Cell therapy for treating systemic autoimmune diseases

Abstract

Provided herein are adoptive cell therapy methods and uses involving administration of a dose of T cells expressing a CD19-directed chimeric antigen receptor for treating a subject having a systemic autoimmune disease; and related methods, compositions, uses, and articles of manufacture.

Description

This application is a divisional application of Chinese patent application No. 202480027461.X (filed on February 28, 2024, entitled "Cell Therapy for Treating Systemic Autoimmune Diseases"). Cross-reference to related applications

[0001] This application claims priority to the following patent applications: U.S. Provisional Application No. 63 / 487,612, filed February 28, 2023; U.S. Provisional Application No. 63 / 466,671, filed May 15, 2023; U.S. Provisional Application No. 63 / 522,085, filed June 20, 2023; U.S. Provisional Application No. 63 / 608,166, filed December 8, 2023; U.S. Provisional Application No. 63 / 618,271, filed January 5, 2024; U.S. Provisional Application No. 63 / 624,745, filed January 24, 2024; and U.S. Provisional Application No. 63 / 553,586, filed February 14, 2024, each entitled “Cellular Therapy for the Treatment of Systemic Autoimmune Diseases”, the contents of which are incorporated herein by reference in their entirety. Reference to electronic sequence listing

[0002] This application is submitted together with an electronic sequence list. The sequence list is provided as a file named 735042027040SeqList.xml, created on February 23, 2024, and is 200,426 bytes in size. The electronic information of the sequence list is incorporated herein by reference in its entirety. Technical Field

[0003] In some respects, this disclosure relates to adoptive cell therapy, which involves administering a dose of T cells expressing a CD19-directed chimeric antigen receptor for the treatment of a subject with a systemic autoimmune disease; and related methods, compositions, uses, and articles thereof. Background Technology

[0004] Systemic autoimmune diseases involve a variety of illnesses and disorders characterized by immune system dysregulation. These include systemic lupus erythematosus (SLE), an abnormal immune disorder presenting with a range of clinical manifestations, most notably renal involvement, namely lupus nephritis. Many patients eventually relapse or become refractory to available therapies, and second-, third-, and especially fourth-line treatments are limited. There is a need for effective therapies for patients with SLE (such as severe refractory SLE) who have experienced failure to one or more prior therapies. Methods and uses to meet this need are provided. Summary of the Invention

[0005] This article provides a method for treating a subject with a systemic autoimmune disease, the method comprising administering to a subject with or suspected of having a severe systemic autoimmune disease a dose of CD19-directed genetically modified T cells derived from a composition comprising engineered T cells expressing a chimeric antigen receptor (CAR), wherein the dose of the T cells is positive for expression of the CD19-binding CAR, and the dose is from 1 × 10⁻⁶. 6 Up to 50 × 10 6 One CAR-positive live T cell.

[0006] This article also provides a method for treating a subject with a systemic autoimmune disease, the method comprising administering to a subject with or suspected of having a moderate systemic autoimmune disease a dose of CD19-directed genetically modified T cells, wherein the dose of the T cells is positive for expression of a CD19-binding chimeric antigen receptor (CAR), and the dose is from 1 × 10⁻⁶. 6 Up to 50 × 10 6 One CAR-positive live T cell.

[0007] In some embodiments, the systemic autoimmune disease is selected from systemic lupus erythematosus (SLE), Sjögren'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, Behçet's disease, rheumatoid arthritis, IgA nephropathy, pemphigus vulgaris, myasthenia gravis, autoimmune hemolytic anemia, immune thrombocytopenic purpura, IgG4-related disease, membranous nephropathy, and skin diseases. Skin lupus erythematosus, sarcoidosis, light chain amyloidosis, 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, Rh hemolytic disease, Still's disease, type 1 diabetes mellitus, urticaria, capillary leak syndrome, cytokine release syndrome, erythema multiforme, pyoderma gangrenosa, X-linked agammaglobulinemia, antiphospholipid syndrome, and chronic inflammatory demyelinating polyneuropathy (also known as inflammatory demyelinating polyradiculopathy).

[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 thrombocytopenic purpura. 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 polyradiculopathy. 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 prior therapies targeting the systemic autoimmune disease. In some embodiments, the subject is refractory to treatment with two or more prior therapies targeting the systemic autoimmune disease. In some embodiments, the systemic autoimmune disease is a severe disease.

[0010] This article also provides a method for treating a subject with severe systemic lupus erythematosus (SLE), the method comprising administering to a subject with or suspected of having severe systemic lupus erythematosus (SLE) a dose of CD19-directed genetically modified T cells, wherein the dose of the T cells is positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and the dose is from 1 × 10⁻⁶. 6 Up to 50 × 10 6 One CAR-positive live T cell.

[0011] This article also provides a method for reducing disease activity in systemic lupus erythematosus (SLE), the method comprising administering a dose of CD19-directed genetically modified T cells to a subject who has or is suspected of having severe systemic lupus erythematosus (SLE), wherein the dose of the T cells is positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and the dose is from 1 × 10⁻⁶. 6 Up to 50 × 10 6 One CAR-positive live T cell.

[0012] In some implementations, the subject's SLE has one or more of the following: kidney, central nervous system, or blood involvement.

[0013] In some implementations, the subject has at least one organ system classified as Category A (“BILAG A”) according to the British Isles Lupus Assessment Group 2004 (“BILAG”), or at least two organ systems classified as BILAG B.

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

[0015] In some implementations, the subject suffers from lupus nephritis.

[0016] This article also provides a method for treating a subject with lupus nephritis, the method comprising administering to a subject with or suspected of having lupus nephritis a dose of CD19-directed genetically modified T cells, wherein the dose of the T cells is positive for expression of a CD19-binding chimeric antigen receptor (CAR), and the dose is from 1 × 10⁻⁶. 6 Up to 50 × 10 6 One CAR-positive live T cell.

[0017] In some implementations, the subject is refractory to one or more prior therapies for lupus.

[0018] In some implementations, the subject has an inadequate response to one or more prior therapies for the lupus.

[0019] In some implementations, the two or more prior therapies for lupus include glucocorticoids, antimalarial drugs, immunosuppressants, anti-CD20 antibodies, or soluble B lymphocyte stimulator (BLyS) inhibitors.

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

[0021] In some implementations, the subject does not have drug-induced SLE, clinically significant CNS symptoms, associated systemic autoimmune diseases, and / or SLE overlap syndromes.

[0022] In some implementations, the subject does not have any relevant systemic autoimmune diseases, including but not limited to multiple sclerosis, psoriasis, and inflammatory bowel disease.

[0023] In some embodiments, the subject does not have SLE overlap syndromes, 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 implementations, the method reduces the activity of the subject's systemic autoimmune disease.

[0025] In some implementations, reducing the subject's disease activity includes reducing the subject's inflammation.

[0026] In some implementations, the method reduces the SLE disease activity of the subject.

[0027] In some implementations, reducing the subject's SLE disease activity includes: a BILAG-based Comprehensive Lupus Assessment (BICLA) response; a reduction in the subject's CLASI score compared to the subject's pre-treatment SLE area and severity index (CLASI) score; a reduction in the subject's pre-treatment tender and swollen joint count compared to the subject's pre-treatment tender and swollen joint count; a post-treatment BILAG-2004 B score of 1 or higher; a post-treatment BILAG-2004 score of C or better; an improvement in at least one patient-reported outcome (PRO) compared to pre-treatment; and / or a reduction in the subject's SLE flare rate compared to the subject's pre-treatment flare rate.

[0028] In some implementations, reducing SLE disease activity in a subject involves the subject achieving clinical remission as defined by the Systemic Lupus Erythematosus Remission Definition (DORIS). In some implementations, reducing SLE involves the subject achieving a low disease activity state of lupus (LLDAS).

[0029] In some implementations, 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 implementations, 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.

[0031] In some implementations, the subject achieves prolonged remission of lupus.

[0032] This article also provides a method for treating a subject with idiopathic inflammatory myopathy (IIM), the method comprising administering to a subject with or suspected of having idiopathic inflammatory myopathy (IIM) a dose of CD19-directed genetically modified T cells, wherein the dose of the T cells is positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and the dose is from 1 × 10⁻⁶. 6 Up to 50 × 10 6 One CAR-positive live T cell.

[0033] This article also provides a method for reducing disease activity in idiopathic inflammatory myopathy (IIM), the method comprising administering a dose of CD19-directed genetically modified T cells to a subject who has or is suspected of having IIM, wherein the dose of the T cells is positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and the dose is from 1 × 10⁻⁶. 6 Up to 50 × 10 6 One CAR-positive live T cell.

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

[0035] This article also provides a method for treating a subject with systemic sclerosis (SSc), the method comprising administering to a subject with or suspected of having systemic sclerosis (SSc) a dose of CD19-directed genetically modified T cells, wherein the dose of the T cells is positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and the dose is from 1 × 10⁻⁶. 6 Up to 50 × 10 6 One CAR-positive live T cell.

[0036] This article also provides a method for reducing disease activity in systemic sclerosis (SSc), the method comprising administering a dose of CD19-directed genetically modified T cells to a subject who has or is suspected of having systemic sclerosis (SSc), wherein the dose of the T cells is positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and the dose is from 1 × 10⁻⁶. 6 Up to 50 × 10 6 One CAR-positive live T cell.

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

[0038] This article also provides a method for treating a subject with multiple sclerosis (MS), the method comprising administering to a subject with or suspected of having multiple sclerosis (MS) a dose of CD19-directed genetically modified T cells, wherein the dose of the T cells is positive for expression of a CD19-binding chimeric antigen receptor (CAR), and the dose is from 1 × 10⁻⁶. 6 Up to 50 × 10 6 One CAR-positive live T cell.

[0039] This article also provides a method for reducing disease activity in multiple sclerosis (MS), the method comprising administering a dose of CD19-directed genetically modified T cells to a subject who has or is suspected of having MS, wherein the dose of the T cells is positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and the dose is from 1 × 10⁻⁶. 6 Up to 50 × 10 6 One CAR-positive live T cell.

[0040] In some embodiments, the subject is refractory to one or more prior therapies for the MS. In some embodiments, the subject has an inadequate response to one or more prior therapies for the MS. In some embodiments, the subject has or is suspected of having a relapsing form of MS. In some embodiments, the subject has or is suspected of having a progressive form of 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's Extended Disability Status Scale (EDSS) score is ≥ 3.0 and ≤ 5.5 or ≥ 3.0 and ≤ 6.0. In some embodiments, the subject is able to complete a 9-well column test (9-HPT) for each hand in < 240 seconds and a timed 25-foot walk test (T25FWT) in < 150 seconds. In some embodiments, the subject does not have MS lesions or symptoms that would increase the risk of neurotoxicity.

[0043] In some implementations, the method reduces the activity of the subject's autoimmune disease.

[0044] In some implementations, reducing the subject's disease activity includes reducing the subject's inflammation.

[0045] In some implementations, the reduction of the subject's autoimmune disease activity includes: reducing the subject's IMACS score after treatment compared to the subject's IMACS score before treatment; reducing 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 improving at least one patient-reported outcome (PRO) of the subject compared to before treatment.

[0046] In some implementations, the reduction of the subject's autoimmune disease activity includes: reducing the subject's modified Rodnan skin score, European Scleroderma Study Group (EScSG) index, minimum clinically significant difference (MCID), patient-reported brief quality of life assessment (SF-36) physical health score (PCS) and / or mental health score (MCS), or a combination thereof, or improving forced vital capacity.

[0047] In some implementations, reducing the subject's autoimmune disease activity includes improving the subject's score on any of the following tests: Extended Disability Status Scale (EDSS), Disease Steps, Multiple Sclerosis Functional Comprehensive Scale (MSFC), Minimum Clinically Important Difference Value (MCID), Patient-Reported Brief Quality of Life Assessment (SF-36), Physical Health Overall (PCS), and / or Mental Health Overall (MCS), or a combination thereof.

[0048] This article also provides a method for treating a subject with autoimmune vasculitis (AAV), the method comprising administering to a subject with or suspected of having autoimmune vasculitis (AAV) a dose of CD19-directed genetically modified T cells, wherein the dose of the T cells is positive for expression of a CD19-binding chimeric antigen receptor (CAR), and the dose is from 1 × 10⁻⁶. 6 Up to 50 × 10 6 One CAR-positive live T cell.

[0049] This article also provides a method for reducing disease activity in autoimmune vasculitis (AAV), the method comprising administering a dose of CD19-directed genetically modified T cells to a subject who has or is suspected of having autoimmune vasculitis (AAV), wherein the dose of the T cells is positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and the dose is from 1 × 10⁻⁶. 6 Up to 50 × 10 6 One CAR-positive live T cell.

[0050] This article also provides a method for treating a subject with IgA nephropathy, the method comprising administering to a subject with or suspected of having IgA nephropathy a dose of CD19-directed genetically modified T cells, wherein the dose of the T cells is positive for expression of a CD19-binding chimeric antigen receptor (CAR), and the dose is from 1 × 10⁻⁶. 6 Up to 50 × 10 6 One CAR-positive live T cell.

[0051] This article also provides a method for reducing disease activity in IgA nephropathy, the method comprising administering a dose of CD19-directed genetically modified T cells to a subject with or suspected of having IgA nephropathy, wherein the dose of the T cells is positive for expression of a CD19-binding chimeric antigen receptor (CAR), and the dose is from 1 × 10⁻⁶. 6 Up to 50 × 10 6 One CAR-positive live T cell.

[0052] This article also provides a method for treating a subject with pemphigus vulgaris, the method comprising administering to a subject with or suspected of having pemphigus vulgaris a dose of CD19-directed genetically modified T cells, wherein the dose of the T cells is positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and the dose is from 1 × 10⁻⁶. 6 Up to 50 × 10 6 One CAR-positive live T cell.

[0053] This article also provides a method for reducing disease activity in pemphigus vulgaris, the method comprising administering a dose of CD19-directed genetically modified T cells to a subject who has or is suspected of having pemphigus vulgaris, wherein the dose of the T cells is positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and the dose is from 1 × 10⁻⁶. 6 Up to 50 × 10 6 One CAR-positive live T cell.

[0054] This article also provides a method for treating a subject with myasthenia gravis, the method comprising administering to a subject with or suspected of having myasthenia gravis a dose of CD19-directed genetically modified T cells, wherein the dose of the T cells is positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and the dose is from 1 × 10⁻⁶. 6 Up to 50 × 10 6 One CAR-positive live T cell.

[0055] This article also provides a method for reducing disease activity in myasthenia gravis, the method comprising administering a dose of CD19-directed genetically modified T cells to a subject who has or is suspected of having myasthenia gravis, wherein the dose of the T cells is positive for expression of a chimeric antigen receptor (CAR) that binds to CD19, and the dose is from 1 × 10⁻⁶. 6 Up to 50 × 10 6 One CAR-positive live T cell.

[0056] In some embodiments, the dose is or is about 1 × 10⁻⁶. 6 Up to 40 × 10 6 One CAR-positive live T cell.

[0057] In some embodiments, the dose is or is about 1 × 10⁻⁶. 6 Up to 25 × 10 6 One CAR-positive live T cell.

[0058] In some embodiments, the dose is or is about 5 × 10⁻⁶. 6 One CAR-positive live T cell.

[0059] In some embodiments, the dose is or is about 10 × 10⁻⁶. 6 One CAR-positive live T cell.

[0060] In some embodiments, the dose is or is about 25 × 10⁻⁶. 6 One CAR-positive live T cell.

[0061] In some embodiments, the dose is or is about 50 × 10⁻⁶. 6 One CAR-positive live T cell.

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

[0063] In some embodiments, the method further includes obtaining a leukapheresis sample from the subject for manufacturing the composition containing engineered T cells.

[0064] In some implementations, the subject has been pre-conditioned with lymphocyte clearance therapy prior to the administration.

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

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

[0067] In some embodiments, the lymphocyte clearance therapy comprises daily administration of 30 mg / m² 2 Subjects were administered fludarabine by body surface area and daily at 300 mg / m² 2 Cyclophosphamide was applied to the body surface area of ​​the subjects for 3 consecutive days.

[0068] In some implementations, the dose of CD19-directed genetically modified T cells is administered between approximately 48 hours and approximately 9 days after completion of the lymphocyte clearance therapy, including end values.

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

[0070] In some implementations, the CAR includes an extracellular antigen-binding domain that binds to CD19, a transmembrane domain, and an intracellular signal transduction domain.

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

[0072] In some implementations, 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 the variable heavy chain shown in SEQ ID NO: 41 and the variable light chain shown in SEQ ID NO: 42.

[0074] In some implementations, the scFv is as shown in SEQ ID NO: 43.

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

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

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

[0078] In some implementations, the CAR is a single-specific CAR targeting CD19.

[0079] In some embodiments, the CAR is a tandem bispecific CAR targeting CD19 and at least one other antigen expressed on B cells. In some embodiments, the other antigen expressed on B cells is selected from CD20, CD19, CD22, ROR1, BCMA, CD45, CD21, CD5, CD33, Igκ, Igλ, 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 Leu16, C2B8, 11B8, 8G6-5, 2.1.2, and GA101.

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

[0082] In some embodiments, the transmembrane domain is a transmembrane domain derived from CD28, optionally comprising the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence exhibiting at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 8.

[0083] In some implementations, the intracellular signal transduction domain includes a 4-1BB co-stimulatory domain and a CD3ζ activation domain.

[0084] In some embodiments, the CAR comprises, in sequence from the N-terminus to the C-terminus, a single-chain variable fragment (scFv) derived from the FMC63 monoclonal antibody, an IgG4 hinge region, a CD28 transmembrane domain, a 4-1BB (CD137) co-stimulatory domain, and a CD3ζ signaling domain.

[0085] In some embodiments, the 4-1BB co-stimulatory domain is or contains the sequence shown 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 higher sequence identity with SEQ ID NO: 12.

[0086] In some embodiments, the CD3ζ signal transduction domain is or contains the sequence shown 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 higher sequence identity with it.

[0087] In some embodiments, the CAR contains, in sequence from the N-terminus to the C-terminus: an extracellular antigen-binding domain of scFv as shown in SEQ ID NO: 43, a spacer as shown in SEQ ID NO: 1, a transmembrane domain as shown in SEQ ID NO: 8, a 4-1BB co-stimulatory signal transduction domain as shown in SEQ ID NO: 12, and a signal transduction domain of the CD3-ζ (CD3ζ) chain as shown in SEQ ID NO: 13.

[0088] In some embodiments, the CAR comprises the amino acid sequence shown 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 higher sequence identity with it.

[0089] In some embodiments, the composition is produced by a manufacturing process comprising: (i) stimulating an input composition containing primary T cells from the subject with an oligomeric stimulating agent to generate a stimulated population, wherein the oligomeric stimulating agent comprises multiple cross-linked tetramers of streptavidin or a streptavidin mutant protein, and wherein the streptavidin or streptavidin mutant protein reversibly binds to a first agent containing an anti-CD3 antibody or an antigen-binding fragment thereof and a second agent containing an anti-CD28 antibody or an antigen-binding fragment thereof; (ii) introducing a heteropolynucleotide encoding the CAR targeting CD19 into the T cells of the stimulated population to generate a population of transformed cells; (iii) incubating the population of transformed cells for up to 96 hours; and (iv) Harvesting T cells from the population of the transformed cells to produce a composition of CD19-directed genetically modified T cells, wherein the harvesting is performed between 24 and 120 hours after the onset of exposure to the stimulating agent, including end values.

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

[0091] In some embodiments, the first and second agents each comprise a streptavidin-binding peptide that reversibly binds the first and second agents to the oligomeric particle reagent, optionally wherein the streptavidin-binding peptide comprises an amino acid sequence shown in any one of SEQ ID NO: 78-82.

[0092] In some embodiments, the streptavidin mutant protein molecule is a tetramer of a streptavidin mutant protein containing the amino acid residues Val44-Thr45-Ala46-Arg47 or Ile44-Gly45-Ala46-Arg47, optionally wherein the streptavidin mutant protein contains the sequence shown in any one of 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 mutant protein tetramers, including end values.

[0094] In some embodiments, the method further includes adding biotin or a biotin analogue before harvesting the cells, after or during the incubation.

[0095] In some implementations, the harvest is conducted between 48 and 120 hours after the initial exposure to the irritant, including the end value.

[0096] In some implementations, the dose of autologous CD19-directed genetically modified T cells is cryopreserved before being administered to the subject.

[0097] In some embodiments, the cryopreserved dose of autologous CD19-directed genetically modified T cells is thawed before being administered to the subject.

[0098] In some implementations, the dose of autologous CD19-directed genetically modified T cells is administered to the subject within approximately two hours of thawing.

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

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

[0101] In some embodiments, the preparation contains albumin, optionally human albumin.

