID NO: 93; a CD8α transmembrane domain of SEQ ID NO: 96; a 4-1BB costimulatory domain of SEQ ID NO: 12; and a CD3ζ signaling domain of SEQ ID NO: 13. In some embodiments, the CAR has a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of the above sequences. In some embodiments, the CAR has a sequence of amino acids with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 97. In some embodiments, the CAR has the sequence set forth in SEQ ID NO: 97. In some embodiments, the CAR is a CD19 CAR as present in tisagenlecleucel.

[0448] In some embodiments, the CAR contains, in order from N-terminus to C-terminus, an extracellular antigen-binding domain that is an scFv comprising the variable heavy chain region of FMC63 set forth in SEQ ID NO: 41 and the variable light chain region of FMC63 set forth in SEQ ID NO: 42, such as the scFv set forth in SEQ ID NO: 43; a CD28 hinge domain of SEQ ID NO: 94; a CD28 transmembrane domain of SEQ ID NO: 8 or 9; a CD28 costimulatory domain of SEQ ID NO: 10; and a CD3ζ signaling domain of SEQ ID NO: 13. In some embodiments, the CAR has a sequence of amino acids having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 98. In some embodiments, the CAR has the sequence set forth in SEQ ID NO: 98. In some embodiments, the CAR is a CD19 CAR such as that present in axicabtagenecilloeucel.

[0449] In some embodiments, the CAR contains an extracellular binding domain composed of an scFv derived from the anti-CD19 antibody known as Hu19. In some embodiments, the CAR contains an scFv derived from Hu19, a CD8a hinge and transmembrane domain (e.g., SEQ ID NO: 111), a CD28 costimulatory domain (e.g., SEQ ID NO: 10), and a CD3ζ signaling domain (e.g., SEQ ID NO: 13). In some embodiments, the scFv designated Hu19 contains a light chain variable region (SEQ ID NO: 112), a linker peptide [GSTSGSGKPGSGEGSTKG (SEQ ID NO: 113)], and a heavy chain variable region (SEQ ID NO: 114). The scFv may also include a human CD8α leader sequence (SEQ ID NO: 115). In some embodiments, the CAR has the sequence set forth in SEQ ID NO: 116. In some embodiments, the CAR has the sequence set forth in SEQ ID NO: 117. In some embodiments, the CAR cont...

Claims

1. 1. A method of treating a subject having a systemic autoimmune disease, comprising administering to a subject having or suspected of having a severe systemic autoimmune disease a dose of CD19-directed genetically modified T cells from a composition comprising engineered T cells that express a chimeric antigen receptor (CAR), wherein said dose of T cells is positive for expression of a CAR that binds to CD19, and wherein said dose is greater than or equal to 1 x 10 ６ ~50 x 10 ６ The method of claim 1, wherein the CAR-positive viable T cells are

2. 1. A method of treating a subject having a systemic autoimmune disease, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having a moderate systemic autoimmune disease, wherein said T cells in said dose are positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and wherein said dose is greater than or equal to 1 x 10 ６ ~50 x 10 ６ The method of claim 1, wherein the CAR-positive viable T cells are

3. The systemic autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), Sjogren's syndrome, progressive systemic sclerosis (i.e., scleroderma); idiopathic inflammatory myositis (IIM), including dermatomyositis, polymyositis, and necrotizing myositis; mixed connective tissue disorder (MCTD), highly active relapsing-remitting multiple sclerosis, primary progressive MS, ANCA-associated vasculitis (AAV), Crohn's disease, myasthenia gravis, Behcet's disease, rheumatoid arthritis, IgA nephropathy, pemphigus vulgaris, myasthenia gravis, autoimmune hemolytic anemia, immune thrombocytopenia, IgG4-related disease, membranous nephropathy, cutaneous lupus erythematosus, sarcoidosis, and light chain amyloid.

3. The method of claim 1 or claim 2, wherein the inflammatory bowel disease is selected from the group consisting of eczema, acute respiratory distress syndrome, atopic eczema, hereditary angioedema, hidradenitis suppurativa, inclusion body myositis, inflammatory bowel disease, mastocytosis, multifocal motor neuropathy, necrotizing myopathy, neuromyelitis optica spectrum disorder, mixed connective tissue disorder, POEMS syndrome, primary biliary cholangitis, psoriasis, rhesus hemolytic disease, Still's disease, type 1 diabetes, urticaria, capillary leak syndrome, cytokine release syndrome, erythema multiforme, pyoderma gangrenosum, X-linked agammaglobulinemia, antiphospholipid antibody syndrome, and chronic inflammatory demyelinating polyneuropathy.