[0102] In some embodiments, the dose of T cells contains CD4 expressing the CAR at a ratio between approximately 1:5 and approximately 5:1. + T cells and CD8 cells expressing the CAR + T cells.

[0103] In some embodiments, the dose of T cells contains CD4 expressing the CAR at a ratio between approximately 1:3 and approximately 3:1. +T cells and CD8 cells expressing the CAR + T cells.

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

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

[0106] In some embodiments, at least 25% of the T cells in the composition are CAR+ T cells. In 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 composition contains between 5% and 30% CAR. + T cells, optionally CARs in the composition at or between 10% and 15%. + T cells express apoptosis markers, more optionally said apoptosis markers being annexin V or active caspase 3.

[0108] In some embodiments, less than 10% of the T cells in the composition express apoptosis markers. 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 apoptosis markers. In some embodiments, less than 10% of the CAR+ T cells in the composition express apoptosis markers. 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 apoptosis markers. In some embodiments of any of these embodiments, the apoptosis marker is annexin V or active caspase 3.

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

[0110] In some implementations of any given implementation, viability is determined by staining with acridine orange (AO) and propidium iodide (PI).

[0111] In some embodiments, the composition contains at least or at least about 80% CAR + T cells exhibit naive or central memory phenotypes.

[0112] In some implementations, the markers expressed on naive or central memory T cells are selected from 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% 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+.

[0114] In some embodiments, the composition contains at least or at least about 80% CARs with a naive or central memory phenotype. + T cells have a phenotype selected from the following: CCR7 + CD45RA + CCR7+CD45RA - CD27 + CCR7 + Or CD62L - CCR7 + .

[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. - .

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

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

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

[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 an apoptosis marker, optionally wherein the apoptosis marker 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 an apoptosis marker, optionally wherein the apoptosis marker 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 an apoptosis marker, optionally wherein the apoptosis marker is annexin V or active caspase 3; and / or more than or more 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 an apoptosis marker, optionally wherein the apoptosis marker 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 an apoptosis marker, optionally wherein the apoptosis marker 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 an apoptosis marker, optionally wherein the apoptosis marker is annexin V or active caspase 3; and / or at least 20% of the CAR+ T cells in the composition are CCR7+CD45RA-. Attached Figure Description

[0126] Figure 1A and Figure 1B T cell memory subtypes in CAR+CD4+ and CAR+CD8+ were described for non-amplification and amplification processes, respectively.

[0127] Figure 2 A- Figure 2 C depicts the fold increase of T cells in T cell compositions generated through amplification and non-amplification processes in a long-term stimulation assay following CAR stimulation with anti-idiotype antibodies, serving as an indicator of persistence and amplification potential. The fold increase of T cell compositions from different donors was calculated over time 10 days after stimulation with anti-idiotype antibodies against CAR and depicted as fold increase cell counts. Figure 2 A) Area under the curve for amplification fold ( Figure 2 B) and the amplification fold of CAR T cells generated through non-amplification processes divided by the donor-matched amplification fold of CAR T cells generated through amplification processes (B) Figure 2 C).

[0128] Figure 3 A- Figure 3 D describes as follows Figure 2 A- Figure 2 As described in section C, after 10 days of CAR stimulation with anti-idiotype antibodies, cytokine production in CAR+CD4+ T cells and CAR+CD8+ T cells was observed in a long-term stimulation assay via amplification and non-amplification processes. Results showed an effect on IL-2 (… Figure 3 A), IFN-γ ( Figure 3 B), TNFα ( Figure 3 C) or IL-2, IFNγ and TNFα ( Figure 3 D) The percentage of positive CAR+CD4+ T cells or CAR+CD8+ T cells.

[0129] Figure 4 A and Figure 4 B depicts the time-dependent CD19+ target cell-specific lysis of CAR+ T cells generated from amplification and non-amplification processes. Figure 4 A) and the area under the curve of cleavage over time ( Figure 4B).

[0130] Figure 5 A- Figure 5 F depicts a scene using 10 x 10 6 Or 25 x 10 6 The level of CAR transgenes in human patients after treatment with anti-CD19 CAR cells produced through a non-amplification process ( Figure 5 A) Serum IgG ( Figure 5 B), serum IgA ( Figure 5 C) Neutrophil count ( Figure 5 D) Total number of lymphocytes ( Figure 5 E) and platelet count ( Figure 5 F).

[0131] Figure 6 A depicts the cumulative population multiplier (PDL) at harvest time of engineered cell compositions compared to the time before post-stimulation transduction during the process of manufacturing anti-CD19 from donor human subjects (including SLE subjects). Figure 6 B describes the cell viability at harvest of engineered cell compositions used in the production of anti-CD19 from donor human subjects, including SLE subjects. Detailed Implementation

[0132] This document provides methods and uses for treating subjects with engineered cells (e.g., T cells) and / or combinations thereof, typically or including severe or moderate systemic autoimmune diseases. In embodiments of the provided methods, a therapeutic T-cell composition containing engineered cells is administered to a subject with a severe or moderate systemic autoimmune disease, for example, via adoptive cell therapy (such as adoptive T-cell therapy). In some aspects, the disease or condition is a systemic autoimmune disease. Systemic autoimmune diseases are a class of abnormal immune disorders that share similar clinical presentations and are generally treatable using similar methods. Besides 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 disorder (MCTD), relapsing-remitting multiple sclerosis, ANCA-associated vasculitis (AAV), Crohn's disease, myasthenia gravis, Bethie's disease, rheumatoid arthritis, multiple sclerosis (MS), IgA nephropathy, pemphigus vulgaris, myasthenia gravis, autoimmune hemolytic anemia, immune thrombocytopenic purpura, IgG4-related disease, membranous nephropathy, cutaneous lupus erythematosus, sarcoidosis, light chain amyloidosis, rheumatoid arthritis, bullous pemphigoid, and chronic inflammatory demyelinating polyneuropathy. In any specific embodiment of the provided method and use, T cells are engineered with a chimeric antigen receptor (CAR) targeting differentiation cluster 19 (CD19).

[0133] In some embodiments, the methods and uses include administering, in adoptive cell therapy, to a subject T cells expressing a genetically engineered (recombinant) cell surface receptor, typically a chimeric receptor (such as a chimeric antigen receptor (CAR)) that recognizes CD19. In some embodiments, CD19 is expressed by cells (e.g., B cells) that play a role in the manifestation of systemic autoimmune diseases. In some embodiments, CD19 is expressed by cells that are associated with and / or specific to the following manifestations: SLE, IIM, SSc, AAV, systemic sclerosis, highly active relapsing-remitting multiple sclerosis (MS), primary progressive MS, IgA nephropathy, pemphigus vulgaris, myasthenia gravis, demyelinating polyradiculopathy, autoimmune hemolytic anemia, immune thrombocytopenic purpura, IgG4-related disease, membranous nephropathy, primary Sjögren's syndrome, cutaneous lupus erythematosus, sarcoidosis, light chain amyloidosis, rheumatoid arthritis. This condition is associated with conditions such as arthritis, bullous pemphigoid, acute respiratory distress syndrome, atopic dermatitis, 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, Rh hemolytic disease, Still's disease, type 1 diabetes mellitus, urticaria, capillary leak syndrome, cytokine release syndrome, erythema multiforme, pyoderma gangrenosa, antiphospholipid syndrome, or X-linked agammaglobulinemia. In some embodiments, CD19 is expressed by cells that are associated with and / or specific to the following manifestations: SLE, IIM, AAV, systemic sclerosis, highly active relapsing-remitting multiple sclerosis (MS), primary progressive MS, IgA nephropathy, pemphigus vulgaris, or myasthenia gravis.

[0134] In certain embodiments, CD19 is expressed by cells associated with and / or specific to the manifestations of SLE (such as severe refractory SLE). In certain embodiments, CD19 is expressed by cells associated with and / or specific to the manifestations of idiopathic inflammatory myopathy (IIM). In certain embodiments, CD19 is expressed by cells associated with and / or specific to the manifestations of systemic sclerosis (SSc). In certain embodiments, CD19 is expressed by cells associated with and / or specific to the manifestations of multiple sclerosis (MS). In certain embodiments, CD19 is expressed by cells associated with and / or specific to the manifestations of rheumatoid arthritis (RA). In certain embodiments, CD19 is expressed by cells associated with and / or specific to the manifestations of active secondary progressive MS (aSPMS). In certain embodiments, CD19 is expressed by cells associated with and / or specific to the manifestations of myositis. In certain embodiments, CD19 is expressed by cells associated with and / or specific to the manifestations of myasthenia gravis. In certain embodiments, CD19 is expressed by cells associated with and / or specific to the manifestations of bullous pemphigoid. In certain embodiments, CD19 is expressed by cells associated with and / or specific to the manifestations of immune thrombocytopenic purpura. In certain embodiments, CD19 is expressed by cells associated with and / or specific to the manifestations of autoimmune hemolytic anemia. In certain embodiments, CD19 is expressed by cells associated with and / or specific to the manifestations of pemphigus vulgaris. In certain embodiments, CD19 is expressed by cells associated with and / or specific to the manifestations of demyelinating polyradiculopathy. In certain embodiments, CD19 is expressed by cells associated with and / or specific to the manifestations of membranous nephropathy.

[0135] In some embodiments, the systemic autoimmune disease is SLE, IIM, MS, or SSc. In some aspects, the disease or condition is intermediate SLE. In some aspects, the disease or condition is severe refractory SLE. In particular, this document provides methods and uses for treating subjects with engineered cells (e.g., T cells) and / or combinations thereof. 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, via adoptive cell therapy (such as adoptive T cell therapy). In specific embodiments of such methods and uses, the T cells are engineered with a chimeric antigen receptor (CAR) targeting differentiation cluster 19 (CD19).

[0136] In some respects, such as compared to certain alternative methods, the methods and uses provide or achieve improved responses and / or more durable responses or efficacy and / or reduced risk of toxicity or other side effects, for example, in a specific group of subjects treated. In some embodiments, the methods are advantageous because of: administering a specified number or relative number of engineered cells, administering a defined ratio of a specific type of cell, administering a particularly high percentage of poorly differentiated cells (e.g., naive or central memory cells or cells in an early differentiation state, such as CCR7+CD27+ cells), treating a specific patient population (such as those with a specific risk status, stage, and / or prior treatment history) and / or combinations thereof.

[0137] Genetically engineered T cells are typically administered in a composition formulated for application; the method generally involves administering one or more doses of cells to a subject, the one or more doses including a specific number or relative number of cells or engineered cells. In some cases, the CD19-directed CAR+ engineered cells in the composition comprise a defined ratio or composition of two or more subtypes (such as CD4 and CD8 T cells) within the composition.

[0138] In a particular embodiment, the composition for use or application of cells in the provided method comprises primary T cells engineered to express CD19-directed CAR, said T cells (i) containing a low percentage (e.g., less than 40%, less than 30%, less than 20%, or less than 10%) of exhausted cells and / or cells exhibiting exhaustion-related markers or phenotypes; and / or (ii) containing 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, such as naive T cells, central memory T cells, or long-lived memory T cells.

[0139] In the provided embodiments, compared to other CD19-targeted CAR T-cell therapies involving the administration of other CD19-targeted CAR T-cell therapies containing a higher percentage of exhausted cells and / or a higher number of cells exhibiting exhaustion-related phenotypes, and / or containing a lower percentage of certain T cells (such as naive T cells, central memory T cells, or long-lived memory T cells). In the provided embodiments, compared to other CD19-targeted CAR T-cell therapies involving the administration of other CD19-targeted CAR T-cell therapies, the characteristics of the composition and the provided methods result in improved therapeutic efficacy, such as an increased percentage of patients achieving complete remission (CR), where other CD19-targeted CAR T-cell therapies contain a higher percentage of exhausted cells and / or a higher number of cells exhibiting exhaustion-related phenotypes, and / or contain a lower percentage of certain T cells (such as naive T cells, central memory T cells, or long-lived memory T cells). In the provided method, compared with other CD19-targeted CAR T-cell therapies involving the administration of other CD19-targeted CAR T-cell therapies, the characteristics of the composition and the provided method result in improved clinical durability of treatment response (such as CR) (e.g., a response that persists for a period of time after the start of therapy), which contain a higher percentage of exhausted cells and / or a higher number of cells exhibiting exhaustion-related phenotypes, and / or contain a lower percentage of memory-like T cells (such as naive T cells, central memory T cells, or long-lived memory T cells).

[0140] In certain embodiments, the use or administration of the provided CD19-directed CAR T-cell composition in the provided method may be achieved at a cell dose more than 2 times (e.g., 5 times or 10 times lower) than that of a reference CD19-directed CAR T-cell composition (e.g., engineered with the same or similar CAR, such as having the same antigen-binding domain), but wherein the reference CD19-directed CAR T-cell composition contains a higher percentage of exhausted cells and / or a higher number of cells exhibiting exhaustion-related phenotypes, and / or contains a lower percentage of memory-like T cells (e.g., naive 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 in vitro by a process involving the step of culturing cells under conditions for expansion, such as causing cell proliferation or population doubling during the process for cell production (e.g., the number of cells in the population doubling 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times compared to the start of the process).

[0141] In some embodiments, the CD19-directed CAR T-cell composition for use in the provided methods and uses is produced through a relatively short process that does not include a step of culturing cells under conditions designed for expansion or proliferation. Different processes can be used to generate compositions containing populations of genetically engineered T cells, including those for generating engineered T cells expressing CAR, and these processes typically include steps designed for or intended to: culture cells to expand cells or increase cell proliferation. However, in certain aspects, some of these processes may require a long or relatively long time to generate engineered cells. Additionally, in various aspects, the amount of time required for some existing processes to successfully generate engineered T cells suitable for cell therapy may vary, making it difficult to coordinate the administration of the cell therapy. In some aspects, some of these processes may produce cell populations comprising a relatively high percentage or amount of exhausted cells, differentiated cells, or cells with low potency. The provided CD19-directed CAR T-cell composition for use in the provided methods addresses one or more of these problems.

[0142] In certain embodiments, the provided method is used in conjunction with a process for efficiently generating or producing engineered cells suitable for use in cell therapy. In some embodiments, the provided composition containing CD19-directed CAR-engineered T cells is generated by a process that requires no additional steps for cell expansion, e.g., no expansion unit manipulation and / or no steps designed to induce cell expansion. Regarding the process for generating the CD19-directed CAR T cell composition, the process includes one or more steps for stimulating and genetically engineering T cells (e.g., transforming, transducing, or transfecting) to generate an engineered T cell population that can be collected or formulated for use as a composition for cell therapy. In certain embodiments, the process includes the step of transducing cells with a viral vector (e.g., a lentiviral vector) containing nucleic acid encoding a CD19-directed CAR. In some aspects, the provided process results in the stable integration of the heterologous nucleic acid (expressed from the viral vector) into the cell's genome. In some aspects, the provided process produces engineered CD19-directed CAR T cells with enhanced potency compared to engineered T cell compositions produced from alternative processes, such as those involving cell expansion.

[0143] In certain aspects, the duration of the process for producing the provided composition can be measured from the moment when the input cell population or the cells to which the composition is input (e.g., T cells) first come into contact with or are exposed to a stimulating condition (e.g., as described herein, for example, in sections II-C), referred herein as the start of stimulation or stimulating, and also herein as exposure to a stimulating agent, e.g., at the start of exposure to a stimulating agent. In some embodiments, the duration of time required to harvest or collect an output population containing engineered cells (also herein referred to as the output composition or the composition of engineered cells (e.g., engineered T cells)) is measured from the start of stimulation. In certain embodiments, the duration of the process is, is about or less than 120 hours, 108 hours, 96 hours, 84 hours, 72 hours, 60 hours, 48 ​​hours, 36 hours, or 30 hours. In certain embodiments, the duration of the process is, is about or less than 5 days, 4 days, 3 days, 2 days, or 1 day. In certain embodiments, the engineered cells (e.g., cells that output a composition or population) are more potent, more durable, or more immature than cells engineered by processes requiring longer durations and larger quantities. In some aspects, the duration of the provided process (e.g., the amount of time required to generate or produce an engineered T cell population) is shorter than, by about or at least 2, 3, 4, 5, 6, 7, or more than 7 days, than the duration of some existing processes. In some embodiments, the duration of the provided process is, by about or less than 75%, 60%, 50%, 40%, 30%, 25%, 15%, or 10% of that of alternative or existing processes.

[0144] In some embodiments, the provided process is performed on a population of cells (e.g., CD3+, CD4+, and / or CD8+ T cells) isolated, enriched, or selected from a biological sample. In some aspects, the provided method can generate or produce a composition of engineered T cells within a shortened time frame compared to other methods or processes, starting from the time the biological sample is collected from the subject. In some embodiments, the provided method can generate or produce engineered T cells within approximately 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, or 2 days, or within approximately 120 hours, 96 hours, 72 hours, or 48 hours (including any or all of the time during which the biological sample or the enriched, isolated, or selected cells are cryopreserved and stored) from the time the biological sample is collected from the subject until the collection, harvesting, or formulation of engineered T cells (e.g., for cryopreservation or administration).

[0145] In certain embodiments, the process for generating or engineering a population of T cells includes a step of stimulating the cells, such as prior to transduction with a viral vector. In aspects of the provided process, stimulation is performed with an oligomeric stimulating agent (such as a streptavidin mutant protein oligomer), and one or more stimulating binders (e.g., anti-CD3 / anti-CD28) are immobilized or attached to the oligomeric stimulating agent. Existing reagents for stimulating T cells in vitro (e.g., in the absence of exogenous growth factors or in low amounts of exogenous growth factors) are known (see, for example, U.S. Patent 6,352,694 B1 and European Patent EP 0 700 430 B1). Typically, such reagents can be in the form of beads (e.g., magnetic beads) with a diameter greater than 1 µm, to which various binders (e.g., anti-CD3 antibodies and / or anti-CD28 antibodies) are immobilized. However, in some cases, such magnetic beads are difficult to integrate, for example, into methods for stimulating cells under conditions required for clinical trials or therapeutic purposes, because it must be ensured that these magnetic beads are completely removed before the expanded T cells are administered to the subject. In some respects, this removal (such as by exposing cells to a magnetic field) may reduce the yield of live cells that can be used for cell therapy. In certain cases, such reagents (e.g., irritants containing magnetic beads) must be incubated with cells for a minimum amount of time to allow T cells to fully detach from the irritant.

[0146] The process utilizing oligomeric irritant agents (e.g., streptavidin mutant protein polymers) overcomes such potential limitations. For example, in some embodiments, the provided process avoids or reduces the risk of residual irritant agents (e.g., agents containing magnetic beads) in the output cells generated or produced by said process. In some embodiments, this also means that a GMP-compliant process can be more easily established compared to other methods (e.g., those requiring additional steps to ensure the final engineered T cell population is bead-free). In some embodiments, this can be readily accomplished in the embodiments of the invention by adding a substance (e.g., a competing agent) that dissociates the oligomeric irritant agent from the cells, e.g., by simple rinsing or washing of the cells, e.g., by centrifugation. Therefore, in some aspects, the removal or separation of oligomeric irritant agents from cells (e.g., by adding a substance or a competing agent) results in almost no cell loss compared to the removal or separation of bead-based irritant agents. In some aspects, the timing of the removal or separation of oligomeric irritant agents is unrestricted or less restricted than that of the removal or separation of bead-based irritant agents. Therefore, in some respects, oligomeric irritants can be removed or isolated from cells at any time or stage during the provided process.

[0147] In some respects, the use of oligomeric stimulants (e.g., anti-CD3 / anti-CD28 streptavidin mutant oligomers) can result in an overall reduced stimulatory signal compared to alternative stimulants (e.g., anti-CD3 / anti-CD28 paramagnetic beads). Processes involving weaker or reduced stimulation may produce engineered CAR+ T cells that are as potent, durable, or effective, or even more potent, durable, or effective, as, those produced by processes involving stronger stimulatory conditions or higher amounts or concentrations of stimulants (as may be present after stimulation with anti-CD3 / anti-CD28 paramagnetic beads). Furthermore, in some embodiments, stimulating cells with lower or relatively low amounts of oligomeric stimulants can increase the potency, efficacy, or durability of the resulting engineered cell population, as compared to processes using higher amounts of oligomeric stimulants. Such embodiments contemplate that such effects may persist during and after the process, even at doses low enough to reduce the expression of activation markers or the share of cells positive for activation markers.

[0148] In some embodiments, engineered T cells (e.g., output compositions or populations of T cells expressing recombinant receptors such as chimeric antigen receptors) generated or produced by the provided process are particularly effective or potent when used as cells for cell therapy. For example, in some aspects, output compositions containing engineered T cells (e.g., CAR+ T cells) generated from the provided process have a significantly higher degree of potency and / or proliferative capacity than engineered T cells generated or produced by alternative existing processes. In some aspects, output compositions containing engineered T cells (e.g., CAR+ T cells) generated by the provided process have enhanced immune activity compared to engineered T cells (e.g., CAR+ T cells) produced by alternative or existing methods.

[0149] In certain embodiments, processes for generating the provided CD19-directed T-cell composition that do not include the step of expanding cells to a threshold amount or concentration have other advantages. In some aspects, protocols that do not rely on expanding cells to increase the number or concentration of cells from a starting cell population (e.g., an input population) do not require incubation or cultivation that may vary between cell populations. For example, some embodiments consider that cell populations obtained from different subjects (e.g., subjects with different diseases or disease subtypes, particularly patients with SLE, including high-risk, aggressive, and / or severely refractory SLE) may divide or expand at different rates. In some aspects, eliminating potentially variable steps requiring cell expansion allows for tight control over the duration of the entire process. In some embodiments, the variability in process duration is reduced or eliminated, which may, in some aspects, allow for improved coordination of appointments and treatments between physicians, patients, and technicians to facilitate autologous cell therapy.

[0150] In some embodiments, the provided method includes treating a specific group or subset of subjects, for example, subjects identified as having a high-risk disease (e.g., a systemic autoimmune disease, such as severe systemic autoimmune disease). In some embodiments, subjects with a systemic autoimmune disease to be treated (as described herein) have relapsed or are refractory (R / R) to standard therapy for treating systemic autoimmune diseases and / or have a poor prognosis. In some aspects, the method treats subjects with severe disease that has relapsed or are refractory (R / R) to standard therapy. In some embodiments, the provided method includes treating a specific group or subset of subjects, for example, subjects identified as having a high-risk disease (e.g., SLE, such as severe refractory SLE). In some aspects, the method treats subjects with some form of aggressive and / or poorly prognostic SLE (e.g., relapsed or refractory (R / R) to standard therapy and / or have a poor prognosis). In some aspects, the method treats subjects with severe SLE that has relapsed or are refractory (R / R) to standard therapy.

[0151] In certain aspects, the engineered cells are autologous to the subject and are administered after generation via a shortened ex vivo process compared to existing methods, which does not include or involve a culture step for cell expansion during the method of generating the engineered cells, and / or is capable of producing a CAR-engineered T-cell composition with a low degree of differentiation, thereby allowing for the administration of lower doses. As a result, the provided methods are advantageous compared to existing methods because they can shorten the time until engineered T-cell therapy is available to the patient, particularly in patients requiring treatment, such as in subjects whose treatment has relapsed or is refractory after one or more other prior therapies for treating a disease or condition. In some aspects, the provided methods, compositions, uses, and articles achieve improved and superior responses to available therapies. In some embodiments, the improved or superior response is against the current standard of care (SOC).

[0152] CD19 is a member of the immunoglobulin superfamily and a component of the B-cell surface signal transduction complex, which actively regulates signal transduction via B-cell receptors. Most B-cell malignancies express CD19 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 conversion therapy for severe refractory lupus and other related diseases. CD19 CAR T-cell therapy has demonstrated conversion efficacy and a favorable safety profile in severe SLE.

[0153] In a particular implementation, the method provided herein is based on the administration of CD19-directed CAR T-cell therapy, wherein the CAR contains a CD19-directed scFv antigen-binding domain (e.g., derived from FMC63). The CAR also contains an intracellular signaling domain comprising a signaling domain derived from CD3ζ, and is further incorporated with a 4-1BB co-stimulatory domain, which is associated with a lower incidence of cytokine release syndrome (CRS) and neurotoxicity (NE) compared to constructs containing CD28 (Lu et al. J ClinOncol. 2018;36:3041).

[0154] The provided method is based on the finding that a lower differentiation state of adoptive T cells can affect their ability to persist and promote durable immune activity. In some embodiments, the provided CD19-directed CAR+ engineered T cell compositions are produced by culturing cells under non-expansion conditions, thereby limiting or reducing the population multiplication rate of the final engineered output composition and resulting in a less differentiated product. However, the provided compositions are also produced via a process that results in a stable integrated vector copy number (iVCN) to ensure consistent and reliable CAR expression, resulting in consistent cell products for administration to subjects and low variability in the dose of CAR-expressing cells administered. In contrast, most T cell engineering protocols routinely expand T cells in vitro for 9 to 14 days or longer. The data provided, illustrated herein, support a model in which CAR T cell products with an increased composition of less differentiated memory T cells can exhibit enhanced durable immune activity. These findings suggest that strategies aimed at minimizing effector differentiation in CAR T cell products may lead to improved clinical efficacy. Embodiments that can achieve such objectives are provided herein.

[0155] In particular, the results of this study demonstrate the following advantageous effect: CD19-directed CAR T cells can induce immune reset following targeted cytotoxic killing of CD19-expressing B cells. In some embodiments, such as those demonstrated in Example 2 in the case of relapsed or refractory (R / R) non-Hodgkin lymphoma (NHL), compositions containing anti-CD19 CAR T cells can suppress B cell overactivation, thereby leading to immune reset and restoration of homeostatic immune system function. Therefore, these results support the use of CD19-expressing CAR T cells to achieve the same effect of resetting the immune system in autoimmune diseases by removing overactive B cells, and allow for reduction of autoimmune disease activity and clinical remission. Although attempts have been made to deplete B cells or reset the immune system with other treatments (such as HSCT or antibody therapies targeting 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), none have been successful in effectively reducing circulating B cells to decrease disease activity (as observed in this paper through the cytotoxic activity of T cells expressing CD19-directed CARs), and / or simultaneously minimizing the toxicity of the therapy to the subjects.

[0156] In some implementations, the results presented in this paper unexpectedly showed reduced disease activity in subjects with autoimmune or inflammatory diseases, as demonstrated by results from treated subjects with SLE. (Using only 10 x 10) 6 A relatively low dose of CD19-directed CAR T cells (including CD4+ and CD8+ CAR+ T cells) was observed to reduce disease activity. This dose is several orders of magnitude lower than the doses administered with other CD19-directed CAR T cell products. Furthermore, the doses used in this study are generally administered as a flat dose (not a weight-based dose based on subject weight), which has the added benefit of improving dosing consistency and reducing the risk of toxic side effects that weight-based dosing strategies might cause due to over-administration of cells in some subjects. The results in this study showed no serious toxicities observed, thus demonstrating the safety of the T-cell therapy presented. It is worth noting that while administering relatively low doses of cells (e.g., CAR-expressing T cells) may reduce the risk of toxic adverse events, relatively low doses of cells manufactured by other methods may not be entirely effective in treating the 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 methods and compositions provided.

[0157] The provided implementation schemes also support the ability to successfully treat subjects without any other immunosuppression. Typically, successful treatment of autoimmune indications usually requires sustained immunosuppression. However, as described herein, increased hospitalization and drug side effects (such as chronic oral corticosteroids (OCS or glucocorticoids and other immunosuppressive therapies)) can increase the disease burden in subjects with autoimmune indications such as SLE. The results of this study support the possibility of achieving remission of disease activity by a single infusion of a dose of CD19-CAR-directed T cells without further administration of immunosuppressants (e.g., corticosteroids, glucocorticoids, or other immunosuppressive therapies) following the administration of said dose of T cells. In some implementation schemes, subjects achieve prolonged remission through treatment according to the provided method. In some implementation schemes, further treatment of the disease is unnecessary, and subjects maintain remission after the said dose of CD19-CAR-directed T cells. For example, in the provided implementation, after administration of CD19-CAR-directed T cells, the subject maintains remission and is not given another treatment (e.g., methotrexate, mycophenolate mofetil, cyclophosphamide, tocilizumab, IVIg, rituximab, nintedanib, or an immunosuppressant).

[0158] The observations in this article support the treatment of subjects with high-risk diseases using CD19-targeted CAR T-cell therapy according to the provided methods. For example, subjects with systemic autoimmune diseases (such as severe or moderate systemic autoimmune diseases) can be treated using the provided methods. In some embodiments, subjects with SLE (including those with severe SLE or certain high-risk characteristics, such as those with relapsed / 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 already undergone extensive pretreatment (e.g., with one, two, three, four, or more prior therapies for treating the disease). Any references in this article to methods for treating human or animal bodies by surgical or therapeutic means to compounds, compositions, or agents used in said methods.

[0159] All publications (including patent documents, scientific articles, and databases) mentioned in this application are incorporated herein by reference in their entirety for all purposes, as if each individual publication were incorporated individually by reference. Where the definitions described herein contradict or otherwise are inconsistent with those set forth in patents, applications, publications, and other publications incorporated herein by reference, the definitions described herein shall prevail over those incorporated herein by reference.

[0160] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described. I. Methods and applications of CD19-targeted cell therapy in systemic autoimmune diseases

[0161] This document provides treatment methods involving the administration of engineered cells (such as engineered T cells) or compositions containing engineered cells. In some embodiments, this document provides methods and uses of 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) who have failed at least two or more prior therapies. In a particular embodiment, the method includes administering to the subject a dose of T cells comprising CD4+ and CD8+ T cells, wherein the T cells contain a chimeric antigen receptor (CAR) that specifically binds to CD19. In a particular embodiment, the method includes administering to the subject a dose of T cells comprising CD4+ and CD8+ T cells, wherein the T cells contain a chimeric antigen receptor (CAR) that specifically binds to CD19.

[0162] In some implementation schemes, immune diseases include, but are not limited to, Addison's disease, allergies, ankylosing spondylitis, asthma, atherosclerosis, autoimmune diseases of the ear, autoimmune diseases of the eye, autoimmune hepatitis, autoimmune mumps, colitis, coronary heart disease, diabetes (including type 1 and / or type 2 diabetes), epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, immune responses to recombinant drug products, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, spondyloarthritis, thyroiditis, transplant rejection, vasculitis, AIDS, atopic dermatitis, bronchial asthma, eczema, leprosy, schizophrenia, chronic fatigue syndrome, Alzheimer's disease, Parkinson's disease, myocardial infarction, stroke, autism, epilepsy, Addison's phenomenon, and allergic reactions. In some implementations, systemic autoimmune diseases include, but are not limited to, systemic lupus erythematosus (SLE) and severe SLE, rheumatoid arthritis (RA), and systemic sclerosis. In some implementations, systemic autoimmune diseases may include systemic lupus erythematosus (SLE), Sjögren'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, Behçet's disease, rheumatoid arthritis, primary progressive MS, IgA nephropathy, pemphigus vulgaris, myasthenia gravis, autoimmune hemolytic anemia, immune thrombocytopenic purpura, IgG4-related disease, membranous nephropathy, cutaneous lupus erythematosus, sarcoidosis, etc. Light chain amyloidosis, acute respiratory distress syndrome, atopic dermatitis, 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, Rh hemolytic disease, Still's disease, type 1 diabetes mellitus, urticaria, capillary leak syndrome, cytokine release syndrome, erythema multiforme, pyoderma gangrenosa, X-linked agammaglobulinemia, antiphospholipid syndrome, and chronic inflammatory demyelinating polyneuropathy (also known as chronic inflammatory demyelinating polyradiculopathy).

[0163] In some embodiments, the systemic autoimmune disease is SLE, such as moderate or severe refractory SLE, idiopathic inflammatory myopathy, systemic sclerosis, rheumatoid arthritis (RA), or multiple sclerosis. The provided methods include treatment methods involving administering engineered cells (such as engineered T cells) or compositions containing engineered cells to a subject suffering from SLE, including severe refractory SLE. Methods and uses of the provided CD19-directed CAR-engineered cells (e.g., T cells) and / or compositions thereof are also provided, including methods involving administering engineered cells and / or compositions thereof for treating a subject suffering from SLE, including severe refractory SLE. In some embodiments, the subject suffers from severe refractory SLE. In some embodiments, subjects are selected or identified as having severe refractory SLE, such as by indicating the presence of certain characteristics or clinical manifestations 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 combinations thereof include methods for treating subjects with severe refractory SLE who have failed at least two or more prior therapies. In a particular embodiment, the method includes administering to the subject a dose of T cells comprising CD4+ and CD8+ T cells, wherein the T cells contain a chimeric antigen receptor (CAR) that specifically binds to CD19.

[0164] This document also discloses a method for treating systemic autoimmune diseases, the method comprising administering to a subject suffering from or suspected of having a severe or moderate systemic autoimmune disease a composition comprising engineered T cells expressing a CAR targeting CD19, the composition being generated by initiating a manufacturing process of an output composition exhibiting predetermined characteristics, wherein the repeated generation of said manufacturing process produces a plurality of output compositions, optionally derived from human biological samples comprising a plurality of different individual subjects, wherein the predetermined characteristics of said output compositions in the plurality of output compositions are selected in any combination from characteristics of the compositions disclosed in sections II-C and III, including the percentage of CD3+ cells, the ratio of CD4+ / CD8+ cells or the ratio of CD4+CAR+ / CD8+CAR+ cells, the percentage of cells expressing apoptosis markers, the percentage of poorly differentiated cells, and iVCN values ​​and iVCN / VCN values.

[0165] In some embodiments, the methods and uses include administering, in adoptive cell therapy, cells expressing a genetically engineered (recombinant) cell surface receptor to a subject, said genetically engineered (recombinant) cell surface receptor typically being a chimeric receptor (such as a chimeric antigen receptor (CAR)) that recognizes CD19 expressed by a cell type derived from CD19, is associated with, and / or is specific to said cell type. The cells are typically administered in a composition formulated for administration. In some embodiments, cells are collected from the subject prior to treatment for the purpose of engineering said cells with a CD19-directed recombinant receptor (e.g., CAR). In some embodiments, cells are collected by apheresis. In some embodiments, cells have already been collected by apheresis. In some aspects, cells are engineered by an ex vivo method that does not involve culturing cells for amplification (hereinafter also referred to as a non-amplification process). Exemplary non-amplification processes for engineering provided therapeutic compositions expressing CARs are described in Sections II-C.

[0166] In some embodiments, the subject has received one or more prior therapies for the treatment of an autoimmune disease, such as two or more prior therapies. In some embodiments, the subject has received one prior therapy for the treatment of a systemic autoimmune disease. In some embodiments, the subject has received two prior therapies for the treatment of a systemic autoimmune disease. In some embodiments, the subject has received three prior therapies for the treatment of a systemic autoimmune disease.

[0167] In some embodiments, the systemic autoimmune disease is a refractory disease. In some embodiments, a refractory disease is characterized by a lack of response to one or more prior therapies (such as one or more standard therapies). In some embodiments, a refractory disease is characterized by the absence of complete remission to one or more prior therapies (such as one or more standard therapies). In some embodiments, the subject is refractory to treatment with one or more prior therapies for treating systemic autoimmune diseases. In some embodiments, the subject is refractory to treatment with two or more prior therapies for treating systemic autoimmune diseases.

[0168] In some implementations, systemic autoimmune disease is severe autoimmune disease. In some implementations, severe systemic autoimmune disease is a disease in which the subject has achieved a response to standard therapy, but the response is inadequate or partial. In some implementations, severe systemic autoimmune disease is a disease in which the subject's response can only be achieved in subjects using a combination of standard therapy drugs.

[0169] In some implementations, one or more prior therapies (such as two or more prior therapies) are standard treatments for autoimmune diseases. In some implementations, the standard therapy is an anti-inflammatory drug, a steroid (such as a corticosteroid), an analgesic (e.g., acetaminophen or codeine), or an immunosuppressant, or a combination thereof.

[0170] In some embodiments, the subject has not previously received CAR T-cell therapy before administering CD19-targeted engineered CAR T cells according to the provided method. In some embodiments, the subject has not received genetically modified T-cell therapy. In some embodiments, the subject has not received 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 is not hypersensitive to fludarabine and / or cyclophosphamide.

[0171] In certain implementations, subjects were administered or had received lymphocyte depletion chemotherapy prior to the administration of the dose of CD19-directed engineered CAR T cells. Lymphocyte depletion 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 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). Furthermore, LD chemotherapy can further reduce the risk and severity of cytokine release syndrome (CRS).

[0172] Therefore, in some embodiments, the method includes administering a preconditioning agent, such as a lymphocyte scavenger or a chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof, to the subject prior to the administration of engineered cells. For example, the preconditioning agent may be administered to the subject at least 2 days prior to the administration of engineered cells (e.g., at least 3, 4, 5, 6, 7, 8, or 9 days prior). In some embodiments, the preconditioning agent may be administered to the subject no more than 9 days prior to the administration of engineered cells (e.g., no more than 8, 7, 6, 5, 4, 3, or 2 days prior).

[0173] In some embodiments, subjects are preconditioned with cyclophosphamide at a dose between or equal to about 20 mg / kg and 100 mg / kg of the subject's body weight (e.g., between or equal to about 40 mg / kg and 80 mg / kg). In some aspects, subjects are preconditioned or administered cyclophosphamide at or equal to about 60 mg / kg. In some embodiments, cyclophosphamide may be administered as a single dose or in multiple doses, such as daily, every other day, or every three days. In some embodiments, cyclophosphamide is administered once daily for one or two days. In some embodiments, where the lymphocyte scavenger contains cyclophosphamide, at or equal to about 100 mg / kg of the subject's body weight, cyclophosphamide is administered at or equal to about 100 mg / kg of the subject's body weight. 2 With 500 mg / m 2 Between the subject's body surface area (e.g., at or around 200 mg / m²) 2 With 400 mg / m 2 Between, or 250 mg / m 2 With 350 mg / m 2 Cyclophosphamide was administered to the subject at doses ranging from the midpoint (including the endpoints). In some cases, approximately 100 mg / m² was administered to the subject. 2 Cyclophosphamide. In some cases, subjects were administered approximately 150 mg / m². 2 Cyclophosphamide. In some cases, subjects were administered approximately 200 mg / m². 2 Cyclophosphamide. In some cases, subjects were administered approximately 250 mg / m². 2 Cyclophosphamide. In some cases, subjects were administered approximately 300 mg / m². 2 Cyclophosphamide. In some embodiments, cyclophosphamide may be administered as a single dose or in multiple doses, such as daily, every other day, or every three days. In some embodiments, cyclophosphamide is administered daily, such as for 1-5 days, for example, for 3 to 5 days. In some cases, approximately 300 mg / m² is administered to the subject daily prior to initiating cell therapy. 2 Cyclophosphamide was administered to the subject's body surface area for 3 days. In some embodiments, a total of approximately 300 mg / m² was administered to the subject before initiating 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 / m2 1800 mg / m 2 2000 mg / m 2 2500mg / 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 Cyclophosphamide or cyclophosphamide within the range defined by any of the foregoing values.

[0174] In some implementations, where the lymphocyte scavenger contains fludarabine, the concentration is at or approximately 1 mg / m². 2 With or approximately 100 mg / m 2 Between (e.g., at or approximately 10 mg / m²) 2 With or approximately 75 mg / m 2 Between, or approximately 15 mg / m 2 With or approximately 50 mg / m 2 Between, or approximately 20 mg / m 2 With or approximately 40 mg / m 2 Between, or approximately 24 mg / m 2 With or approximately 35 mg / m 2 Fludarabine was administered to the subject at doses ranging from (including extreme values). In some cases, the dose administered to the subject was 10 mg / m² or approximately 10 mg / m². 2 Fludarabine. In some cases, subjects were administered doses of approximately 15 mg / m². 2 Fludarabine. In some cases, subjects were administered doses of approximately 20 mg / m². 2 Fludarabine. In some cases, subjects were administered doses of approximately 25 mg / m². 2 Fludarabine. In some cases, subjects were administered doses of approximately 30 mg / m². 2 Fludarabine. In some embodiments, fludarabine may be administered as a single dose or in multiple doses, such as daily, every other day, or every three days. In some embodiments, fludarabine is administered daily, such as for 1-5 days, for example, for 3 to 5 days. In some cases, at or approximately 30 mg / m² is administered to the subject daily before initiating cell therapy. 2 Fludarabine was administered to the subject's body surface area for 3 days. In some implementations, a total of approximately 10 mg / m² was administered to the subject before initiating cell therapy. 2 20 mg / m2 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 cyclophosphamide, or cyclophosphamide within the range defined by any of the foregoing values.

[0175] In some embodiments, the lymphocyte scavenger comprises a single agent, such as cyclophosphamide or fludarabine. In some embodiments, only cyclophosphamide is administered to the subject, without fludarabine or other lymphocyte scavengers. In some embodiments, the subject has received lymphocyte scavenging therapy prior to administration, which includes daily administration of approximately 200-400 mg / m². 2 Subject body surface area, optionally 300 mg / m² 2 Cyclophosphamide is administered for 2-4 days. In some embodiments, only fludarabine is administered to the subject, for example, without cyclophosphamide or other lymphocyte scavenging agents. In some embodiments, the subject has received lymphocyte scavenging therapy prior to this administration, which includes daily administration of approximately 20-40 mg / m². 2 Subject body surface area, optionally 30 mg / m² 2 Fludarabine, lasting 2-4 days.