4. The method according to any one of claims 1 to 3, wherein the systemic autoimmune disease is rheumatoid arthritis.

5. The method of any one of claims 1 to 3, wherein the systemic autoimmune disease is myositis.

6. 4. The method of claim 1, wherein the systemic autoimmune disease is myasthenia gravis.

7. 4. The method of claim 1, wherein the systemic autoimmune disease is bullous pemphigoid.

8. The method according to any one of claims 1 to 3, wherein the systemic autoimmune disease is immune thrombocytopenia.

9. The method according to any one of claims 1 to 3, wherein the systemic autoimmune disease is autoimmune hemolytic anemia.

10. The method according to any one of claims 1 to 3, wherein the systemic autoimmune disease is pemphigus vulgaris.

11. The method according to any one of claims 1 to 3, wherein the systemic autoimmune disease is demyelinating polyneuropathy.

12. The method according to any one of claims 1 to 3, wherein the systemic autoimmune disease is membranous nephropathy.

13. The method according to any one of claims 1 to 12, wherein the systemic autoimmune disease is an intractable disease.

14. 14. The method of any of claims 1 to 13, wherein the subject is refractory to treatment with one or more previous therapies for the systemic autoimmune disease.

15. 15. The method of any of claims 1-14, wherein the subject is refractory to treatment with two or more previous therapies for the systemic autoimmune disease.

16. 16. The method of any of claims 1 to 15, wherein the systemic autoimmune disease is a severe disease.

17. 1. A method of treating a subject with severe systemic lupus erythematosus (SLE), comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having severe systemic lupus erythematosus (SLE), wherein said T cells in said dose are positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and wherein said dose is greater than or equal to 1 x 10 ６ ~50 x 10 ６ The method of claim 1, wherein the CAR-positive viable T cells are

18. 1. A method for reducing systemic lupus erythematosus (SLE) disease activity, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having severe systemic lupus erythematosus (SLE), wherein said T cells in said dose are positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and wherein said dose is greater than or equal to 1 x 10 ６ ~50 x 10 ６ The method of claim 1, wherein the CAR-positive viable T cells are

19. 19. The method of claim 17 or claim 18, wherein the SLE in the subject has one or more of renal, central nervous system, or hematological complications.

20. 20. The method of any of claims 17-19, wherein the subject has at least one organ system categorized as British Isles Lupus Assessment Group 2004 ("BILAG") Category A ("BILAG A") or at least two organ systems categorized as BILAG B.

21. 21. The method of any of claims 17 to 20, wherein the subject meets the 2019 American College of Rheumatology (ACR) / European League Against Rheumatism (EULAR) classification criteria for SLE and / or the subject has detectable anti-dsDNA, anti-histone, anti-chromatin or anti-Sm antibodies in their blood.

22. 21. The method of any of claims 17 to 20, wherein the subject meets the 2019 American College of Rheumatology (ACR) / European League Against Rheumatism (EULAR) classification criteria for SLE.

23. 22. The method of any of claims 17 to 21, wherein the subject has detectable anti-dsDNA, anti-histone, anti-chromatin or anti-Sm antibodies in their blood.

24. 24. The method of any of claims 17 to 23, wherein the subject has lupus nephritis.

25. 1. A method of treating a subject with lupus nephritis, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having lupus nephritis, wherein said T cells in said dose are positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and wherein said dose is greater than or equal to 1 x 10 ６ ~50 x 10 ６ The method of claim 1, wherein the CAR-positive viable T cells are

26. 26. The method of any of claims 17-25, wherein the subject is refractory to treatment with one or more previous therapies for the lupus.

27. 27. The method of any of claims 17-26, wherein the subject has achieved an inadequate response to one or more previous treatments for the lupus.

28. 28. The method of claim 26 or claim 27, wherein the two or more previous treatments for lupus comprise a glucocorticoid, an antimalarial, an immunosuppressant, an anti-CD20 antibody, or an inhibitor of soluble B-lymphocyte stimulator (BLyS).

29. 29. The method of any of claims 26 to 28, wherein the two or more previous treatments are selected from any two or more of mycophenolate mofetil (MFF), cyclophosphamide (cyc), belimumab, rituximab, anifrolumab, azathioprine, methotrexate, cyclosporine (csp), or voclosporin.

30. 30. The method of any of claims 17-29, wherein the subject does not have drug-induced SLE, clinically significant CNS pathology, associated systemic autoimmune disease, and / or an SLE overlap syndrome.

31. 31. The method of claim 30, wherein the subject does not have an associated systemic autoimmune disease, including but not limited to multiple sclerosis, psoriasis, and inflammatory bowel disease.

32. 31. The method of claim 30, wherein the subject does not have an SLE overlap syndrome, including but not limited to rheumatoid arthritis, scleroderma, and mixed connective tissue disease.