[0176] In some embodiments, the lymphocyte scavenger comprises a combination of agents, such as cyclophosphamide and fludarabine. Therefore, the combination of agents may include cyclophosphamide at any dosage or administration regimen (as described above) and fludarabine at any dosage or administration regimen (as described above). For example, in some aspects, a dose of approximately 60 mg / kg (approximately 2 g / m²) is administered to the subject prior to the first or subsequent doses. 2 ) cyclophosphamide and 3 to 5 doses of 25 mg / m 2 Fludarabine. In some cases, fludarabine (30 mg / m²) was administered intravenously to the subject prior to cell administration. 2 / day, for 3 consecutive days) and cyclophosphamide (300 mg / m 2 / day, for 3 days (flu / cy). In some implementations, a reduced, delayed, or eliminated dose of one or more doses of one or more lymphocyte scavengers is administered to the subject.

[0177] In some embodiments, the subject is given a prophylactic medication, for example, to minimize the risk of infusion reaction. In some aspects, the prophylactic medication includes the administration of an analgesic and / or an antihistamine. In some embodiments, the prophylactic medication includes the administration of acetaminophen and / or diphenhydramine or another H1-antihistamine. In some embodiments, the patient receives acetaminophen (e.g., 650 mg orally) and diphenhydramine (e.g., 25-50 mg, IV or orally) or another H1-antihistamine approximately 30 to 60 minutes prior to or after treatment with cell therapy.

[0178] In any implementation of the provided method, the subject is a human subject. A. Exemplary Diseases

[0179] In some implementations, the methods provided herein are used to treat autoimmune diseases caused by, associated with, and / or specific to CD19-expressing cells, 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 polyradiculopathy, autoimmune hemolytic anemia, immune thrombocytopenic purpura, IgG4-related diseases, membranous nephropathy, primary Sjögren's syndrome, cutaneous lupus erythematosus, and nodular rash. Diseases, light chain amyloidosis, 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, Rh hemolytic disease, Still's disease, type 1 diabetes mellitus, urticaria, capillary leak syndrome, cytokine release syndrome, erythema multiforme, pyoderma gangrenosa, antiphospholipid syndrome, or X-linked agammaglobulinemia.

[0180] 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.

[0181] In some implementations, the methods provided herein are used to treat rheumatoid arthritis. In some implementations, the systemic autoimmune disease is rheumatoid arthritis.

[0182] In some implementations, the methods provided herein are used to treat myositis. In some implementations, the systemic autoimmune disease is myositis.

[0183] In some implementations, the methods described herein are used to treat myasthenia gravis. In some implementations, the systemic autoimmune disease is myasthenia gravis.

[0184] In some implementations, the methods provided herein are used to treat bullous pemphigoid. In some implementations, the systemic autoimmune disease is bullous pemphigoid.

[0185] In some implementations, the methods described herein are used to treat immune thrombocytopenic purpura. In some implementations, the systemic autoimmune disease is immune thrombocytopenic purpura.

[0186] In some implementations, the methods provided herein are used to treat autoimmune hemolytic anemia. In some implementations, the systemic autoimmune disease is autoimmune hemolytic anemia.

[0187] In some implementations, the methods provided herein are used to treat pemphigus vulgaris. In some implementations, the systemic autoimmune disease is pemphigus vulgaris.

[0188] In some embodiments, the methods provided herein are used to treat demyelinating polyradiculopathy. In some embodiments, the systemic autoimmune disease is demyelinating polyradiculopathy.

[0189] In some implementations, the methods provided herein are used to treat membranous nephropathy. In some implementations, the systemic autoimmune disease is membranous nephropathy. 1. Systemic lupus erythematosus (SLE)

[0190] Systemic lupus erythematosus (SLE) is a systemic autoimmune disease caused by abnormal activity of the immune system, resulting in variable clinical symptoms. SLE is characterized by the production of autoantibodies against nuclear and cytoplasmic antigens, which can affect several different organs, causing a wide range of clinical and immunological abnormalities, and is characterized by a clinical course of relapse and remission. (Yu H, Nagafuchi Y, Fujio K. Clinical and Immunological Biomarkers for Systemic Lupus Erythematosus. Biomolecules. 2021-06-22;11(7):928.). SLE presents with a range of clinical manifestations, including renal, dermatological, neuropsychiatric, and cardiovascular symptoms. The complexity, heterogeneity, and variability of lupus have historically led to a focus on symptom-based treatment rather than disease-based treatment. The basis for the heterogeneity of lupus disease includes genetics, pathogenesis (pathways, autoantibodies), demographics, ethnicity and race, and socioeconomic factors. This has led to a variety of challenges, including prognosis, optimization of therapies, efficacy and safety, and clinical trial design. (Bazzan M, Vaccarino A, Marletto F. Systemic lupus erythematosus and thrombosis. ThrombJ. 2015 Apr 23;13:16. doi: 10.1186 / s12959-015-0043-3.)

[0191] In some embodiments, the systemic autoimmune disease is SLE, such as moderate SLE or severe refractory SLE. The provided methods include treatments involving administering engineered cells (such as engineered T cells) or compositions containing engineered cells to a subject suffering from SLE, including severe refractory SLE. Methods and uses of the provided CD19-directed CAR-engineered cells (e.g., T cells) and / or compositions thereof are also provided, including methods involving administering engineered cells and / or compositions thereof for treating a subject suffering from SLE, including severe refractory SLE. In some embodiments, the subject suffers from severe refractory SLE. In some embodiments, the subject is selected or identified as having severe refractory SLE, such as by indicating the presence of certain characteristics or clinical manifestations of severe refractory SLE. Exemplary selection criteria are further described herein. In some embodiments, the provided 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 suffering from severe refractory SLE who has failed at least two or more prior therapies. In a particular embodiment, the method includes administering to a subject a dose of T cells comprising CD4+ and CD8+ T cells, wherein the T cells contain a chimeric antigen receptor (CAR) that specifically binds to CD19.

[0192] Clinical features of severe SLE during an attack and during its progression include, but are not limited to, erythema lutea, arthritis, nephropathy, photosensitivity, thrombosis, Sjögren's syndrome, serositis, nephropathy, neurological involvement, oral ulcers, thrombocytopenia, lymphadenopathy, discoid lesions, livedo reticularis, thrombosis, myositis, hemolytic anemia, pulmonary involvement, 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). Mar 1993;72(2):113-24.PMID: 8479324). These disease manifestations contribute to a significant disease burden and can lead to decreased physical function, unemployment, reduced health-related quality of life (QoL), and a 10-year reduction in lifespan. Increased hospitalizations and side effects from medications, including chronic oral corticosteroids (OCS or glucocorticoids and other immunosuppressive therapies), increase the disease burden of SLE.

[0193] In some cases, the subject develops lupus nephritis. Lupus nephritis (LN) is one of several proteinuric nephropathy conditions in which kidney inflammation is caused by systemic lupus erythematosus (SLE), which accounts for up to 60% of SLE patients. LN is a debilitating and expensive disease that often leads to kidney failure, requiring dialysis or a kidney transplant, and frequently results in death. In fact, patients with kidney failure have a more than 60-fold increased risk of premature death compared to general SLE patients. The clinical sign of LN is the leakage of blood proteins into the urine, and the disease can be diagnosed by many factors, including the urine protein / creatinine ratio (UPCR), where a UPCR greater than 0.5 mg / mg indicates active disease. In addition, certain markers in the blood can also be diagnostic, such as complement 3 (C3), complement 4 (C4), and anti-dsDNA antibodies.

[0194] Treating SLE is challenging due to the limited efficacy and poor tolerability of standard therapies. All current treatments for SLE have well-known side effect profiles, and there is a medical need to identify new targeted therapies, particularly those that can reduce the need for corticosteroids and nonspecific cytotoxic agents.

[0195] In the approximately 50 years since hydroxychloroquine was approved for use in discoid lupus and SLE, the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have approved only one new treatment for SLE (belimumab). However, belimumab is not approved everywhere and its use is limited. Many agents currently used to treat SLE, such as azathioprine, cyclophosphamide, and mycophenolate mofetil (MMF) / mycophenolic acid, are not approved for the aforementioned disease. Furthermore, these drugs have well-documented safety issues and are not effective for all manifestations of lupus in all patients. Antimalarial drugs (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. It is often difficult to gradually wean subjects with moderate or severe disease off OCS completely, which can lead to long-term morbidity and potentially early cardiovascular death. Even low daily doses of prednisone, such as 5 to 10 mg, can increase the risk of side effects (such as cataracts, osteoporosis, and coronary artery disease) with long-term use.

[0196] Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used for the symptomatic management of joint pain, mild arthritis, myalgia, serositis, and fever in patients with SLE. They do not have any immunosuppressive properties. NSAIDs should only be used for short periods and are not suitable for patients with kidney involvement, hypertension, or a confirmed heart condition. NSAIDs may cause fluid retention, kidney damage, and interstitial nephritis.

[0197] Mycophenolate mofetil (MMF) is a specific inhibitor of inosine monophosphate dehydrogenase. MMF impairs de novo purine synthesis. Inosine monophosphate dehydrogenase is an essential pathway for activation in lymphocytes. Therefore, MMF inhibits both T and B lymphocyte proliferation and reduces antibody synthesis.

[0198] In some cases, rituximab has been used to treat subjects with SLE, particularly lupus nephritis. Rituximab is a chimeric anti-CD20 monoclonal antibody. Rituximab is an effective treatment for a variety of autoimmune diseases, including rheumatoid arthritis and ANCA vasculitis. A small number of uncontrolled trials in lupus nephritis suggest that rituximab may also be potentially effective in patients with lupus nephritis.

[0199] In some implementations, inhibitors of type I interferon (IFN) have been used to treat SLE. Type I interferon (IFN) is a cytokine that forms a key link between innate and adaptive immunity, and is associated with SLE based on genetic susceptibility data and the upregulation of interferon-stimulated gene expression in most SLE patients. Sifamumab is an anti-interferon-alpha monoclonal antibody. Efficacy and safety of sifamumab have been observed in some subjects, but treatment efficacy is generally limited. Anilumab (MEDI-546) is a monoclonal antibody that binds to IFNAR. Compared to placebo, anilumab reduced disease activity in patients with moderate to severe SLE; however, its efficacy has not met all primary endpoints.

[0200] Many patients with SLE (including severe SLE) exhibit inadequate or refractory responses to existing treatments, such as those using any two or more of the following: MMF, CYC, belimumab, rituximab, anilumab, azathioprine, mTX, csp, and vorticol. Patients with severe refractory SLE are often young adults facing lifelong treatment, frequent relapses, and cumulative organ dysfunction over time. Despite advances in SLE therapy, a large proportion of patients with severe SLE remain unresponsive and / or relapsed, and are at high risk of organ failure or death. There is a significant unmet need for SLE therapies with better efficacy and safety profiles than currently available treatments, particularly among patients with severe refractory SLE.

[0201] In some implementations, a diagnosis of SLE can be made based on 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 the presence of clinical features including: discoid rash, oral ulcers, arthritis, serositis, renal disorders, neurological disorders, hematologic disorders, and immune disorders. If a mammal (e.g., a human) scores at least 10 points according to the weighted criteria, he or she can be clinically classified as having SLE. In some implementations, a diagnosis of SLE can be made based on the presence of detectable SLE-related antibodies in the subject's blood. In some implementations, these antibodies include anti-dsDNA, anti-histone, anti-chromatin, and / or anti-Sm antibodies. In some implementations, the subject has severe SLE, characterized by at least one organ system being classified as BILAG A or at least two organ systems being classified 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 the treating physician’s intention to treat (Isenberg et al., 2005). The “organ system” criteria used in conjunction with BILAG refer to the following nine systems considered in the BILAG 2004 index: constitution, mucocutaneous system, central nervous system, musculoskeletal system, cardiovascular / respiratory system, abdomen, kidneys, and blood. The BILAG 2004 assessment consists of 101 questions (plus 5 additional items primarily used to calculate glomerular filtration rate). Each question is answered as: 0 = absent; 1 = improved; 2 = same; 3 = worsened; 4 = new. The index records disease activity occurring over the past 4 weeks compared to the previous 4 weeks. Based on the scores for each of these questions, a predefined algorithm specific to each system provides a disease activity score ranging from A to E for each system.

[0202] A = 12, defined as severe disease requiring moderate / high doses of corticosteroids (>20 mg prednisolone or equivalent) and / or initiation or addition of immunosuppressive drugs, or high-dose anticoagulation therapy (INR > 3) (Yee et al., Rheumatology, 2010). In some implementations, grade A indicates very active disease requiring immunosuppressive drugs and / or a dose of prednisolone > 20 mg / day or equivalent;

[0203] B = 8 is defined as disease activity requiring slightly lower doses of immunosuppressants (e.g., < 20 mg prednisolone) and / or specific medications (such as antimalarial drugs, antiepileptic drugs, antidepressants, and NSAIDs) or topical steroids. In some implementations, B indicates moderate disease activity requiring lower doses of corticosteroids, topical steroids, topical immunosuppressants, antimalarial drugs, or NSAIDs.

[0204] C = 1 is defined as mild, persistent, active disease requiring only symptomatic treatment (e.g., analgesics or NSAIDs). In some implementations, C indicates mild, stable disease.

[0205] D = 0, defined as an organ or system that was once active but is no longer active. In some implementations, D indicates no disease activity, but the system was previously affected; and

[0206] E = 0, defined as an organ or system that has never been active. In some implementations, E level indicates the absence of current or prior disease activity.

[0207] In some implementations, the subject has OCS-related organ damage. OCS may include prednisone, prednisolone, and / or methylprednisolone. In some implementations, the subject may be selected as having SLE that is unresponsive to OCS treatment.

[0208] In some implementations, the subject's SLEDAI disease activity score is ≥ 10, which is an indicator of SLE disease severity.

[0209] In some implementations, severe illness is based on the presence of major organ involvement (at least one of kidney, nervous system, cardiovascular or respiratory system involvement) and the need for treatment with >7.5 mg / day of corticosteroids or immunosuppressants.

[0210] In some embodiments, the subject has previously received prior treatment with the following: glucocorticoids, antimalarial drugs, immunosuppressants, anti-CD20 antibodies, IFN inhibitors, or soluble B-lymphocyte stimulating agents (BLyS) inhibitors. In some embodiments, the immunosuppressants are azathioprine, cyclosporine (CSP), cyclophosphamide (CYC), imidazolidinedione, mycophenolate mofetil (MFF), mycophenolic acid, and / or methotrexate (MTX). In some embodiments, the glucocorticoids are oral corticosteroids such as prednisone, prednisolone, and / or methylprednisolone. In some embodiments, the antimalarial drug is hydroxychloroquine. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, the IFN inhibitor is anilumab. In some embodiments, the BLyS inhibitor is belimumab.

[0211] In some embodiments, the subject is refractory to treatment with two or more prior therapies. In some embodiments, the two or more prior therapies (e.g., 2, 3, 4, 5 or more prior therapies) are selected from any two or more of the following: mycophenolic acid or a derivative thereof, cyclophosphamide (CYC), belimumab, rituximab, anilumab, azathioprine, methotrexate (mtx), cisplatin (CSP), ostotuzumab, cyclosporine, tacrolimus, and / or vorticol. In some embodiments, methotrexate and azathioprine are counted as 1 for the purpose of determining the number of failed treatments. In some embodiments, the two or more prior therapies (e.g., 2, 3, 4, 5 or more prior therapies) are selected from any two or more of the following: mycophenolic acid esters (MFF), cyclophosphamide (CYC), belimumab, rituximab, anilumab, azathioprine, methotrexate, cyclosporine (csp), or vorticol. In some embodiments, the subject has received two or more prior treatments for lupus (e.g., 2, 3, 4, 5 or more prior treatments), resulting in an inadequate response (e.g., as measured by SLE disease activity). In some embodiments, the subject has an inadequate response to two prior treatments. In some embodiments, the subject has an inadequate response to three prior treatments. In some embodiments, the subject has an inadequate response to four or more prior treatments. In some embodiments, the subject has failed to achieve clinical remission after treatment with any two or more prior treatments for lupus (e.g., three months after a given treatment). In any embodiment, the subject is identified or selected as having an inadequate response to prior treatment prior to apheresis associated with the engineered CD19-directed CAR T-cell composition. An inadequate response to treatment is defined as a lack of response to an appropriate dose, insufficient response, or lack of sustained response. Intolerance is not considered an inadequate response.

[0212] In some implementations, the subject does not have drug-induced SLE. In some implementations, the subject does not have other systemic autoimmune diseases, including but not limited to multiple sclerosis, psoriasis, and / or inflammatory bowel disease. In some implementations, the subject does not have SLE overlap syndromes, including but not limited to rheumatoid arthritis, scleroderma, and / or mixed connective tissue disease. In some implementations, the subject does not have clinically significant CNS symptoms. a. Reactions and Efficacy

[0213] In some implementations, the methods and uses provided for administering anti-CD19 CAR T-cell therapy reduce SLE disease activity in subjects.

[0214] In some implementations, treatment effectively reduces lupus disease activity. In some implementations, lupus disease activity is measured by selecting from the following disease activity scores: the British Isles Lupus Assessment Group 2004 (BILAG), the SLE Disease Activity Index (SLEDAI-2K), the SLEDAI-2K Response Index 50 (SRI-50), the Composite SLE Response Index (cSR), the Minimum Clinically Important Difference (MCID), the Patient-Reported Brief Quality of Life Assessment (SF-36), the Physical Health Overall Assessment (PCS) and / or the Mental Health Overall Assessment (MCS), and the Lupus-Specific Quality of Life Scale (Lupus-QOL) or a combination thereof.

[0215] In some implementations, reducing SLE disease activity in a subject may include one or more of the following: a subject’s response to the BILAG-based Comprehensive Lupus Assessment (BICLA); a reduction in the subject’s CLASI score compared to the subject’s pre-treatment SLE area and severity index (CLASI) score; a reduction in the subject’s pre-treatment tender and swollen joint count compared to the subject’s pre-treatment tender and swollen joint count; a subject having a BILAG-2004 B score of 1 or higher after treatment; a subject having a BILAG-2004 score of C or better after treatment; an improvement in at least one patient-reported outcome (PRO) compared to the subject’s pre-treatment score; or a reduction in the subject’s SLE flare rate compared to the subject’s pre-treatment flare rate.

[0216] In some implementations, the subject's BILAG score may be measured before and after administration of CD19-targeted cell therapy. In some implementations, patient-reported outcomes (PROs) may be measured in subjects before and after administration of CD19-targeted cell therapy. PROs may include the subject's Functional Assessment of Treatment for Chronic Disease-Fatigue (FACIT-F), SF-36-v2 Mental Health Overall Score (MCS), and / or SF-36 Physical Health Overall Score (PCS).

[0217] In some implementations, the treatment results in a decrease of one point in any organ system classified as BILAG A or at least two organ systems classified as BILAG B in a subject who at baseline has at least one organ system classified as BILAG A or at least two organ systems classified as BILAG B, without any other organ system deteriorating to BILAG A or B.

[0218] In some implementations, reducing SLE disease activity in a subject includes a BILAG-based Comprehensive Lupus Assessment (BICLA) response. In some implementations, reducing SLE disease activity in a subject includes a BICLA response up to at least week 4 of treatment. In some implementations, reducing SLE disease activity includes a BICLA response up to at least week 8 of treatment. In some implementations, a BICLA response can persist in the subject for at least 52 weeks. In some implementations, a BICLA response includes a reduction in the subject's BILAG-2004 A and B domain scores to B / C / D and C / D, respectively.

[0219] In some implementations, the treatment results in subjects who at baseline had at least one organ system classified as BILAG A or at least two organ systems classified as BILAG B having all organ systems classified as BILAG C or BILAG D / E after treatment.

[0220] In some implementations, the minimum clinically important difference (MCID) in SRI-50 caused by treatment is one.

[0221] In some implementations, treatment results in subjects with at least one BILAG A or at least two BILAG B organ systems at baseline being classified as BILAG C or BILAG D / E after treatment, and showing no worsening as measured by SLEDAI-2K after treatment. In some implementations, the SLE Disease Activity Index “SLEDAI-2K” (also known as “SLEDAI”) is an empirical tool developed as a comprehensive assessment of disease activity in SLE patients (Gladman et al., 2002). It represents the consensus of an expert group in the field of lupus research. SLEDAI-2K assesses 24 descriptors (sixteen clinical manifestations and eight laboratory measures) across nine organ systems. Descriptors are given different weights based on clinical importance, using a dichotomous score (presence / absence in the previous 30 days). A descriptor must be classified as active SLE or should not be scored. SLEDAI-2K is designed to assess current lupus activity rather than chronic lesions. In some implementations, in the case of SLEDAI-2K, worsening means an increase in disease activity as measured by SLEDAI-2K.

[0222] In some implementations, disease activity is monitored using the SRI-50. The SRI-50 is the SLE Disease Activity Index, which contains the same 24 descriptors covering nine organ systems. It generates a total score that reflects disease activity over the previous 30 days, similar to SLEDAI-2K (Touma et al., 2012). Each of the SRI-50 descriptors is defined to identify a 50% or greater improvement, and a score is generated for that descriptor. Overall, the SRI-50 is an index developed to reflect some significant improvement in disease activity between visits.

[0223] In some implementations, treatment results in an SRI (Systemic Lupus erythematosus Response Index) > 4 or an SRI(4). A subject achieves an SRI(4) if all of the following criteria are met: a decrease of > 4 points in SLEDAI-2K relative to baseline; no new organ systems affected, as defined by using one or more BILAG-2004 A items or two or more BILAG-2004 B items compared to baseline using BILAG-2004; and no worsening of lupus disease activity relative to baseline, with worsening defined as an increase of > 0.30 points in the 3-point PGA VAS.

[0224] In some implementations, SRI(X) (X = 5, 6, 7, or 8) is defined as the proportion of subjects who meet the following criteria: a decrease in SLEDAI-2K score relative to baseline of >X points; no new organ systems affected, as defined by using one or more BILAG-2004 A items or two or more BILAG-2004 B items compared to baseline using BILAG-2004; and no worsening of lupus disease activity relative to baseline, with worsening defined as an increase in PGA VAS score of >0.30 points.

[0225] In some implementations, disease activity is monitored by the “composite SLE response index” (cSRI), an SLE disease index that incorporates two different systems: BILAG and SLEDAI-2K, defined as a substantial response as measured by BILAG 2004 and no exacerbation as measured by SLEDAI-2K.

[0226] In some implementations, treatment results in a subject’s SELENA-SLEDAI score of 4 or greater, wherein the subject has no new organ system classified as BILAG A or no more than one organ system classified as BILAG B, and wherein the subject’s physician comprehensive assessment score increases by less than 0.3.

[0227] In some implementations, the CLASI (Cut Lupus Erythematosus Area and Severity Index) is used to monitor disease activity. The 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 treatment responder. In certain implementations, treatment with CD19-targeted cell therapy, as provided, results in a CLASI score reduction of at least 50% compared to the subject's baseline score. In some implementations, the CLASI is an empirically validated index for assessing cutaneous lesions of SLE and consists of two separate scores: a first score summarizing the inflammatory activity of the disease; and a second score measuring the damage caused by the disease. The activity score considers erythema, scaling / hypertrophy, mucosal lesions, recent alopecia, and non-scarring alopecia. The damage score represents depigmentation, scarring / atrophy / panniculitis, and scalp scarring. Subjects are asked if their depigmentation has lasted for 12 months or longer; in such cases, the depigmentation score is doubled. Each of the parameters above was measured at 13 different anatomical locations, which were specifically included because they are most commonly involved in cutaneous lupus erythematosus (CLE). The most severe lesions in each region were measured.

[0228] In some implementations, treatment leads to subjects achieving a low-disease-activity status (LLDAS) for lupus. LLDAS is a comparable, validated target for SLE to measure low disease activity. LLDAS is defined as (1) an SLE Disease Activity Index (SLEDAI)-2K ≤ 4 with no activity in major organ systems, (2) no new lupus disease activity, (3) a SELENA-SLEDAI Physician Comprehensive Assessment (scale 0-3) ≤ 1, (4) a current prednisolone (or equivalent) dose ≤ 7.5 mg / day, and (5) a well-tolerated standard maintenance dose of immunosuppressive drugs and approved biologics (Franklyn et al., 2015).

[0229] In some implementations, treatment resulted in significant changes in the subject's SF-36 PCS and / or MCS relative to baseline. In some implementations, the Patient-Reported Brief Quality of Life Assessment (SF-36) is a widely validated, universal patient questionnaire that has shown sensitivity to changes in a variety of chronic conditions: hypertension and cardiovascular disease, diabetes, lung 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 scored on a separate "domain" scale: physical functioning, physical role, bodily pain, general health status, vitality, social functioning, emotional role, and mental health (Ware et al., Medical Care, 1992). These eight scales can be combined into two aggregate measures: the overall physical (PCS) and mental (MCS) health score.

[0230] In some implementations, treatment resulted in a significant change in the subject's Health Assessment Questionnaire-Disability Index (HAQ-DI) relative to baseline. In some implementations, the patient-reported quality of life assessment was a widely validated generic patient questionnaire that measures the difficulty of performing activities of daily living. These questions were rated on a 0-3 scale, where 0 indicated "no difficulty" and 3 indicated "cannot complete" (Allanore et al., 2020).

[0231] In some implementations, reducing SLE disease activity in a subject results in an improvement of at least 50% in the subject's tender and swollen joint counts compared to pre-treatment counts. In some implementations, the swollen and tender joint counts are based on the left and right shoulder, elbow, wrist, metacarpophalangeal joints (MCP1, MCP2, MCP3, MCP4, MCP5), proximal interphalangeal joints (PIP1, PIP2, PIP3, PIP4, PIP5) of the upper extremities, and the left and right knees of the lower extremities. Active joints used for joint count assessment are defined as joints exhibiting tenderness and swelling.

[0232] In some implementations, reducing SLE disease activity in subjects includes preventing subject outbreaks. In some implementations, an outbreak may be defined as ≥ 1 new BILAG-2004 A score or ≥ 2 new (worsening) BILAG-2004 B score compared to the subject's score in the previous month.

[0233] In some implementations, the treatment resulted in an increase in the time to first confirmation of a severe SLE outbreak or the time to first confirmation of a major SLE outbreak.

[0234] In some implementations, treatment results in an increased time to the first confirmed severe SLE outbreak, and severe SLE outbreak includes the subject having any new organ system classified as BILAG A or any two new organ systems classified as BILAG B.

[0235] In some implementations, the treatment results in an increased time to the first confirmed major SLE outbreak as defined by Fortin, which includes the initiation or increase of immunosuppressive or high-dose corticosteroid therapy, hospitalization, or death from SLE.

[0236] In some embodiments, the treatment method reduces the dose of OCS administered to the subject compared to the pre-treatment dose of oral corticosteroids (OCS). In some embodiments, reducing SLE disease activity in a subject is characterized by a reduction in the subject's flare rate compared to the pre-treatment flare rate, wherein said method includes reducing the dose of OCS administered to the subject compared to the pre-treatment dose of OCS. In some embodiments, the OCS includes prednisone, prednisolone, and / or methylprednisolone.

[0237] In some implementations, treatment results in significant changes in the cumulative damage index of subjects, as measured by the Systemic Lupus International Collaboration Clinic / American College of Rheumatology Damage Index (SLICC / ACR DI). In some implementations, the SLICC / ACR is an index of cumulative organ damage (Dayal et al., Lupus 2002). SLE damage is defined as irreversible changes that have been present in an organ or system for at least 6 months.

[0238] In some implementations, the treatment resulted in significant changes in the subject's daily glucocorticoid dose.

[0239] In some implementations, the treatment resulted in a significant improvement in the lupus QOL of the subjects.

[0240] In some implementations, the treatment resulted in a significant improvement in a comprehensive assessment of disease activity based on the minimum clinically significant difference (MCID). In some implementations, MCID refers to a patient-source score that reflects a change in clinical intervention that was meaningful to the patient.

[0241] In some implementations, the method reduces SLE disease activity in the subject, such as by reducing the subject's anti-dsDNA level.

[0242] In some implementations, subjects treated according to the provided methods are evaluated or monitored after a period of time to determine whether a complete or partial remission has occurred. In some implementations, subjects are evaluated or monitored to assess whether the remission achieved according to measurements has been maintained.

[0243] In some implementations, remission is monitored using the Definition of Remission in Systemic Lupus Erythematosus (DORIS) (Revised: 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 implementations, remission is defined as a SLE Disease Activity Index (SLEDAI) score of 0 and a comprehensive evaluator score < 0.5 (0–3). Subjects may use stable antimalarial drugs, immunosuppressants, biologics, and / or low-dose glucocorticoids (5 mg / day or less prednisolone).

[0244] In some implementations, the subject has lupus nephritis. In some implementations, efficacy may be evaluated based on the urinary protein / creatinine ratio (UPCR), where a ratio ≤ 0.5 mg / mg indicates complete remission; alternatively or additionally, it may indicate an eGFR ≥ 60 mL / min / 1.73 m2, or no decrease relative to baseline and an eGFR ≥ 20%. Other indicators of complete remission include not requiring rescue medication, such as intravenous steroids, cyclophosphamide, or requiring ≤ 10 mg of prednisone for more than three consecutive days or more than seven days in total. In some implementations, complete remission (CR) is defined as: a confirmed protein / creatinine ratio ≤ 0.5 mg / mg and an eGFR ≥ 60 mL / min / 1.73 m2 or no confirmed eGFR decrease relative to baseline ≥ 20%. Partial remission is defined as: a UPCR decrease of 50% relative to baseline.

[0245] In some embodiments, treatment according to the provided method results in clinical remission of SLE in the subject, and the clinical remission lasts for more than 3 months. In some embodiments, treatment according to the provided method results in clinical remission of SLE in the subject, and the clinical remission lasts 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 longer. In some embodiments, treatment according to the provided method results in clinical remission of SLE in the subject, and the clinical remission lasts for more than 6 months. In some embodiments, treatment according to the provided method results in clinical remission of SLE in the subject, and the clinical remission lasts for more than 12 months. In some embodiments, treatment according to the provided method results in clinical remission of SLE in the subject, and the clinical remission lasts for more than 24 months. In some embodiments, treatment according to the provided method results in clinical remission of SLE in the subject, and the clinical remission lasts for more than 3 years. In some embodiments, treatment according to the provided method results in clinical remission of SLE in the subject, and the clinical remission lasts for more than 4 years. In some implementations, treatment according to the provided method results in clinical remission of SLE in the subject, and the clinical remission is maintained for more than 5 years.

[0246] In some implementations, treatment according to the provided method results in prolonged remission. In some implementations, prolonged remission is defined as five consecutive years without disease activity (SLE Disease Activity Index, SLEDAI = 0) and without treatment (corticosteroids, antimalarial drugs, or immunosuppressants).

[0247] In some cases, the pharmacokinetics of the administered cells (e.g., adoptive cells) are determined to assess the utilization, such as bioavailability, of the administered cells. Methods for determining the pharmacokinetics of adoptive cells may include drawing peripheral blood from a subject who has been administered engineered cells and determining the number or ratio of the engineered cells in the peripheral blood. Methods for selecting and / or isolating cells may 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 in the CAR, thereby directly evaluating the CAR using Strep-Tag binding reagents (Liu et al. (2016) Nature Biotechnology, 34:430; International Patent Application Publication No. WO 2015095895); and monoclonal antibodies that specifically bind to CAR peptides (see International Patent Application Publication No. WO 2014190273). In some cases, engineered cell therapies may be combined with non-inherent marker genes to allow for the detection or selection of cells, and in some cases, to promote cell suicide. In some cases, truncated epidermal growth factor receptor (EGFRt) can be co-expressed with a target transgene (CAR) in transduced cells (see, for example, U.S. Patent No. 8,802,374). EGFRt may contain an epitope recognized by an antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibody or binding molecule, which 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 isolate cells expressing the receptor. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. April 2016; 34(4): 430-434.

[0248] In some implementations, the presence of CAR in a biological sample (e.g., blood) obtained from the patient can be determined some time after cell therapy administration. + The number of T cells, for example, to determine the pharmacokinetics of the cells. In some embodiments, CARs are detectable in the blood of the subject or in most subjects so treated by said method. + T cells (optionally CAR) + CD8 + T cells and / or CAR + CD4 +The number of T cells is greater than 1 cell / µL, greater than 5 cells / µL, or greater than 10 cells / µL. 2. Idiopathic inflammatory myopathy (IIM)

[0249] Idiopathic inflammatory myopathy (IIM) is a group of chronic autoimmune disorders that primarily affect proximal muscles. IIM includes dermatomyositis, polymyositis, and other conditions such as immune-mediated necrotizing myopathy (IMNM), many of which involve antisynthetic enzyme syndrome (aSS). aSS is characterized by autoantibodies against aminoacyltransferase synthases that overlap with interstitial lung disease (ILD), myositis, and other conditions. IIM manifestations include skin lesions, muscle fatigue, and weakness, leading to a significantly reduced quality of life and a high risk of developing various serious long-term complications. For example, 10%–25% of IIM cases also have ILD, with 5% of these cases being acute. 15%–25% of patients with IIM have or will develop malignancies. One-third of patients with IIM will develop myocarditis, and the risk of congestive heart failure is increased. The 10-year survival rate for IIM is 70% across multiple indications. Currently, only two drugs are approved for IIM, including IVIg and Acthar gel; no drugs are approved for aSS. There is strong evidence that B cells are involved, and IVIg is approved for dermatomyositis, while rituximab is used as an off-label treatment. Evidence also suggests that B cells play a role in disease pathogenesis, including complete remission of aSS following anti-CD19 CAR T-cell therapy in patients refractory to steroids, rituximab, tacrolimus, and cyclophosphamide.

[0250] 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 antisynthetic enzyme syndrome (aSS). The provided methods include treatments involving administering engineered cells (such as engineered T cells) or compositions containing engineered cells to a subject suffering from IIM, including dermatomyositis, polymyositis, and / or immune-mediated necrotizing myopathy. Methods and uses of the provided CD19-directed CAR-engineered cells (e.g., T cells) and / or compositions thereof are also provided, including methods involving administering engineered cells and / or compositions thereof for treating a subject suffering from IIM, including dermatomyositis, polymyositis, and / or immune-mediated necrotizing myopathy. In some embodiments, the subject has dermatomyositis, polymyositis, and / or immune-mediated necrotizing myopathy. In some embodiments, the subject is selected or identified as having dermatomyositis, polymyositis, and / or immune-mediated necrotizing myopathy, such as by indicating the presence of certain characteristics or clinical manifestations of dermatomyositis, polymyositis, and / or immune-mediated necrotizing myopathy. In some embodiments, the methods and uses of the provided CD19-directed CAR-engineered cells (e.g., T cells) and / or combinations thereof include methods for treating subjects with dermatomyositis, polymyositis, and / or immune-mediated necrotizing myopathy who have failed at least two or more prior therapies. In a particular embodiment, the method includes administering to the subject a dose of T cells comprising CD4+ and CD8+ T cells, wherein said T cells contain a chimeric antigen receptor (CAR) that specifically binds to CD19.

[0251] In any embodiment herein, at or immediately prior to the administration of the composition comprising engineered T cells, the subject relapses after remission following treatment with one or more prior therapies targeting IIM, or becomes refractory to the one or more prior therapies.

[0252] In any embodiment herein, at or immediately prior to administration of the composition comprising engineered T cells, the subject relapsed after remission following treatment with one or more prior therapies targeting IIM, or became refractory to said one or more prior therapies. In any embodiment herein, at or immediately prior to administration of the composition comprising engineered T cells, the subject relapsed after treatment with one or more prior therapies targeting IIM, or became refractory to said one or more prior therapies. In any embodiment herein, the one or more prior therapies targeting IIM do not include an additional dose of CAR-expressing cells.

[0253] In any embodiment of this document, one or more prior therapies for IIM may include corticosteroids, Octagam (IVIg), Acthar, or rituximab. In any embodiment of this document, one or more prior therapies for IIM may include corticosteroids, Octagam, Acthar, or rituximab. In any embodiment of this document, CD19-directed CAR-engineered cells (e.g., T cells) and / or combinations thereof are used to treat patients with IIM who are refractory to prior therapies. a. Reactions and Efficacy

[0254] In some implementations, the methods and uses provided for administering anti-CD19 CAR T-cell therapy reduce IIM disease activity in subjects.

[0255] In some implementations, treatment effectively reduces IIM disease activity. In some implementations, IIM disease activity is measured by a disease activity score selected from the following: International Myositis Assessment and Clinical Study Group (IMACS), Minimum Clinically Important Difference (MCID), Patient-Reported Brief Quality of Life Assessment (SF-36) Physical Health Overall (PCS) and / or Mental Health Overall (MCS), or a combination thereof.

[0256] In some implementations, reducing IIM disease activity in a subject may include one or more of the following: a reduction in the subject's IMACS score after treatment compared to the subject's IMACS score before treatment; a reduction in 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 an improvement in at least one patient-reported outcome (PRO) of the subject compared to before treatment.

[0257] In some implementations, the IMACS score of the subject can be measured before and after administration of CD19-targeted cell therapy. In some implementations, patient-reported outcomes (PROs) are measured in the subject before and after administration of CD19-targeted cell therapy. PROs may include the subject's Functional Assessment of Treatment for Chronic Disease-Fatigue (FACIT-F), SF-36-v2 Mental Health Overall Score (MCS), and / or SF-36 Physical Health Overall Score (PCS).

[0258] In some implementations, treatment resulted in a significant change in the subject's Health Assessment Questionnaire-Disability Index (HAQ-DI) relative to baseline. In some implementations, the patient-reported quality of life assessment was a widely validated generic patient questionnaire that measures the difficulty of performing activities of daily living. These questions were rated on a 0-3 scale, where 0 indicated "no difficulty" and 3 indicated "cannot complete" (Allanore et al., 2020).

[0259] In some implementations, the treatment resulted in a significant improvement in a comprehensive assessment of disease activity based on the minimum clinically significant difference (MCID). In some implementations, MCID refers to a patient-source score that reflects a change in clinical intervention that was meaningful to the patient.

[0260] In some embodiments, treatment according to the provided method results in clinical remission of IIM in the subject, and the clinical remission lasts for more than 3 months. In some embodiments, treatment according to the provided method results in clinical remission of IIM in the subject, and the clinical remission lasts 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 longer. In some embodiments, treatment according to the provided method results in clinical remission of IIM in the subject, and the clinical remission lasts for more than 6 months. In some embodiments, treatment according to the provided method results in clinical remission of IIM in the subject, and the clinical remission lasts for more than 12 months. In some embodiments, treatment according to the provided method results in clinical remission of IIM in the subject, and the clinical remission lasts for more than 24 months. In some embodiments, treatment according to the provided method results in clinical remission of IIM in the subject, and the clinical remission lasts for more than 3 years. In some embodiments, treatment according to the provided method results in clinical remission of IIM in the subject, and the clinical remission lasts for more than 4 years. In some implementations, treatment according to the provided method results in clinical remission of IIM in the subject, and the clinical remission is maintained for more than 5 years.

[0261] In some implementations, treatment according to the provided method results in prolonged remission. In some implementations, prolonged remission is defined as five consecutive years of disease-free and treatment-free (corticosteroids, IVIg, rituximab, or immunosuppressants).

[0262] In some embodiments, the treatment results in a reduction of muscle weakness or a decrease in the progression of muscle weakness. In some embodiments, the treatment results in improved muscle strength. In some embodiments, the treatment results in a reduction of muscle weakness in the upper extremities or a decrease in the progression of muscle weakness. In some embodiments, the treatment results in a reduction of muscle weakness in the lower extremities or a decrease in the progression of muscle weakness. In some embodiments, the treatment results in a reduction of muscle weakness in the neck flexors or a decrease in the progression of muscle weakness. In some embodiments, the treatment results in a reduction of muscle weakness in the proximal muscles or a decrease in the progression of muscle weakness.

[0263] In some implementations, the treatment results in a reduction of skin lesions. In some implementations, the treatment results in a reduction of positive rashes. In some implementations, the treatment results in a reduction of Gottron's papules. In some implementations, the treatment results in a reduction of Gottron's sign.

[0264] In some implementations, the treatment results in a reduction of dysphagia or esophageal motility. In other implementations, the treatment results in an improvement in swallowing or esophageal motility.

[0265] In some embodiments, the treatment reduces the presence of anti-Jo-a (anti-histyl-tRNA synthetase) autoantibodies. In some embodiments, the treatment results in undetectable anti-Jo-a (anti-histyl-tRNA synthetase) autoantibodies. In some embodiments, the treatment results in decreased serum levels of creatine kinase, lactate dehydrogenase, aspartate aminotransferase, and / or alanine aminotransferase.

[0266] In some embodiments, the treatment reduces endomysial infiltration of mononuclear cells surrounding but not penetrating muscle fibers. In some embodiments, the treatment reduces perimysial and / or perivascular infiltration of mononuclear cells. In some embodiments, the treatment reduces perifascicular atrophy. In some embodiments, the treatment reduces rimmed vacuoles present in muscle biopsies.