33. 33. The method of any of claims 17 to 32, wherein the subject is at high risk of organ failure.

34. 18. The method of any of claims 1 to 17, wherein the method reduces the systemic autoimmune disease activity in the subject.

35. 35. The method of claim 34, wherein reducing disease activity in the subject comprises reducing inflammation in the subject.

36. 34. The method of any of claims 17 to 33, wherein the method reduces SLE disease activity in the subject.

37. 37. The method of any of claims 18-24 and 34-36, wherein reducing SLE disease activity in the subject comprises a BILAG-based Composite Lupus Assessment (BICLA) response in the subject, a reduction in the subject's Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI) score compared to the subject's CLASI score before treatment, a reduction in the subject's tender and swollen joint count compared to the subject's tender and swollen joint count before treatment, the subject having a BILAG-2004 B score of up to 1 after treatment, the subject having a BILAG-2004 score of C or higher after treatment, the subject having an improvement in at least one patient-reported outcome (PRO) compared to before treatment, and / or a reduction in the subject's SLE flare rate compared to the subject's flare rate before treatment.

38. 37. The method of any of claims 18-24 and 34-36, wherein reducing SLE disease activity in the subject comprises the subject achieving clinical remission as defined by the Definition of Remission in Systemic Lupus Erythematosus (DORIS) and / or the subject achieving a lupus low disease activity status (LLDAS).

39. 39. The method of any of claims 2-33 and 37-38, wherein the subject achieves clinical remission of the lupus within 3 months or within 6 months of administration of the dose of CD19-directed genetically modified T cells.

40. 40. The method of claim 38 or claim 39, wherein the clinical remission is maintained for at least about 6 months, at least about 12 months, at least about 24 months, at least about 3 years, at least about 4 years, or at least about 5 years.

41. 41. The method of any of claims 2-33 and 36-40, wherein the subject achieves long-term remission of the lupus.

42. 1. A method of treating a subject having idiopathic inflammatory myopathy (IIM), comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having idiopathic inflammatory myopathy (IIM), wherein said T cells in said dose are positive for expression of a chimeric antigen receptor (CAR) that binds CD19, and wherein said dose is greater than or equal to 1 x 10 ６ ~50 x 10 ６ The method of claim 1, wherein the CAR-positive viable T cells are

43. 1. A method for reducing idiopathic inflammatory myopathy (IIM) disease activity, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having idiopathic inflammatory myopathy (IIM), wherein said T cells in said dose are positive for expression of a chimeric antigen receptor (CAR) that binds CD19, and wherein said dose is greater than or equal to 1 x 10 ６ ~50 x 10 ６ The method of claim 1, wherein the CAR-positive viable T cells are

44. 44. The method of claim 42 or claim 43, wherein the subject is refractory to treatment with one or more prior therapies for the IIM.

45. 44. The method of claim 42 or claim 43, wherein the subject has achieved an inadequate response to one or more previous treatments for the IIM.

46. 1. A method of treating a subject with systemic sclerosis (SSc), comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having systemic sclerosis (SSc), wherein said T cells in said dose are positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and wherein said dose is greater than or equal to 1 x 10 ６ ~50 x 10 ６ The method of claim 1, wherein the CAR-positive viable T cells are

47. 1. A method for reducing systemic sclerosis (SSc) disease activity, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having systemic sclerosis (SSc), wherein said T cells in said dose are positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and wherein said dose is greater than or equal to 1 x 10 ６ ~50 x 10 ６ The method of claim 1, wherein the CAR-positive viable T cells are

48. 48. The method of claim 46 or claim 47, wherein the subject is refractory to treatment with one or more previous therapies for the SSc.

49. 48. The method of claim 46 or claim 47, wherein the subject has achieved an inadequate response to one or more previous treatments for the SSC.

50. 1. A method of treating a subject with multiple sclerosis (MS), comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having multiple sclerosis (MS), wherein said T cells in said dose are positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and wherein said dose is greater than or equal to 1 x 10 ６ ~50 x 10 ６ The method of claim 1, wherein the CAR-positive viable T cells are

51. 1. A method for reducing multiple sclerosis (MS) disease activity, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having multiple sclerosis (MS), wherein said T cells in said dose are positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and wherein said dose is greater than or equal to 1 x 10 ６ ~50 x 10 ６ The method of claim 1, wherein the CAR-positive viable T cells are

52. 52. The method of claim 50 or claim 51, wherein the subject is refractory to treatment with one or more previous therapies for the MS.

53. 52. The method of claim 50 or claim 51, wherein the subject has achieved an inadequate response to one or more previous treatments for the MS.

54. 54. The method of any of claims 50 to 53, wherein the subject has or is suspected of having relapsing MS.