[0267] In some cases, the pharmacokinetics of the administered cells (e.g., adoptive cells) are determined to assess the utilization, such as bioavailability, of the administered cells. Methods for determining the pharmacokinetics of adoptive cells may include drawing peripheral blood from a subject who has been administered engineered cells and determining the number or ratio of the engineered cells in the peripheral blood. Methods for selecting and / or isolating cells may 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 in the CAR, thereby directly evaluating the CAR using Strep-Tag binding reagents (Liu et al. (2016) Nature Biotechnology, 34:430; International Patent Application Publication No. WO 2015095895); and monoclonal antibodies that specifically bind to CAR peptides (see International Patent Application Publication No. WO 2014190273). In some cases, engineered cell therapies may be combined with non-inherent marker genes to allow for the detection or selection of cells, and in some cases, to promote cell suicide. In some cases, truncated epidermal growth factor receptor (EGFRt) can be co-expressed with a target transgene (CAR) in transduced cells (see, for example, U.S. Patent No. 8,802,374). EGFRt may contain an epitope recognized by an antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibody or binding molecule, which 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 isolate cells expressing the receptor. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. April 2016; 34(4): 430-434).

[0268] In some implementations, the presence of CAR in a biological sample (e.g., blood) obtained from the patient can be determined some time after cell therapy administration. + The number of T cells, for example, to determine the pharmacokinetics of the cells. In some embodiments, CARs are detectable in the blood of the subject or in most subjects so treated by said method. + T cells (optionally CAR) + CD8 + T cells and / or CAR + CD4 +The number of T cells is greater than 1 cell / µL, greater than 5 cells / µL, or greater than 10 cells / µL. 3. Systemic sclerosis (SSc)

[0269] Systemic sclerosis (SSc) is an autoimmune disease that primarily affects the skin and can cause complications in organ systems. The disease is characterized by fibrosis affecting both the skin and internal organs, and there are three main types. The first type is localized SSc, which accounts for approximately 60% of cases and is limited to skin involvement, often accompanied by some vascular and lung involvement. The second type is diffuse SSc, the most severe, accounting for approximately 35% of cases. It is characterized by extensive skin involvement and more severe multi-organ involvement, including interstitial lung disease (ILD) and kidney failure. The third type is sine SSc, the rarest, accounting for approximately 5% of cases. It does not involve the skin but has varying levels of organ involvement. SSc disease progression can lead to fibrosis in the heart, lungs, kidneys, and other organs. Patients experience a severe deterioration in quality of life, with a 10-year survival rate of approximately 72%. An estimated 32,000 patients in the United States have diffuse SSc, of whom approximately half develop ILD. B cells are believed to play a role in the development of SSc, and off-label use of rituximab has shown some efficacy.

[0270] In some embodiments, the systemic autoimmune disease is SSc, such as localized SSc, diffuse SSc, or non-dermal sclerosis SSc. The provided methods include treatments involving administering engineered cells (such as engineered T cells) or compositions containing engineered cells to a subject suffering from SSc, including localized SSc, diffuse SSc, or non-dermal sclerosis SSc. Methods and uses of the provided CD19-directed CAR-engineered cells (e.g., T cells) and / or compositions thereof are also provided, including methods involving administering engineered cells and / or compositions thereof for treating a subject suffering from SSc, including localized SSc, diffuse SSc, or non-dermal sclerosis SSc. In some embodiments, the subject has localized SSc. In some embodiments, the subject is selected or identified as having localized SSc, diffuse SSc, or non-dermal sclerosis SSc, such as by indicating the presence of certain characteristics or clinical manifestations of localized SSc, diffuse SSc, or non-dermal sclerosis SSc. In some embodiments, the methods and uses of the provided CD19-directed CAR-engineered cells (e.g., T cells) and / or combinations thereof include methods for treating subjects with SSc who have failed at least two or more prior therapies. In a particular embodiment, the method includes administering to the subject a dose of T cells comprising CD4+ and CD8+ T cells, wherein the T cells contain a chimeric antigen receptor (CAR) that specifically binds to CD19.

[0271] In any embodiment herein, at or immediately prior to the administration of the composition comprising engineered T cells, the subject relapses after remission following treatment with one or more prior therapies targeting SSc, or becomes refractory to the one or more prior therapies.

[0272] In any embodiment herein, at or immediately prior to the administration of the composition comprising engineered T cells, the subject relapsed after remission following treatment with one or more prior therapies targeting SSc, or became refractory to said one or more prior therapies. In any embodiment herein, at or immediately prior to the administration of the composition comprising engineered T cells, the subject relapsed after treatment with one or more prior therapies targeting SSc, or became refractory to said one or more prior therapies. In any embodiment herein, the one or more prior therapies targeting SSc did not include an additional dose of CAR-expressing cells.

[0273] In any embodiment of this document, if the subject does not have ILD, one or more prior therapies against SSc may include mycophenolate mofetil and / or methotrexate. In any embodiment of this document, if the subject does have ILD, one or more prior therapies against SSc may include mycophenolate mofetil, cyclophosphamide, and / or tocilizumab (ACTEMRA). In any embodiment of this document, one or more prior therapies against SSc may include administration of mycophenolate mofetil, methotrexate, cyclophosphamide, and / or tocilizumab (ACTEMRA), followed by administration of B-cell depletion therapy (such as rituximab) and / or a VEGFR inhibitor (such as nintedanib). In any embodiment of this document, CD19-directed CAR-engineered cells (e.g., T cells) and / or combinations thereof are used to treat patients with SSc who are refractory to prior therapy. a. Reactions and Efficacy

[0274] In some implementations, the methods and uses provided for administering anti-CD19 CAR T-cell therapy reduce SSc disease activity in subjects. In some implementations, the treatment effectively reduces SSc disease activity. In some implementations, SSc disease activity is measured by a disease activity score selected from the following: Modified Rodnan Skin Score, Forced Vital Capacity, European Scleroderma Study Group (EScSG) Index, Minimum Clinically Important Difference (MCID), Patient-Reported Brief Quality of Life Assessment (SF-36) Physical Health Overall (PCS) and / or Mental Health Overall (MCS), or a combination thereof.

[0275] In some implementations, reducing SSc disease activity in a subject may include one or more of the following: a reduction in the subject's EScSG score after treatment compared to the subject's pre-treatment EScSG score; a reduction in the subject's skin effects, ILD, and / or pulmonary hypertension compared to the subject's pre-treatment skin effects, ILD, and / or pulmonary hypertension; or an improvement in at least one patient-reported outcome (PRO) in the subject compared to pre-treatment. In some implementations, the EScSG score of the subject may be measured before and after administration of CD19-targeted cell therapy. In some implementations, patient-reported outcomes (PROs) may be measured in the subject before and after administration of CD19-targeted cell therapy. PROs may include the subject's Functional Assessment of Chronic Disease Treatment-Fatigue (FACIT-F), SF-36-v2 Mental Health Overall Score (MCS), and / or SF-36 Physical Health Overall Score (PCS).

[0276] In some implementations, treatment resulted in a significant change in the subject's Health Assessment Questionnaire-Disability Index (HAQ-DI) relative to baseline. In some implementations, the patient-reported quality of life assessment was a widely validated generic patient questionnaire that measures the difficulty of performing activities of daily living. These questions were rated on a 0-3 scale, where 0 indicated "no difficulty" and 3 indicated "cannot complete" (Allanore et al., 2020).

[0277] In some implementations, the treatment resulted in a significant improvement in a comprehensive assessment of disease activity based on the minimum clinically significant difference (MCID). In some implementations, MCID refers to a patient-source score that reflects a change in clinical intervention that was meaningful to the patient.

[0278] In some implementations, the treatment results in a decrease in the modified Rodnan skin score. In some implementations, the treatment results in a reduction in skin thickness. In some implementations, the treatment results in a reduction in skin thickness of the fingers, hands, forearms, upper arms, face, chest, abdomen, thighs, legs, and / or feet. In some implementations, the treatment reduces the EScSG index score.

[0279] In some embodiments, treatment according to the provided method results in clinical remission of SSc in the subject, and the clinical remission lasts for more than 3 months. In some embodiments, treatment according to the provided method results in clinical remission of SSc in the subject, and the clinical remission lasts 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 longer. In some embodiments, treatment according to the provided method results in clinical remission of SSc in the subject, and the clinical remission lasts for more than 6 months. In some embodiments, treatment according to the provided method results in clinical remission of SSc in the subject, and the clinical remission lasts for more than 12 months. In some embodiments, treatment according to the provided method results in clinical remission of SSc in the subject, and the clinical remission lasts for more than 24 months. In some embodiments, treatment according to the provided method results in clinical remission of SSc in the subject, and the clinical remission lasts for more than 3 years. In some embodiments, treatment according to the provided method results in clinical remission of SSc in the subject, and the clinical remission lasts for more than 4 years. In some implementations, treatment according to the provided method results in clinical remission of SSc in the subject, and the clinical remission is maintained for more than 5 years.

[0280] In some implementations, treatment according to the provided method results in prolonged remission. In some implementations, prolonged remission is defined as five consecutive years without disease activity and without treatment (corticosteroids, methotrexate, mycophenolate mofetil, cyclophosphamide, tocilizumab, IVIg, rituximab, nintedanib, or immunosuppressants).

[0281] In some cases, the pharmacokinetics of the administered cells (e.g., adoptive cells) are determined to assess the utilization, such as bioavailability, of the administered cells. Methods for determining the pharmacokinetics of adoptive cells may include drawing peripheral blood from a subject who has been administered engineered cells and determining the number or ratio of the engineered cells in the peripheral blood. Methods for selecting and / or isolating cells may 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 in the CAR, thereby directly evaluating the CAR using Strep-Tag binding reagents (Liu et al. (2016) Nature Biotechnology, 34:430; International Patent Application Publication No. WO 2015095895); and monoclonal antibodies that specifically bind to CAR peptides (see International Patent Application Publication No. WO 2014190273). In some cases, engineered cell therapies may be combined with non-inherent marker genes to allow for the detection or selection of cells, and in some cases, to promote cell suicide. In some cases, truncated epidermal growth factor receptor (EGFRt) can be co-expressed with a target transgene (CAR) in transduced cells (see, for example, U.S. Patent No. 8,802,374). EGFRt may contain an epitope recognized by an antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibody or binding molecule, which 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 isolate cells expressing the receptor. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. April 2016; 34(4): 430-434). In some implementations, the presence of CAR in a biological sample (e.g., blood) obtained from the patient can be determined some time after cell therapy administration. + The number of T cells, for example, to determine the pharmacokinetics of the cells. In some embodiments, CARs are detectable in the blood of the subject or in most subjects so treated by said method. + T cells (optionally CAR) + CD8 + T cells and / or CAR + CD4 +The number of T cells is greater than 1 cell / µL, greater than 5 cells / µL, or greater than 10 cells / µL. 4. Multiple sclerosis (MS)

[0282] Multiple sclerosis (MS) has two main subtypes: relapsing MS and progressive MS. Relapsing MS is associated with immune-dependent damage mechanisms, characterized by a relapse-remission cycle, while progressive MS is associated with non-immune-dependent damage mechanisms, characterized by a steady worsening of symptoms. Early symptoms of MS include fatigue, weakness, and muscle spasms, which can progress to late-stage or severe disease characterized by vision and bladder problems, as well as cognitive changes and physical disability. Approximately 33% of patients will be forced to use a wheelchair within 20 years of diagnosis. Patients with MS have an approximately 80% increased risk of death. B cells are believed to play an important role in the pathogenesis of MS, as demonstrated by the effects of anti-CD20 mAbs used in treatment.

[0283] In some embodiments, the systemic autoimmune disease is MS, such as relapsing MS (RMS) or progressive MS (PMS). In some embodiments, the systemic autoimmune disease is highly active RMS. In some embodiments, MS is clinically isolated syndrome (CIS), relapsing-remitting MS (RRMS), active secondary progressive MS (aSPMS), inactive secondary progressive MS (naSPMS), inactive secondary progressive MS (iSPMS), or primary progressive MS (PPMS). In some embodiments, MS is aSPMS. In some embodiments, the systemic autoimmune disease is aSPMS. The provided methods include treatment methods involving administering engineered cells (such as engineered T cells) or compositions containing engineered cells to a subject suffering from MS (including CIS, RRMS, aSPMS, naSPMS, iSPMS, or PPMS). Methods and uses of the provided CD19-directed CAR-engineered cells (e.g., T cells) and / or combinations thereof are also provided, including methods involving the administration of engineered cells and / or combinations thereof for treating subjects suffering from MS (including CIS, RRMS, aSPMS, naSPMS, iSPMS, or PPMS). In some embodiments, the subject suffers from CIS, RRMS, aSPMS, naSPMS, iSPMS, or PPMS. In some embodiments, the subject suffers from RRMS, aSPMS, iSPMS, or PPMS. In some embodiments, the subject is selected as or identified as having RRMS, aSPMS, iSPMS, or PPMS, such as by indicating the presence of certain characteristics or clinical manifestations of RRMS, aSPMS, iSPMS, or PPMS. In some embodiments, the provided methods and uses of the provided CD19-directed CAR-engineered cells (e.g., T cells) and / or combinations thereof include methods for treating subjects suffering from MS who have failed at least two or more prior therapies. In a particular embodiment, the method includes administering to a subject a dose of T cells comprising CD4+ and CD8+ T cells, wherein the T cells contain a chimeric antigen receptor (CAR) that specifically binds to CD19.

[0284] In any embodiment herein, at or immediately prior to the administration of the composition comprising engineered T cells, the subject relapses after remission following treatment with one or more prior therapies for MS, or becomes refractory to the one or more prior therapies.

[0285] In any embodiment herein, at or immediately prior to the administration of the composition comprising engineered T cells, the subject relapsed after remission following treatment with one or more prior therapies for MS, or became refractory to the one or more prior therapies. In any embodiment herein, at or immediately prior to the administration of the composition comprising engineered T cells, the subject relapsed after treatment with one or more prior therapies for MS, or became refractory to the one or more prior therapies. In any embodiment herein, the one or more prior therapies for MS do not include an additional dose of CAR-expressing cells. In any embodiment herein, the one or more prior therapies comprise two prior disease-modifying therapies (DMTs), wherein one of the prior therapies uses an anti-CD20 antibody.

[0286] In any embodiment of this document, one or more prior therapies for MS may include glucocorticoids, plasma exchange, IVIg, adrenocorticotropic hormone (ACTH), fingolimod, siponimod, ozanimod, natetarizumab, teriflunomide, ozarelizumab, oflamb, alemtuzumab, and dimethyl fumarate. In any embodiment of this document, CD19-directed CAR-engineered cells (e.g., T cells) and / or combinations thereof are used to treat patients with MS who are refractory to prior therapies.

[0287] In any embodiment of this document, the subject is unable to complete a standardized dexterity test when or immediately prior to the administration of the composition containing engineered T cells. In some embodiments, the subject is unable to complete a 9-well column test (9-HPT) for each hand in < 240 seconds, or the subject is unable to perform a timed 25-foot walk test (T25FWT) in < 150 seconds.

[0288] The nine-hole pegboard test described herein involves the following task: A seated subject holds nine wooden pegs (approximately 7 mm in diameter and 32 mm in length) in one hand and uses the other hand to randomly place them one by one into a pegboard with nine holes. Timing begins when the first peg is placed in a hole and ends when the last peg is placed. During the test, the examiner keeps the pegboard stably on the table. The test is conducted with the dominant hand. If the patient drops a peg, the examiner stops the timer, and the patient starts the test again from the beginning.

[0289] The “Timed 25-foot Walk” or “T25FWT” as described herein is a quantitative motor and leg function performance test based on the timed 25-foot walk. The patient is directed to one end of a clearly marked 25-foot route and instructed to walk 25 feet as quickly but safely as possible. Time is counted from the start of the instruction and ends when the patient reaches the 25-foot mark. The task is immediately repeated by having the patient walk the same distance back. Assistive devices may be used by the patient during this task. The T25FWT score is the average of the two tests completed.

[0290] In some implementations, the subject does not have MS lesions or symptoms that could potentially place them at increased risk of neurotoxicity, including but not limited to tumor-like lesions (3 cm or larger within 5 years prior to screening). In some implementations, the subject does not experience a decrease in level of consciousness and / or the presence of active, clinically significant concomitant central nervous system symptoms other than MS, which could affect the ability to interpret study results or complicate the identification or evaluation of neurotoxicity. a. Reactions and Efficacy

[0291] In some implementations, the methods and uses provided for administering anti-CD19 CAR T-cell therapy reduce MS disease activity in subjects. In some implementations, the treatment effectively reduces MS disease activity. In some implementations, MS disease activity is measured by a disease activity score selected from the following: Extended Disability Status Scale (EDSS), disease tiers, Multiple Sclerosis Functional Complex Scale (MSFC), Minimum Clinically Important Difference Value (MCID), Patient-Reported Brief Quality of Life Assessment (SF-36), Physical Health Overall (PCS), and / or Mental Health Overall (MCS), or a combination thereof.

[0292] In some implementations, reducing MS disease activity in a subject may include one or more of the following: a reduction in the subject's EDSS index score after treatment compared to the subject's pre-treatment EDSS index score; improvement in the subject's energy, pain, fatigue, muscle strength, awake distance, mental health, or visual impairment compared to the subject's pre-treatment energy, pain, fatigue, muscle strength, awake distance, mental health, or visual impairment; or improvement in at least one patient-reported outcome (PRO) of the subject compared to pre-treatment.

[0293] In some implementations, the EDSS index score of subjects may be measured before and after administration of CD19-targeted cell therapy. In some implementations, patient-reported outcomes (PROs) may be measured in subjects before and after administration of CD19-targeted cell therapy. PROs may include subjects' chronic disease treatment function assessment-fatigue (FACIT-F), SF-36 version 2 (SF-36-v2) mental health overall score (MCS), and / or SF-36 physical health overall score (PCS).

[0294] In some implementations, treatment resulted in a significant change in the subject's Health Assessment Questionnaire-Disability Index (HAQ-DI) relative to baseline. In some implementations, the patient-reported quality of life assessment was a widely validated generic patient questionnaire that measures the difficulty of performing activities of daily living. These questions were rated on a 0-3 scale, where 0 indicated "no difficulty" and 3 indicated "cannot complete" (Allanore et al., 2020).

[0295] In some implementations, the treatment resulted in a decrease in the subject's EDSS score. In some implementations, the treatment resulted in a decrease in the subject's disease stage score. In some implementations, the treatment resulted in a decrease in the subject's MSFC score.

[0296] In some implementations, the treatment results in a significant improvement in a comprehensive assessment of disease activity based on the minimum clinically significant difference (MCID) in the subject. In some implementations, MCID refers to a patient-source score reflecting a change in clinical intervention meaningful to the patient. In some implementations, the subject's walking speed increases after treatment. In some implementations, the treatment results in increased dexterity in the subject (e.g., arm or hand). In some implementations, the treatment results in improved cognitive function (e.g., mathematical calculation), or as measured by a paced auditory serial addition test. In some implementations, the treatment results in increased energy in the subject. In some implementations, the treatment results in reduced pain in the subject. In some implementations, the treatment results in reduced visual impairment. In some implementations, the treatment results in improved bowel and bladder control.

[0297] In some embodiments, treatment according to the provided method results in clinical remission of MS in the subject, and the clinical remission lasts for more than 3 months. In some embodiments, treatment according to the provided method results in clinical remission of MS in the subject, and the clinical remission lasts 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 longer. In some embodiments, treatment according to the provided method results in clinical remission of MS in the subject, and the clinical remission lasts for more than 6 months. In some embodiments, treatment according to the provided method results in clinical remission of MS in the subject, and the clinical remission lasts for more than 12 months. In some embodiments, treatment according to the provided method results in clinical remission of MS in the subject, and the clinical remission lasts for more than 24 months. In some embodiments, treatment according to the provided method results in clinical remission of MS in the subject, and the clinical remission lasts for more than 3 years. In some embodiments, treatment according to the provided method results in clinical remission of MS in the subject, and the clinical remission lasts for more than 4 years. In some implementations, treatment according to the provided method results in clinical remission of MS in the subject, and the clinical remission is maintained for more than 5 years.

[0298] In some implementations, treatment according to the provided method results in prolonged remission. In some implementations, prolonged remission is defined as five consecutive years without disease activity and without treatment (fingolimod, sinimod, ozamod, natetuzumab, dimethyl fumarate, teriflunomide, ozenafil, olfamolumab, alemtuzumab, anti-CD20 antibody, or immunosuppressant).

[0299] In some cases, the pharmacokinetics of the administered cells (e.g., adoptive cells) are determined to assess the utilization, such as bioavailability, of the administered cells. Methods for determining the pharmacokinetics of adoptive cells may include drawing peripheral blood from a subject who has been administered engineered cells and determining the number or ratio of the engineered cells in the peripheral blood. Methods for selecting and / or isolating cells may 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 in the CAR, thereby directly evaluating the CAR using Strep-Tag binding reagents (Liu et al. (2016) Nature Biotechnology, 34:430; International Patent Application Publication No. WO 2015095895); and monoclonal antibodies that specifically bind to CAR peptides (see International Patent Application Publication No. WO 2014190273). In some cases, engineered cell therapies may be combined with non-inherent marker genes to allow for the detection or selection of cells, and in some cases, to promote cell suicide. In some cases, truncated epidermal growth factor receptor (EGFRt) can be co-expressed with a target transgene (CAR) in transduced cells (see, for example, U.S. Patent No. 8,802,374). EGFRt may contain an epitope recognized by an antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibody or binding molecule, which 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 isolate cells expressing the receptor. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. April 2016; 34(4): 430-434).

[0300] In some implementations, the presence of CAR in a biological sample (e.g., blood) obtained from the patient can be determined some time after cell therapy administration. + The number of T cells, for example, to determine the pharmacokinetics of the cells. In some embodiments, CARs are detectable in the blood of the subject or in most subjects so treated by said method. + T cells (optionally CAR) + CD8 + T cells and / or CAR + CD4 +The number of T cells is greater than 1 cell / µL, greater than 5 cells / µL, or greater than 10 cells / µL. 5. Rheumatoid Arthritis (RA)

[0301] In some implementations, the systemic autoimmune disease is rheumatoid arthritis (RA). Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease affecting 1% of the population. Disease progression is characterized by destructive inflammation of the joints, which can lead to progressive disability and shortened life expectancy. In RA, the synovium is infiltrated by activated immune cells (mostly macrophages and T cells), leading to the chronic production of pro-inflammatory cytokines and matrix metalloproteinases, thereby causing inflammation and cartilage and bone degradation (Choy EH and Panayi GS, N Engl Jmed. 2001; 344:907-916).

[0302] Patients awaiting treatment may have RA as defined by the 1987 ACR criteria. Prior to treatment, patients may test positive for rheumatoid factor (RF) and / or anti-cyclic citrullinated peptide (CCP) IgG antibodies. A positive RF and anti-CCP antibody status confirms the diagnosis of RA. Patients may have had RA for at least 5 years or at least 7 years (e.g., between 5 and 10 years).

[0303] In some embodiments, the methods and uses of the provided CD19-directed CAR-engineered cells (e.g., T cells) and / or combinations thereof include methods for treating subjects with MS who have failed at least two or more prior therapies. In a particular embodiment, the method includes administering to the subject a dose of T cells comprising CD4+ and CD8+ T cells, wherein the T cells contain a chimeric antigen receptor (CAR) that specifically binds to CD19.

[0304] In any embodiment herein, at or immediately prior to the administration of the composition comprising engineered T cells, the subject relapses after remission following treatment with one or more prior therapies for RA, or becomes refractory to the one or more prior therapies.

[0305] In any embodiment herein, at or immediately prior to the administration of the composition comprising engineered T cells, the subject relapsed after remission following treatment with one or more prior therapies for RA, or became refractory to said one or more prior therapies. In any embodiment herein, at or immediately prior to the administration of the composition comprising engineered T cells, the subject relapsed after treatment with one or more prior therapies for RA, or became refractory to said one or more prior therapies. In any embodiment herein, the one or more prior therapies for RA do not include an additional dose of CAR-expressing cells.

[0306] In some implementations, the methods and uses provided for administering anti-CD19 CAR T-cell therapy reduce RA disease activity in a subject. In some implementations, the reduction in RA disease activity is significant, provided that the subject experiences a clinical benefit following administration of anti-CD19 CAR T-cell therapy.

[0307] One measure of RA control is the Disease Activity Score (DAS) (Fransen & van Riel Clin Exp Rheumatol 23:S93-S99 2005). The DAS is calculated by a practicing physician based on several validated measures of disease activity, including physical symptoms of RA. A lower DAS reflects a decrease in disease severity. A DAS less than 2.6 indicates disease remission. A DAS between 2.6 and 3.2 indicates low disease activity. A DAS greater than 3.2 indicates increased disease activity, and at this level, a patient's therapy may need to be reviewed to determine if a change in therapy is necessary. A DAS greater than 5.1 indicates severe disease activity. Calculating differences in DAS may include assessing different numbers of joints in the patient and monitoring different blood components. DAS28 is a disease activity score where 28 joints in the body are assessed to determine the number of tender joints and swollen joints (Prevoo et al. Arthritis Rheum 38:44-48 1995). When DAS28 calculations include a measurement of C-reactive protein (CRP) instead of erythrocyte sedimentation rate (ESR), it is referred to as 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 is associated with radiological progression of RA (van Leeuwen MA et al. Br J Rheumatol 32(Supplement 3):9-13 1993) and is considered at least as effective as ESR in measuring RA disease activity (Mallya RK et al. J Rheumatol 9:224-8 1982; Wolfe F. J Rheumatol 24: 1477-85 1997).

[0308] The American College of Rheumatology (ACR) has developed a set of criteria for classifying rheumatoid arthritis (RA). The most commonly used criteria are the ACR 1987 revised criteria (Arnett et al., Arthritis Rheum. 31:315-324 1988). Diagnosing RA according to the ACR criteria requires patients to meet a minimum number of listed criteria, such as a count of tender or swollen joints, stiffness, pain, radiographic indications, and a measurement of serum rheumatoid factor. ACR 20, ACR 50, and ACR 70 are commonly used measures of efficacy for RA treatment, particularly in clinical trials. ACR 20 indicates a 20% improvement in the measured ACR criteria. Similarly, ACR 50 indicates a 50% improvement, and ACR 70 indicates a 70% improvement. A separate patient-reported disability measure for RA patients is the Health Assessment Questionnaire-Disability Index (HAQ-DI). The HAQ-DI score indicates a patient's reported physical function in terms of their ability to perform daily tasks, including the level of difficulty they experience while performing activities. The HAQ-DI score can be used as a measure of a patient's quality of life by recording their ability to perform daily activities.

[0309] Clinical benefits can include relief of RA. Typically, relief is defined as a DAS28-CRP level of less than 2.6.

[0310] Clinical benefit can be an improvement in treatment efficacy of at least 20%, at least 50%, or at least 70% as determined by the 1987 ACR criteria, i.e., clinical benefit can be achieving ACR 20, ACR 50, or ACR 70, respectively.

[0311] One form of clinical benefit particularly valuable to RA patients is improved ability to perform daily activities. The methods of this disclosure may include improvements in patient-self-assessed disabilities (referred to as HAQ-DI) measured by a health assessment questionnaire. Methods for providing clinical benefits to RA patients (wherein the clinical benefits include improved physical function in RA patients, as determined by HAQ-DI) and compositions and kits used in such methods are aspects of this disclosure. Clinical benefits may include improved physical function in RA patients, as determined by HAQ-DI. In some embodiments, a statistically significant improvement in HAQ-DI is achieved within twelve, ten, eight, or six weeks, or four weeks, or two weeks of initiating treatment according to this disclosure. The improvement may be an improvement of at least 0.25 in HAQ-DI, i.e., a reduction of 0.25 or more in the patient's HAQ-DI score. In some embodiments, the improvement is an improvement of at least 0.30, 0.40, or 0.45 in the HAQ-DI score. Improvements are generally measured relative to the patient's baseline mean HAQ-DI score prior to treatment with an inhibitor according to this disclosure.

[0312] Patients can be monitored during and / or after treatment with 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. The methods may include determining the achievement of clinical benefit, such as a specified reduction in DAS28-CRP and / or the achievement of ACR 20, ACR50, or ACR 70, and / or an improvement in HAQ-DI score, as discussed elsewhere herein. B. Administration

[0313] In some embodiments, a dose of engineered cells is administered to a subject according to the provided method and / or using the provided article or composition. In some embodiments, the dose size or timing is determined based on the subject's specific disease or condition. In some cases, the dose size or timing for a specific disease may be determined empirically based on the provided description.

[0314] In some of the provided embodiments, the dose of T cells (such as engineered T cells expressing recombinant receptors) comprises, enriches, or contains a cell composition or population enriched with CD3+ T cells, CD4+ T cells, CD8+ T cells, or both CD4+ T cells and CD8+ T cells. In some of these embodiments, the dose of T cells contains greater than or equal to about 70%, 75%, 80%, 85%, 90%, 95%, or 98% of the cells being CD3+ T cells, CD4+ T cells, CD8+ T cells, or both CD4+ T cells and CD8+ T cells. In some of these embodiments, the dose of T cells contains greater than or equal to about 70%, 75%, 80%, 85%, 90%, 95%, or 98% of the cells being CD3+ T cells. In some of these embodiments, the dose of T cells contains both CD4+ and CD8+ cells. In some of these implementations, more than 70%, 75%, 80%, 85%, 90%, 95%, or 98% of the T cells in the dose are CD4+ T cells and CD8+ T cells.

[0315] In some embodiments, the cell dose is contained in a concentration of 0.1 x 10⁻⁶. 5 The ratio of CD19-directed CAR-engineered cells per kilogram of subject body weight (cells / kg) is approximately 2 x 10⁻⁶. 6 Between cells / kg, such as at or approximately 0.1 x 10⁻⁶. 5 Cells / kg and approximately 0.5 x 10 5 Between [number] cells / kg, and approximately 0.5 x 10 [units]. 5 Cells / kg and approximately 1 x 105 Between [number] cells / kg, and approximately 1 x 10 [units]. 5 Cells / kg or approximately 1.5 x 10 5 Between [number] cells / kg, and approximately 1.5 x 10 [units]. 5 Cells / kg and approximately 2 x 10 5 Between [number] cells / kg, and approximately 2 x 10 [units]. 5 Cells / kg or approximately 2.5 x 10 5 Between [number] cells / kg, and approximately 2.5 x 10 [units]. 5 Cells / kg and approximately 3 x 10 5 Between [number] cells / kg, and approximately 3 x 10 [units]. 5 Cells / kg or approximately 3.5 x 10 5 Between [number] cells / kg, and approximately 3.5 x 10 [units]. 5 Cells / kg or approximately 4 x 10 5 Between [number] cells / kg, and approximately 4 x 10 [units]. 5 Cells / kg and approximately 4.5 x 10 5 Between [number] cells / kg, and approximately 4.5 x 10 [units]. 5 Cells / kg or approximately 5 x 10 5 Between 10 cells / kg, or approximately 5 x 10⁻⁶ 5 Cells / kg and approximately 5.5 x 10 5 Between [number] cells / kg, and approximately 5.5 x 10 [units]. 5 Cells / kg and approximately 6 x 10 5 Between [number] cells / kg, and approximately 6 x 10 [units]. 5 Cells / kg or approximately 6.5 x 10 5 Between [number] cells / kg, and approximately 6.5 x 10 [units]. 5 Cells / kg or approximately 7 x 10 5 Between 10 cells / kg, or approximately 7 x 10 5 Cells / kg or approximately 7.5 x 10 5 Between [number] cells / kg, and approximately 7.5 x 10 [units]. 5 Cells / kg and approximately 8 x 10 5 Between [number] cells / kg, or approximately 8 x 10 [units]. 5 Cells / kg or approximately 10 x 10 5 The dosage is between [number] cells / kg. In some embodiments, the cell dose contains no more than 2 x 10 [units / kg]. 5The number of CD19-directed CAR-engineered cells per kilogram of subject weight (cells / kg) should not exceed or exceed approximately 3 x 102 5 Cells / kg, not exceeding or not exceeding about 4 x 10 5 Cells / kg, not exceeding or not exceeding about 5 x 10 5 Cells / kg, not exceeding or not exceeding about 6 x 10 5 Cells / kg, not exceeding or not exceeding about 7 x 10 5 Cells / kg, not exceeding or not exceeding about 8 x 10 5 Cells / kg, not exceeding or not exceeding about 9 x 10 5 Cells / kg, not exceeding or not exceeding about 1 x 10 6 Cells / kg, or not exceeding or not exceeding about 2 x 10 6 Cells / kg. In some embodiments, the cell dose comprises at least or at least about or about 0.1 x 10⁻⁶ cells / kg. 5 CD19-directed CAR-engineered cells per kilogram of subject body weight (cells / kg), such as at least or at least about or about 0.2 x 10⁻⁶ cells / kg. 5 Cells / kg, at least or at least about or about 0.3 x 10 5 Cells / kg, at least or at least about or about 0.4 x 10 5 Cells / kg, at least or at least about or about 0.5 x 10 5 Cells / kg, at least or at least about or about 0.6 x 10 5 Cells / kg, at least or at least about or about 0.7 x 10 5 Cells / kg, at least or at least about or about 0.8 x 10 5 Cells / kg, at least or at least about or about 0.9 x 10 5 Cells / kg, at least or at least about or about 0.1 x 10 6 Cells / kg, or at least about 0.2 x 10⁻⁶ 6 Cells / kg. In some embodiments, the number of cells is the number of such cells as live cells (e.g., live T cells, such as live CD3+ cells expressing CD19-directed CAR).

[0316] In some implementations, a separate population of cells or cell subtypes is administered to the subject in the following ranges: approximately 100,000 to approximately 100 billion cells and / or that amount of cells per kilogram of the subject's body weight, such as, for example, approximately 100,000 to approximately 50 billion cells (e.g., approximately 5 million cells, approximately 25 million cells, approximately 500 million cells, approximately 1 billion cells, approximately 5 billion cells, approximately 20 billion cells, approximately 300 million cells, etc.). 0 billion cells, or about 40 billion cells, or a range defined by any two of the foregoing values), or about 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to The number of cells is approximately 100 billion (e.g., approximately 20 million, approximately 30 million, approximately 40 million, approximately 60 million, approximately 70 million, approximately 80 million, approximately 90 million, approximately 10 billion, approximately 25 billion, approximately 50 billion, approximately 75 billion, approximately 90 billion, or any two of the foregoing values). The dosage is defined as a range, and in some cases, from about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value between these ranges and / or per kilogram of the subject's body weight. The dosage may vary depending on the disease or impairment and / or patient- and / or other treatment-specific properties. In some embodiments, such values ​​refer to the number of cells expressing the recombinant receptor; 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 is the number of such cells as living cells.

[0317] In some implementations, the cell dose is a flat or fixed dose, such that the cell dose is independent of or based on the subject's body surface area or weight. In some implementations, administering a higher number of cytotoxic cells based on the subject's weight may increase the subject's risk of toxicity (such as neurotoxicity).

[0318] In some implementations, the dose of genetically engineered cells comprises from 1 x 10⁻⁶ to about 1 x 10⁻⁶. 5 One to approximately 1 x 10 8Total T cells expressing CD19-directed CAR, from approximately 1 x 10-1 5 One to approximately 1.0 x 10 7 Total T cells expressing CD19-directed CAR, from approximately 1 x 10-1 5 One to approximately 1.0 x 10 6 Total T cells expressing CD19-directed CAR, from approximately 1 x 10-1 6 One to approximately 1.0 x 10 8 Total T cells expressing CD19-directed CAR, from approximately 1 x 10-1 6 One to approximately 1.0 x 10 7 Total T cells expressing CD19-directed CAR, from approximately 5 x 10-1 6 One to approximately 1.0 x 10 8 Total T cells expressing CD19-directed CAR, from approximately 5 x 10-1 6 One to approximately 1.0 x 10 7 Total T cells expressing CD19-directed CAR, from approximately 10 x 10-1 6 One to approximately 1.0 x 10 8 Total T cells expressing CD19-directed CAR. In some implementations, the number of cells is the number of such cells as living cells (such as live T cells).

[0319] In some implementations, the dose of genetically engineered cells comprises from 1 x 10⁻⁶ to about 1 x 10⁻⁶. 5 One to approximately 1 x 10 8 Total live T cells expressing CD19-directed CAR, from approximately 1 x 103 5 One to approximately 1.0 x 10 7 Total live T cells expressing CD19-directed CAR, from approximately 1 x 103 5 One to approximately 1.0 x 10 6 Total live T cells expressing CD19-directed CAR, from approximately 1 x 103 6 One to approximately 1.0 x 10 8 Total viable T cells expressing CD19-directed CAR, from approximately 1 x 10 6 One to approximately 1.0 x 10 7 Total live T cells expressing CD19-directed CAR, from approximately 5 x 10-1 6 One to approximately 1.0 x 10 8 Total live T cells expressing CD19-directed CAR, from approximately 5 x 10-1 6One to approximately 1.0 x 10 7 Total live T cells expressing CD19-directed CAR, from approximately 10 x 10⁻⁶ cells. 6 One to approximately 1.0 x 10 8 Total live T cells expressing CD19-directed CAR.

[0320] In some embodiments, the cell dose is a relatively low dose. In some embodiments, the anti-CD19 CAR T cell composition used in the provided embodiments comprises cells with a low degree of differentiation, wherein most cells have an naive or central memory cell phenotype. Furthermore, in the provided embodiments, the composition comprises a T cell population in which more than 25% of the T cells (e.g., CD3+ T cells) express CAR, such as more than 30%, 35%, 40%, 45%, or 50% of the T cells (e.g., CD3+ T cells) express CAR. In some embodiments, the composition comprises a T cell population in which more than 50% of the T cells (e.g., CD3+ T cells) express CAR, such as more than 60%, more than 70%, or more than 80% of the T cell composition express CAR. Not wishing to be bound by theory, compositions having the characteristics provided herein ensure that cells exhibit higher potency and greater persistence in subjects, while minimizing or reducing the potential toxicity of CAR-expressing T cells. In some embodiments, the dose of anti-CD19 CAR T cells exhibits higher potency, durability, and / or lower toxicity compared to cells in an alternative composition containing a higher percentage of more differentiated cells (e.g., effector T cells with a higher percentage). In some embodiments, the dose of anti-CD19 CAR T cells exhibits higher potency, durability, and / or lower toxicity compared to cells in an alternative composition containing a lower percentage of CAR-expressing cells. In some embodiments, the dose of genetically engineered cells may be less than 10 x 10⁻⁶. 7 The total amount of total T cells expressing CD19-directed CAR was administered.

[0321] In some implementations, the dose of genetically engineered cells can be less than 9 x 10⁻⁶. 7 The dosage is one total T cell expressing CD19-directed CAR. In some implementations, the dose of genetically engineered cells can be less than 8 x 10⁻⁶. 7 The dosage is one total live T cell expressing CD19-directed CAR. In some embodiments, the dose of genetically engineered cells can be less than 7.5 x 10⁻⁶. 7 The dosage is one total live T cell expressing CD19-directed CAR. In some embodiments, the dose of genetically engineered cells can be less than 7.0 x 10⁻⁶.7 The dosage is one total live T cell expressing CD19-directed CAR. In some embodiments, the dose of genetically engineered cells can be less than 6.0 x 10⁻⁶. 7 The dosage is one dose of total live T cells expressing CD19-directed CAR. In some embodiments, the dose of genetically engineered cells is from approximately 1 x 10⁻⁶ cells. 6 One is approximately 50 x 10 6 Total live T cells expressing CD19-directed CAR, from approximately 1 x 103 6 Each is approximately 40 x 10 6 Total viable T cells expressing CD19-directed CAR, approximately 1 x 10-1 6 Each is approximately 30 x 10 6 Total viable T cells expressing CD19-directed CAR, approximately 1 x 10-1 6 Each is approximately 20 x 10 6 Total viable T cells expressing CD19-directed CAR, approximately 1 x 10-1 6 One is approximately 10 x 10 6 Total viable T cells expressing CD19-directed CAR, approximately 1 x 10-1 6 One to approximately 5 x 10 6 Total viable T cells expressing CD19-directed CAR, approximately 1 x 10-1 6 One is approximately 2.5 x 10 6 Total live T cells expressing CD19-directed CAR, from approximately 2.5 x 10⁻⁶. 6 Each is approximately 50 x 10 6 Total live T cells expressing CD19-directed CAR, from approximately 2.5 x 10⁻⁶. 6 Each is approximately 40 x 10 6 The total number of live T cells expressing CD19-directed CARs is approximately 2.5 x 10^6. 6 Each is approximately 30 x 10 6 The total number of live T cells expressing CD19-directed CARs is approximately 2.5 x 10^6. 6 Each is approximately 20 x 10 6 The total number of live T cells expressing CD19-directed CARs is approximately 2.5 x 10^6. 6 One is approximately 10 x 10 6 The total number of live T cells expressing CD19-directed CARs is approximately 2.5 x 10^6. 6 One to approximately 5 x 10 6Total live T cells expressing CD19-directed CAR, from approximately 2.5 x 10⁻⁶. 6 Each is approximately 50 x 10 6 Total live T cells expressing CD19-directed CAR, from approximately 5 x 10-1 6 Each is approximately 50 x 10 6 Total live T cells expressing CD19-directed CAR, from approximately 5 x 10-1 6 Each is approximately 40 x 10 6 Total viable T cells expressing CD19-directed CAR, approximately 5 x 10-1 6 Each is approximately 30 x 10 6 Total viable T cells expressing CD19-directed CAR, approximately 5 x 10-1 6 Each is approximately 20 x 10 6 Total viable T cells expressing CD19-directed CAR, approximately 5 x 10-1 6 One is approximately 10 x 10 6 Total live T cells expressing CD19-directed CAR, from approximately 10 x 10⁻⁶ cells. 6 Each is approximately 50 x 10 6 Total live T cells expressing CD19-directed CAR, from approximately 10 x 10⁻⁶ cells. 6 Each is approximately 40 x 10 6 Total live T cells expressing CD19-directed CAR, approximately 10 x 10⁻⁶ 6 Each is approximately 30 x 10 6 Total live T cells expressing CD19-directed CAR, approximately 10 x 10⁻⁶ 6 Each is approximately 20 x 10 6 Total live T cells expressing CD19-directed CAR, from approximately 20 x 10 6 Each is approximately 50 x 10 6 Total live T cells expressing CD19-directed CAR, from approximately 20 x 10 6 Each is approximately 40 x 10 6 Total live T cells expressing CD19-directed CAR, approximately 20 x 10⁻⁶ 6 Each is approximately 30 x 10 6 Total live T cells expressing CD19-directed CAR, from approximately 30 x 10 6 Each is approximately 50 x 10 6 Total live T cells expressing CD19-directed CAR, from approximately 30 x 10 6 Each is approximately 40 x 106 Total live T cells expressing CD19-directed CAR, or approximately 40 x 10 6 Each is approximately 50 x 10 6 Total live T cells expressing CD19-directed CAR.