55. 54. The method of any of claims 50 to 53, wherein the subject has or is suspected of having progressive MS.

56. 55. The method of any of claims 50-54, wherein the subject has or is suspected of having highly active relapsing-remitting MS.

57. 54. The method of any of claims 50 to 53, wherein the subject has or is suspected of having primary progressive MS.

58. 54. The method of any of claims 50 to 53, wherein the subject has or is suspected of having active secondary progressive MS (aSPMS).

59. 54. The method of any of claims 50 to 53, wherein the subject has or is suspected of having inactive secondary progressive MS (iSPMS).

60. 60. The method of any of claims 50-59, wherein the subject has an Expanded Disability Status Scale (EDSS) of ≧3.0 and ≦5.5, or ≧3.0 and ≦6.

0.

61. 61. The method of any of claims 50-60, wherein the subject is able to complete the 9-Hole Peg Test (9-HPT) for each hand in <240 seconds and the subject is able to perform the 25-foot Timed Walk Test (T25FWT) in <150 seconds.

62. 62. The method of any of claims 50-61, wherein the subject does not have MS lesions or symptoms that may increase their risk of neurotoxicity.

63. 63. The method of any of claims 39-62, wherein the method reduces the autoimmune disease activity in the subject.

64. 64. The method of any of claims 32-63, wherein reducing disease activity in the subject comprises reducing inflammation in the subject.

65. 64. The method of claim 63, wherein reducing the autoimmune disease activity in the subject comprises reducing the subject's IMACS score after treatment compared to the subject's IMACS score before treatment, alleviating the subject's skin lesions, muscle fatigue, and / or weakness compared to the subject's skin lesions, muscle fatigue, and / or weakness before treatment, or the subject having an improvement in at least one patient-reported outcome (PRO) compared to before treatment.

66. 65. The method of claim 64, wherein reducing the autoimmune disease activity in the subject comprises reducing the subject's Modified Rodnan Skin Score, European Scleroderma Study Group (EScSG) Index, Minimal Clinically Important Difference (MCID), Patient-Reported Short Form Quality of Life Assessment (SF-36) Physical Summary Score (PCS) and / or Mental Summary Score (MCS), or a combination thereof, or improving forced vital capacity.

67. 66. The method of claim 65, wherein reducing the autoimmune disease activity in the subject comprises improving the subject's score on any of the following tests: Expanded Disability Status Scale (EDSS), disease stage, Multiple Sclerosis Functional Composite (MSFC), Minimal Clinically Important Change (MCID), Patient-Reported Short Form Quality of Life (SF-36) Physical Summary Score (PCS) and / or Mental Summary Score (MCS), or a combination thereof.

68. 1. A method of treating a subject having autoimmune vasculitis (AAV), comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having autoimmune vasculitis (AAV), wherein said T cells in said dose are positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and wherein said dose is greater than or equal to 1 x 10 ６ ~50 x 10 ６ The method of claim 1, wherein the CAR-positive viable T cells are

69. 1. A method for reducing autoimmune vasculitis (AAV) disease activity, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having autoimmune vasculitis (AAV), wherein said T cells in said dose are positive for expression of a chimeric antigen receptor (CAR) that binds CD19, and wherein said dose is greater than or equal to 1 x 10 ６ ~50 x 10 ６ The method of claim 1, wherein the CAR-positive viable T cells are

70. 1. A method of treating a subject with IgA nephropathy, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having IgA nephropathy, wherein said T cells in said dose are positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and wherein said dose is greater than or equal to 1 x 10 ６ ~50 x 10 ６ The method of claim 1, wherein the CAR-positive viable T cells are

71. 1. A method for reducing IgA nephropathy disease activity, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having IgA nephropathy, wherein said T cells in said dose are positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and wherein said dose is greater than or equal to 1 x 10 ６ ~50 x 10 ６ The method of claim 1, wherein the CAR-positive viable T cells are

72. 1. A method of treating a subject with pemphigus vulgaris, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having pemphigus vulgaris, wherein said T cells in said dose are positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and wherein said dose is greater than or equal to 1 x 10 ６ ~50 x 10 ６ The method of claim 1, wherein the CAR-positive viable T cells are

73. 1. A method for reducing pemphigus vulgaris disease activity, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having pemphigus vulgaris, wherein said T cells in said dose are positive for expression of a chimeric antigen receptor (CAR) that binds CD19, and wherein said dose is greater than or equal to 1 x 10 ６ ~50 x 10 ６ The method of claim 1, wherein the CAR-positive viable T cells are

74. 1. A method of treating a subject having myasthenia gravis, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having myasthenia gravis, wherein said T cells in said dose are positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, said dose comprising 1 x 10 ６ ~50 x 10 ６ The method of claim 1, wherein the CAR-positive viable T cells are

75. 1. A method for reducing myasthenia gravis disease activity, comprising administering a dose of CD19-directed genetically modified T cells to a subject having or suspected of having myasthenia gravis, wherein said T cells in said dose are positive for expression of a chimeric antigen receptor (CAR) that binds CD19, said dose comprising 1 x 10 ６ ~50 x 10 ６ The method of claim 1, wherein the CAR-positive viable T cells are

76. The dose is 1 x 10 ６ ~40 x 10 ６ or about 1 x 10 ６ ~40 x 10 ６ 76. The method of any one of claims 1 to 75, wherein the CAR-positive live T cells are of the type described above.

77. The dose is 1 x 10 ６ ~25 x 10 ６ or about 1 x 10 ６ ~25 x 10 ６ 76. The method of any one of claims 1 to 75, wherein the CAR-positive live T cells are of the type described above.

78. The dose is 5 x 10 ６ or about 5 x 10 ６ 76. The method of any one of claims 1 to 75, wherein the CAR-positive live T cells are of the type described above.

79. The dose is 10 x 10 ６ or about 10 x 10 ６ 76. The method of any one of claims 1 to 75, wherein the CAR-positive live T cells are of the type described above.

80. The dose is 25 x 10 ６ or about 25 x 10 ６ 76. The method of any one of claims 1 to 75, wherein the CAR-positive live T cells are of the type described above.

81. The dose is 50 x 10 ６ or about 50 x 10 ６ 76. The method of any one of claims 1 to 75, wherein the CAR-positive live T cells are of the type described above.

82. 82. The method of any of claims 1-81, wherein the T cells are autologous to the subject.

83. 82. The method of any of claims 1-81, further comprising obtaining a leukapheresis sample from the subject to produce the composition comprising engineered T cells.

84. 84. The method of any of claims 1 to 83, wherein prior to said administering, said subject has undergone preconditioning with lymphodepleting therapy.

85. 85. The method of any of claims 1 to 84, further comprising administering lymphodepleting therapy to the subject immediately prior to said administration of said dose of CD19-directed genetically modified T cells, wherein said lymphodepleting therapy comprises administration of fludarabine and / or cyclophosphamide.

86. 86. The method of any of claims 1 to 85, wherein said dose of CD19-directed genetically modified T cells and / or said administration of said lymphodepletion therapy is performed by outpatient delivery.

87. said lymphodepletion therapy comprising administering to said subject a lymphocyte-depleting therapy to a patient of said subject on a daily basis over 1 m of body surface area of ​​said subject; ２ and 30 mg of fludarabine per m of body surface area of ​​said subject daily. ２ 87. The method of any of claims 84 to 86, comprising administration of 300 mg of cyclophosphamide per day for each of three days.

88. 87. The method of any of claims 84-86, wherein said dose of CD19-directed genetically modified T cells is administered between at or about 48 hours and at or about 9 days, inclusive, after completion of lymphodepletion therapy.

89. 89. The method of any of claims 1 to 88, wherein said dose of CD19-directed genetically modified T cells is administered to said subject by intravenous infusion.

90. 90. The method of any one of claims 1 to 89, wherein the CAR comprises an extracellular antigen-binding domain that binds to CD19, a transmembrane domain, and an intracellular signaling domain.

91. 91. The method of claim 90, wherein the CAR comprises a hinge spacer between the extracellular antigen-binding domain and the transmembrane domain, optionally wherein the hinge spacer is an immunoglobulin hinge or a CD8a hinge.

92. 92. The method of claim 90 or claim 91, wherein the extracellular antigen-binding domain is a single-chain variable fragment (scFv) derived from the FMC63 monoclonal antibody.

93. 93. The method of any of claims 90 to 92, wherein the extracellular antigen-binding domain comprises a variable heavy chain set forth in SEQ ID NO: 41 and a variable light chain set forth in SEQ ID NO:

42.

94. The method of claim 92 or claim 93, wherein the scFv is set forth in SEQ ID NO:

43.

95. 92. The method of claim 90 or claim 91, wherein the extracellular antigen-binding domain is a Hu19 single-chain variable fragment (scFv).

96. 96. The method of any of claims 90, 91 and 95, wherein the extracellular antigen-binding domain comprises a variable heavy chain set forth in SEQ ID NO: 114 and a variable light chain set forth in SEQ ID NO:

112.

97. 97. The method of claim 95 or 96, wherein the extracellular antigen-binding domain comprises, in order, a variable light chain set forth in SEQ ID NO: 112, a linker peptide set forth in SEQ ID NO: 113, and a variable heavy chain set forth in SEQ ID NO:

114.

98. 98. The method of any one of claims 1 to 97, wherein the CAR is a monospecific CAR directed against CD19.

99. 98. The method of any one of claims 1 to 97, wherein the CAR is a tandem bispecific CAR directed against CD19 and at least one other antigen expressed on B cells.

100. 100. The method of claim 99, wherein the other antigen expressed on B cells is selected from the group consisting of CD20, CD19, CD22, ROR1, BCMA, CD45, CD21, CD5, CD33, Ig kappa, Ig lambda, CD79a, CD79b, or CD30.

101. 101. The method of claim 99 or claim 100, wherein the other antigen expressed on B cells is CD20.

102. 102. The method of claim 101, wherein the extracellular antigen-binding domain comprises a variable heavy chain and a variable light chain derived from a CD20 antibody selected from the group consisting of Leu16, C2B8, 11B8, 8G6-5, 2.1.2, and GA101.

103. 103. The method of any of claims 90 to 102, wherein the transmembrane domain is a CD28 transmembrane domain.

104. 104. The method of any of claims 90 to 103, wherein the transmembrane domain is a transmembrane domain from CD28, optionally comprising the sequence of amino acids set forth in SEQ ID NO: 8, or a sequence of amino acids exhibiting at least, or at least about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:

8.

105. The method of any of claims 101 to 104, wherein the intracellular signaling domain comprises a 4-1BB costimulatory domain and a CD3 zeta activation domain.

106. The method of any one of claims 1 to 105, wherein the CAR comprises, in order from N-terminus to C-terminus, a single-chain variable fragment (scFv) derived from FMC63 monoclonal antibody, an IgG4 hinge region, a CD28 transmembrane domain, a 4-1BB (CD137) costimulatory domain, and a CD3 zeta signaling domain.

107. The method of claim 105 or claim 106, wherein the 4-1BB costimulatory domain is or comprises the sequence set forth in SEQ ID NO: 12, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:

12.

108. 108. The method of any of claims 105 to 107, wherein the CD3 zeta signaling domain is or comprises the sequence set forth in SEQ ID NO: 13, 14 or 15, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

109. The method of any one of claims 1 to 108, wherein the CAR comprises, in order from N-terminus to C-terminus, an extracellular antigen-binding domain which is an scFv shown in SEQ ID NO: 43, a spacer shown in SEQ ID NO: 1, a transmembrane domain shown in SEQ ID NO: 8, a 4-1BB costimulatory signaling domain shown in SEQ ID NO: 12, and a signaling domain of the CD3-zeta (CD3ζ) chain shown in SEQ ID NO:

13.

110. 110. The method of any of claims 1 to 109, wherein the CAR comprises the amino acid sequence set forth in SEQ ID NO: 59, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

111. The composition comprises: (i) generating a stimulated population by stimulating an input composition comprising primary T cells from the subject with an oligomeric stimulating reagent, wherein the oligomeric stimulating reagent comprises a plurality of cross-linked tetramers of streptavidin or a streptavidin mutein, the streptavidin or streptavidin mutein being reversibly bound to a first agent comprising an anti-CD3 antibody or antigen-binding fragment thereof and a second agent comprising an anti-CD28 antibody or antigen-binding fragment thereof; (ii) generating a population of transformed cells by introducing into T cells of the stimulated population a heterologous polynucleotide encoding the CAR that targets CD19; (iii) incubating the population of transformed cells for up to 96 hours; and (iv) producing a composition of CD19-directed genetically modified T cells by harvesting T cells from the population of transformed cells, wherein harvesting occurs between 24 and 120 hours, inclusive, after exposure to the stimulatory reagent is initiated; 111. The method of any one of claims 1 to 110, produced by a manufacturing process comprising:

112. The method of claim 111, wherein the anti-CD3 antibody or antigen-binding fragment is a Fab and the anti-CD28 antibody or antigen-binding fragment is a Fab.

113. 113. The method of claim 111 or claim 112, wherein the first agent and the second agent each comprise a streptavidin-binding peptide that reversibly binds the first agent and the second agent to an oligomeric particle reagent, and optionally the streptavidin-binding peptide comprises a sequence of amino acids set forth in any of SEQ ID NOs: 78-82.

114. A method according to any one of claims 111 to 113, wherein the streptavidin mutant protein molecule is a tetramer of streptavidin mutant protein molecules comprising amino acid residues Val44-Thr45-Ala46-Arg47 or Ile44-Gly45-Ala46-Arg47, and optionally the streptavidin mutant protein comprises the sequence set forth in SEQ ID NO: 69, 84, 87, 88, 90, 85 or 59.

115. 115. The method of any of claims 111 to 114, wherein the oligomeric particle reagent comprises between 1,000 and 5,000 streptavidin mutein tetramers inclusive.

116. 116. The method of any of claims 111 to 115, further comprising adding biotin or a biotin analog after or during the incubation prior to harvesting the cells.

117. 117. The method of any of claims 111 to 116, wherein said harvesting occurs between 48 and 120 hours, inclusive, after exposure to said stimulating agent has commenced.

118. 118. The method of any of claims 1 to 117, wherein the dose of autologous CD19-directed gene-modified T cells is cryopreserved prior to administration to the subject.

119. Freezing of autologous CD19-directed genetically modified T cells 119. The method of claim 118, wherein the stored dose is thawed prior to administration to the subject.

120. 120. The method of claim 119, wherein the dose of autologous CD19-directed genetically modified T cells is administered to the subject within about 2 hours of being thawed.

121. 121. The method of any of claims 1 to 120, wherein the dose of autologous CD19-directed genetically modified T cells is provided in a formulation that includes a cryoprotectant.

122. 122. The method of claim 121, wherein the formulation comprises dimethyl sulfoxide (DMSO).

123. 123. The method of claim 121 or claim 122, wherein the formulation comprises albumin, optionally human albumin.

124. The dose of T cells is a CD4 T cell expressing the CAR. ＋ T cells, and CD8 T cells expressing the CAR. ＋ 124. The method of any preceding claim, comprising T cells.

125. The dose of T cells is a CD4 T cell expressing the CAR. ＋ T cells, and CD8 T cells expressing the CAR. ＋ 125. The method of any preceding claim, comprising T cells at a ratio of about 1.5 to about 5:1, optionally at a ratio of about 1:3 to about 3:

1.

126. At least 90% or at least about 90% of the cells in the composition are CD3 ＋ 126. The method of any one of claims 1 to 125, wherein the cell is a cell.

127. At least 91%, or at least about 91%, at least 92%, or at least about 92%, at least 93%, or at least about 93%, at least 94%, or at least about 94%, at least 95%, or at least about 95%, or at least 96%, or at least about 96% of the cells in the composition are CD3 ＋ 127. The method of any one of claims 1 to 126, wherein the cell is a cell.

128. 128. The method of any of claims 1-127, wherein at least 25% of the T cells in the composition are CAR+ T cells.

129. 129. The method of any of claims 1-128, wherein at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the T cells in the composition are CAR+ T cells.

130. The CAR in the composition ＋ 5% or about 5% to 30% or about 30% of T cells, optionally the CAR in the composition ＋ 130. The method of any preceding claim, wherein 10% or about 10% to 15% or about 15% of the T cells express a marker of apoptosis, and optionally wherein the marker of apoptosis is annexin V or active caspase 3.

131. 128. The method of any of claims 1-127, wherein the T cells in the composition, optionally less than 10% of the CAR+ T cells, express a marker of apoptosis, optionally wherein the marker of apoptosis is annexin V or active caspase 3.

132. 132. The method of any of claims 1-127 and 131, wherein the T cells in the composition, optionally less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, or less than 4% of the CAR+ T cells, express a marker of apoptosis, optionally wherein the marker of apoptosis is annexin V or active caspase 3.

133. 133. The method of any of claims 1-132, wherein at least 80% of the T cells in the composition are viable T cells, optionally wherein viability is determined by acridine orange (AO) and propidium iodide (PI) staining.

134. The CAR in the composition ＋ 131. The method of any of claims 1 to 130, wherein at least 80% or at least about 80% of the T cells are of a naive-like or central memory phenotype.

135. 135. The method of claim 134, wherein the marker expressed on naive-like or central memory T cells is selected from the group consisting of CD45RA, CD27, CD28, and CCR7.

136. 136. The method of any of claims 1-135, wherein at least 85% of the CAR+ T cells in the composition are CCR7+.

137. 137. The method of any of claims 1-136, wherein at least 85% of CD8+ CAR+ T cells in said composition are CCR7+ and at least 90% of CD4+ CAR+ T cells in said composition are CCR7+.

138. 138. The method of any of claims 1-137, wherein 85% to 98% of the CD8+ CAR+ T cells in the composition are CCR7+ and 94% to 99% of the CD4+ CAR+ T cells in the composition are CCR7+.

139. the CAR in the composition is of a naive-like or central memory phenotype. ＋ At least 80% or at least about 80% of the T cells have the phenotype CCR7 ＋ CD45RA ＋ , CCR7 ＋ CD45RA － , CD27 ＋ CCR7 ＋ , or CD62L － CCR7 ＋ 136. The method of any preceding claim, wherein the subject has a phenotype selected from one or more of:

140. 140. The method of any of claims 1-139, wherein at least 40% of the CAR+ T cells are CD45RA+ CCR7+.

141. 141. The method of any of claims 1-140, wherein at least 50% of the CAR+ T cells are CD45RA+ CCR7+.

142. 142. The method of any of claims 1-141, wherein at least 60% of the CAR+ T cells are CD45RA+ CCR7+.

143. 143. The method of any of claims 1-142, wherein at least 70% of the CAR+ T cells are CD45RA+ CCR7+.

144. 144. The method of any of claims 1-143, wherein at least 80% of the CAR+ T cells are CD45RA+ CCR7+.

145. The method of any of claims 1-144, wherein at least 20% of the CAR+ T cells are CD45RA- CCR7+.

146. The method of any of claims 1-145, wherein at least 30% of the CAR+ T cells are CD45RA- CCR7+.

147. The method of any of claims 1-146, wherein at least 40% of the CAR+ T cells are CD45RA- CCR7+.

148. The method of any of claims 1-147, wherein at least 50% of the CAR+ T cells are CD45RA- CCR7+.

149. The method of any of claims 1-148, wherein at least 60% of the CAR+ T cells are CD45RA- CCR7+.

150. At least about 50% of the CD4+ CAR+ T cells in the composition are CCR7+ CD45RA － 150. The method of any one of claims 1 to 149, wherein

151. At least about 60% of the CD4+ CAR+ T cells in the composition are CCR7+ CD45RA － 150. The method of any one of claims 1 to 149, wherein

152. At least about 70% of the CD4+ CAR+ T cells in the composition are CCR7+ CD45RA － 150. The method of any one of claims 1 to 149, wherein

153. At least about 30% of the CD8+ CAR+ T cells in the composition are CCR7+ CD45RA － 153. The method of any one of claims 1 to 152, wherein

154. At least about 40% of the CD8+ CAR+ T cells in the composition are CCR7+ CD45RA － 153. The method of any one of claims 1 to 152, wherein

155. At least about 50% of the CD8+ CAR+ T cells in the composition are CCR7+ CD45RA － 153. The method of any one of claims 1 to 152, wherein

156. (i) at least 60% of the T cells in the composition are viable; (ii) at least 25% of the T cells in the composition are CAR+ T cells; (iii) at least 85% of the CD8+ CAR+ T cells in the composition are CCR7+; and (iv) at least 90% of the CD4+ CAR+ T cells in the composition are CCR7+.

156. The method of any one of claims 1 to 155.

157. (i) at least 80% of the T cells in the composition are viable; (ii) at least 45% of the T cells in the composition are CAR+; (iii) at least 85% of the CD8+ CAR+ T cells in the composition are CCR7+; and (iv) at least 90% of the CD4+ CAR+ T cells in the composition are CCR7+.

156. The method of any one of claims 1 to 155.

158. (i) at least 60% of the T cells in the composition are viable; (ii) at least 25% of the T cells of the composition are CAR+; and (iii) greater than or greater than about 40% of the CAR+ T cells in the composition are CCR7+CD45RA+.

156. The method of any one of claims 1 to 155.

159. (i) at least 80% of the T cells in the composition are viable; (ii) at least 45% of the T cells of the composition are CAR+; and (iii) at least 40% of the CAR+ T cells in the composition are CCR7+CD45RA+.

156. The method of any one of claims 1 to 155.

160. (i) at least 60% of the T cells in the composition are viable; (ii) at least 25% of the T cells of the composition are CAR+; and (iii) greater than 20% of the CAR+ T cells in the composition are CCR7+CD45RA-.

156. The method of any one of claims 1 to 155.

161. (i) at least 80% of the T cells in the composition are viable; (ii) at least 45% of the T cells of the composition are CAR+; and (iii) at least 20% of the CAR+ T cells in the composition are CCR7+CD45RA-.

156. The method of any one of claims 1 to 155.

162. 162. The method of any of claims 1 to 161, wherein less than 10% of the cells of the composition are positive for a marker of apoptosis, and optionally the marker of apoptosis is annexin V or active caspase 3.

163. 162. The method of any of claims 1 to 161, wherein less than 4% of the cells of the composition are positive for a marker of apoptosis, and optionally the marker of apoptosis is annexin V or active caspase 3.

164. The method of any of claims 1 to 163, wherein greater than or about 50%, greater than or about 60%, greater than or about 60%, greater than or about 70%, or greater than or about 80% of the subjects treated according to the method do not exhibit any grade of cytokine release syndrome (CRS).

165. 165. The method of any of claims 1 to 164, wherein greater than or about 40%, greater than or about 50%, or greater than or about 60% of the subjects treated according to the method do not exhibit any grade of neurotoxicity.

166. 166. The method of any of claims 1 to 165, wherein the subject does not receive an immunosuppressant to treat the disease after administration of the dose of CD19-directed genetically modified T cells.

167. 167. The method of any one of claims 1 to 166, wherein the subject is a human.

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