[0322] In some implementations, the dose of genetically engineered cells is 0.1 x 10⁻⁶ or about 0.1 x 10⁻⁶. 6 The total number of T cells expressing CD19-directed CARs was approximately 0.2 x 10^12. 6 The total number of live T cells expressing CD19-directed CARs is approximately 0.25 x 10^6. 6 The total number of live T cells expressing CD19-directed CARs is approximately 0.5 x 10^6. 6 The total number of live T cells expressing CD19-directed CAR was approximately 0.75 x 10^12. 6 Total T cells expressing CD19-directed CAR, approximately 2 x 10-1 6 Total viable T cells expressing CD19-directed CAR, approximately 3 x 10-1 6 Total live T cells expressing CD19-directed CAR, approximately 4 x 10-1 6 Total viable T cells expressing CD19-directed CAR, approximately 6 x 10-1 6 Total viable T cells expressing CD19-directed CAR, approximately 7 x 10-1 6 Total live T cells expressing CD19-directed CAR, approximately 8 x 10⁻⁶ 6 Total viable T cells expressing CD19-directed CAR, approximately 9 x 10-1 6 Total live T cells expressing CD19-directed CAR, approximately 10 x 10⁻⁶ 6 The total number of live T cells expressing CD19-directed CAR is approximately 11 x 10⁻⁶. 6 Total live T cells expressing CD19-directed CAR, approximately 12 x 10⁻⁶. 6 Total viable T cells expressing CD19-directed CAR, approximately 13 x 10⁻⁶. 6 Total live T cells expressing CD19-directed CAR, approximately 14 x 10⁻⁶. 6 Total live T cells expressing CD19-directed CAR, approximately 15 x 10 6 Total live T cells expressing CD19-directed CAR, approximately 16 x 10-1 6 Total viable T cells expressing CD19-directed CAR, approximately 17 x 10-1 6 Total viable T cells expressing CD19-directed CAR, approximately 18 x 10-16 Total viable T cells expressing CD19-directed CAR, approximately 19 x 10⁻⁶. 6 Total viable T cells expressing CD19-directed CAR, approximately 25 x 10 6 Total viable T cells expressing CD19-directed CAR, approximately 35 x 10 6 Total viable T cells expressing CD19-directed CAR, approximately 45 x 10 6 Total viable T cells expressing CD19-directed CAR, approximately 60 x 10⁶ 6 Total viable T cells expressing CD19-directed CAR, approximately 70 x 10⁻⁶ 6 Total T cells expressing CD19-directed CAR, approximately 75 x 10-1 6 Total T cells expressing CD19-directed CAR, approximately 80 x 10⁸ 6 Total viable T cells expressing CD19-directed CAR, approximately 90 x 10⁻⁶ 6 100 x 10 6 Total live T cells expressing CD19-directed CAR.

[0323] In some implementations, the dose of genetically engineered cells is from approximately 5 x 10⁻⁶. 6 One is approximately 50 x 10 6 Total live T cells expressing CD19-directed CAR. In some embodiments, the dose of genetically engineered cells is from approximately 10 x 10⁻⁶ cells. 6 Each is approximately 50 x 10 6 Total live T cells expressing CD19-directed CAR.

[0324] In some implementations, the dose of genetically engineered cells is approximately 5 x 10⁻⁶. 6 Total live T cells expressing CD19-directed CAR. In some implementations, approximately 5 x 10⁶ cells are administered to the subject. 6 A single dose of CD19-directed CAR-expressing T cells.

[0325] In some implementations, the dose of genetically engineered cells is approximately 10 x 10⁻⁶. 6 Total live T cells expressing CD19-directed CAR. In some implementations, approximately 10 x 10⁶ cells are administered to the subject. 6 A single dose of CD19-directed CAR-expressing T cells.

[0326] In some implementations, the dose of genetically engineered cells is approximately 15 x 10⁻⁶. 6Total live T cells expressing CD19-directed CAR. In some implementations, approximately 15 x 10⁶ cells are administered to the subject. 6 A single dose of CD19-directed CAR-expressing T cells.

[0327] In some implementations, the dose of genetically engineered cells is approximately 20 x 10⁻⁶. 6 Total live T cells expressing CD19-directed CAR. In some implementations, approximately 20 x 103 T cells are administered to the subject. 6 A single dose of CD19-directed CAR-expressing T cells.

[0328] In some implementations, the dose of genetically engineered cells is approximately 25 x 10⁻⁶. 6 Total live T cells expressing CD19-directed CAR. In some implementations, approximately 25 x 10⁶ cells are administered to the subject. 6 A single dose of CD19-directed CAR-expressing T cells.

[0329] In some implementations, the dose of genetically engineered cells is approximately 30 x 10⁻⁶. 6 Total live T cells expressing CD19-directed CAR. In some implementations, approximately 30 x 1030 T ... 6 A single dose of CD19-directed CAR-expressing T cells.

[0330] In some implementations, the dose of genetically engineered cells is approximately 40 x 10⁻⁶. 6 Total live T cells expressing CD19-directed CAR. In some implementations, approximately 40 x 10⁶ cells are administered to the subject. 6 A single dose of CD19-directed CAR-expressing T cells.

[0331] In some implementations, the dose of genetically engineered cells is approximately 50 x 10⁻⁶. 6 Total live T cells expressing CD19-directed CAR. In some implementations, approximately 50 x 10⁶ cells are administered to the subject. 6 A single dose of CD19-directed CAR-expressing T cells.

[0332] In some implementations, the quantity is related to CD3. + CD8 + Or CD4+ and CD8+, and in some cases expressing recombinant receptors (e.g., CAR). + The total number of cells. In some implementations, the number of cells is the number of such cells as living cells.

[0333] In some embodiments, the dose of T cells includes CD4.+ T cells, CD8 + T cells or CD4 + and CD8 + T cells.

[0334] In some embodiments, the dose of T cells includes CD4. + T cells, CD8 + T cells or CD4 + and CD8 + T cells.

[0335] In some implementations, the dose of cells (e.g., T cells expressing recombinant receptors) is administered to the subject as a single dose, or administered only once over a period of two weeks, one month, three months, six months, one year, or longer.

[0336] In the case of adoptive cell therapy, the administration of a given “dose” encompasses the administration of a given amount or number of cells as a single composition and / or a single, uninterrupted administration (e.g., as a single injection or continuous infusion), and also encompasses the administration of cells provided in multiple individual compositions or infusions as a fractionated dose or as a given amount or number of compositions over a specified time period (e.g., not exceeding 3 days). Thus, in some cases, the dose is a single or continuous administration of a specified number of cells, given or initiated at a single time point. However, in some cases, the dose is administered over a time period not exceeding three days in the form of multiple injections or infusions, such as once daily for three days or for two days, or by multiple infusions over a single day.

[0337] In certain embodiments, the number and / or concentration of cells refers to the number of cells expressing a recombinant receptor (e.g., CAR). In other embodiments, the number and / or concentration of cells refers to the number or concentration of T cells administered.

[0338] In some embodiments, the subject receives multiple doses (e.g., two or more doses) or multiple consecutive doses of cells. In some embodiments, two doses are administered to the subject. In some embodiments, the subject receives consecutive doses, for example, a 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, such that one or more additional doses are administered after the consecutive doses. In some aspects, the number of cells administered to the subject at additional doses is the same as or similar to the first dose and / or consecutive doses. In some embodiments, one or more additional doses are greater than the previous doses.

[0339] In some respects, the dosage is determined based on one or more criteria, such as the likelihood or incidence of a subject’s response to prior treatment and / or the occurrence of toxic outcomes (e.g., CRS, macrophage activation syndrome, neurotoxicity, and / or host immune response to the administered cells and / or recombinant receptors).

[0340] In some embodiments, the time between the administration of the first dose and the administration of subsequent doses is about 9 to about 35 days, about 14 to about 28 days, or 15 to 27 days. In some embodiments, the administration of subsequent doses is performed at a time point more than about 14 days but less than about 28 days after the administration of the first dose. In some embodiments, the time between the first dose and the subsequent doses is about 21 days. In some embodiments, one or more additional doses (e.g., consecutive doses) are administered after the administration of the subsequent doses. In some embodiments, one or more additional consecutive doses are administered at least about 14 days but less than about 28 days after the administration of the previous dose. In some embodiments, additional doses are 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.

[0341] In some implementations, the cell dose is typically large enough to be effective in reducing the disease burden.

[0342] In certain embodiments, the number and / or concentration of cells refers to the number of cells expressing a recombinant receptor (e.g., CAR). In other embodiments, the number and / or concentration of cells refers to the number or concentration of all cells, T cells, or peripheral blood mononuclear cells (PBMCs) administered.

[0343] In some embodiments, the method further includes administering one or more additional doses of the chimeric antigen receptor (CAR)-expressing cell and / or lymphocyte clearance therapy, and / or repeating one or more steps of the method. 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 differ from the initial dose, for example, being higher, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or more higher than the initial dose; or lower, such as being higher, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or more lower 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 prior treatment and / or the likelihood or incidence of toxic outcomes in the subject (e.g., CRS, macrophage activation syndrome, neurotoxicity, and / or host immune response to the administered cells and / or recombinant receptor). C. Toxicity

[0344] In some embodiments, the provided method is designed to or includes features leading to a lower rate and / or lower degree of treatment-related or serious adverse events (AEs), AEs of particular concern, laboratory abnormalities, or dose-limiting toxicities (DLTs). Adverse events are defined by the Common Terminology Standard for Adverse Events (CTCAE). AEs are rated on a scale from 1 to 5, where 1 is mild and 5 is fatal. In some embodiments, the provided method is designed to or includes features leading to a lower rate and / or lower degree of DLTs. In some embodiments, DLTs are characterized by hematologic, non-hematologic, and / or organ-specific adverse events or side effects relative to baseline.

[0345] In some embodiments, the provided method is designed to or includes characteristics leading to: a lower rate and / or lower degree of toxicity, toxic outcome or symptoms, a profile that promotes toxicity, factors or characteristics (such as those associated with or indicating symptoms or outcomes of cytokine release syndrome (CRS) or neurotoxicity (NT), for example, with the administration of alternative cell therapies (such as alternative CARs). + T-cell compositions) and / or alternative cell administration (e.g., cell administration not administered at a defined ratio). Cytokine release syndrome (CRS) and neurotoxicity can be classified according to the American Society for Transplantation and Cell Therapy (ASTCT) common grading system (see, for example, Lee et al., Biol Blood Marrow Transplant. 2019 Apr;25(4):625-38).

[0346] In some respects, although the engineered T cells applied as part of the methods provided herein are expected to be of a lower differentiation state (e.g., a higher proportion of those with naive or central memory phenotypes, such as those selected from CCR7), + CD45RA + CD27 + CCR7 + Or CD62L - CCR7 +Engineered T cells with a higher degree of differentiation (such as the phenotype of CD19) exhibit higher activity than more differentiated cells, but it has been found that the safety of cell therapy can be successfully managed. In some respects, robust efficacy and high safety are achieved by providing lower doses of the composition (e.g., compared to cell compositions produced through processes involving higher degree of cell differentiation, such as those involving cell expansion). In some respects, it has been found that even higher doses of the provided anti-CD19 CAR composition can be administered to cells while maintaining lower levels of toxicity (such as severe cytokine release syndrome (CRS) or severe neurotoxicity). Therefore, alternative cell therapies, including the administration of alternative CARs with engineered T cells having a higher degree of differentiation than those engineered T cells administered herein, are possible. + Compared to methods using T-cell compositions, the provided method, in some embodiments, includes administering a higher dose of engineered T cells (e.g., greater than 50 × 10⁶). 6 CAR-expressing T cells, such as approximately 100 × 10⁶ 6 (CAR-expressing T cells).

[0347] In some embodiments, the provided method does not result in a high rate or likelihood of toxicity or toxic outcomes such as neurotoxicity (NT) or cytokine release syndrome (CRS), or reduces the rate or likelihood of toxicity or toxic outcomes, as compared to certain other cell therapies. In some embodiments, the method does not result in or increase the risk of severe NT (sNT), severe CRS (sCRS), macrophage activation syndrome, fever of at least or about 38 degrees Celsius for three days or more, and plasma CRP levels of at least or about 20 mg / dL. In some embodiments, greater than or about 30%, 35%, 40%, 50%, 55%, 60% or more of subjects treated according to the provided method do not exhibit any grade of CRS or any grade of neurotoxicity. In some embodiments, no more than 50% of treated subjects (e.g., at least 60%, at least 70%, at least 80%, at least 90% or more of treated subjects) exhibit grade 2 or higher cytokine release syndrome (CRS) and / or grade 2 or higher neurotoxicity. In some embodiments, at least 50% of subjects treated according to the method (e.g., at least 60%, at least 70%, at least 80%, at least 90% or more of the treated subjects) do not exhibit severe toxic outcomes (e.g., severe CRS or severe neurotoxicity), such as not exhibiting grade 3 or higher neurotoxicity and / or not exhibiting severe CRS, or not exhibiting severe toxic outcomes within a certain time period after treatment (e.g., within one week, two weeks, or one month after administration of the cells). In some embodiments, parameters assessed to determine certain toxicities include, but are not limited to, adverse events (AEs), dose-limiting toxicities (DLTs), CRS, and NT.

[0348] Administering 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 occur concurrently with high levels of circulating cytokines, which may be the basis for the observed toxicity.

[0349] In some respects, toxic outcomes are cytokine release syndrome (CRS) or severe CRS (sCRS), or are associated with or indicate cytokine release syndrome (CRS) or severe CRS (sCRS). In some cases, CRS, such as sCRS, may occur after adoptive T-cell therapy and administration of other biologics to the subject. 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.

[0350] Typically, CRS is caused by an excessive systemic immune response mediated by, for example, T cells, B cells, NK cells, monocytes, and / or macrophages. These cells can release large amounts of inflammatory mediators, such as cytokines and chemokines. Cytokines may trigger an acute inflammatory response and / or induce endothelial organ damage, which can lead to microvascular leakage, heart failure, or death. Severe, life-threatening CRS can lead to pulmonary infiltration and lung injury, renal failure, or disseminated intravascular coagulation. Other severe, life-threatening toxicities may include cardiotoxicity, respiratory distress, neurotoxicity, and / or liver failure. In some respects, fever, particularly high fever (≥ 38.5ºC or ≥ 101.3°F), is associated with CRS or its risk. In some cases, the characteristics or symptoms of CRS resemble those of an infection. In some implementations, infection is also considered in subjects presenting with symptoms of CRS, and monitoring by culture and empirical antibiotic therapy may be administered. Other symptoms associated with CRS may include cardiac dysfunction, adult respiratory distress syndrome, kidney and / or liver failure, coagulation disorders, disseminated intravascular coagulation, and capillary leak syndrome.

[0351] CRS can be treated with anti-inflammatory therapies, such as anti-IL-6 therapy (e.g., anti-IL-6 antibodies, such as tocilizumab) or antibiotics or other agents as described herein. Outcomes, signs, and symptoms of CRS are known and include those described herein. In some embodiments, specific outcomes, signs, and symptoms and / or their amount or extent may be specified where a particular dosing regimen or administration affects or does not affect a given CRS-related outcome, sign, or symptom.

[0352] In cases of CAR-expressing cell infusion, CRS typically occurs 6–20 days after infusion of CAR-expressing cells. See Xu et al., Cancer Letters 343 (2014) 172–78. In some cases, CRS occurs less than 6 days or more than 20 days after CAR T cell infusion. The incidence and timing of CRS may be related to baseline cytokine levels at the time of infusion. Typically, CRS involves elevated serum levels of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and / or interleukin (IL)-2. Other cytokines that can be rapidly induced in CRS are IL-1β, IL-6, IL-8, and IL-10.

[0353] Exemplary outcomes associated with CRS include fever, chills, cold extremities, hypotension, dyspnea, acute respiratory distress syndrome (ARDS), encephalopathy, elevated ALT / AST, renal failure, cardiac disturbances, hypoxia, neurological disorders, and death. Neurological complications include delirium, seizure-like activity, confusion, difficulty finding words, aphasia, and / or dulling of consciousness. Other CRS-related outcomes include fatigue, nausea, headache, seizures, tachycardia, myalgia, rash, acute vascular leakage syndrome, liver dysfunction, and renal failure. In some respects, CRS is associated with increases in one or more factors, such as serum ferritin, D-dimer, transaminases, lactate dehydrogenase, and triglycerides, or with hypofibrinogenemia or hepatosplenomegaly. Other exemplary signs or symptoms associated with CRS include hemodynamic instability, febrile neutropenia, increased 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).

[0354] In some implementations, CRS-related outcomes include one or more of the following: persistent fever, such as fever at a specified temperature (e.g., greater than or greater than about 38 degrees Celsius) lasting for two or more days, such as three or more days, such as four or more days, or lasting for at least three consecutive days; fever greater than or greater than about 38 degrees Celsius; an increase in cytokines, such as at least two cytokines (e.g., at least two from the group consisting of interferon-γ (IFNγ), GM-CSF, IL-6, IL-10, Flt-3L, fractal chemokines, and IL-5 and / or tumor necrosis factor-α (TNFα)) with a maximum fold change of, for example, at least or at least about 75, or at least one of such cytokines with a maximum fold change of, for example, at least or at least about 250; and / or at least one clinical sign of toxicity, such as hypotension (e.g., as measured by at least one intravenous vasopressor); hypoxia (e.g., plasma oxygen (PO2) levels below or below about 90%); and / or one or more neurological disorders (including altered mental status, lethargy, and seizures). In some implementations, neurotoxicity (NT) can be observed simultaneously with CRS.

[0355] Exemplary CRS-related outcomes include elevated or high serum levels of one or more factors, including cytokines and chemokines and other CRS-related factors. Exemplary outcomes also include increased synthesis or secretion of one or more such factors. This synthesis or secretion can occur by T cells or cells that interact with T cells, such as innate immune cells or B cells.

[0356] In some embodiments, CRS-related serum factors or CRS-related outcomes include inflammatory cytokines and / or chemokines, including interferon-γ (IFN-γ), IL-7, IL-12, sIL-2Ra, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein (MIP)-1, tumor necrosis factor α (TNFα), IL-6, and IL-10, IL-1β, IL-8, IL-2, MIP-1, Flt-3L, fractal chemokines, and / or IL-5. In some embodiments, the factors or outcomes include C-reactive protein (CRP). In addition to being an early and readily measurable risk factor for CRS, CRP is also a marker of cell proliferation. In some embodiments, subjects measured to have high CRP levels (e.g., ≥ 15 mg / dL) have CRS. In some embodiments, subjects measured to have high CRP levels do not have CRS. In some embodiments, the measurement of CRS includes a measurement of CRP and another factor indicative of CRS.

[0357] In some embodiments, one or more inflammatory cytokines or chemokines are monitored before, during, or after CAR treatment. In some aspects, 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.

[0358] CRS criteria have been developed to show association with CRS onset in order to predict which patients are more likely to be at risk of 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 a group of seven cytokines (IFNγ, IL-5, IL-6, IL-10, Flt-3L, fractal chemokines, and GM-CSF). Other guidelines on the diagnosis and management of CRS are known (see, for example, Lee et al., Blood. 2014;124(2):188-95; Lee et al., Biol Blood Marrow Transplant 2019; 25(4):625-38). In some implementations, the criteria reflecting the CRS grade are those detailed in Table 1 below.

[0359] In some implementations, the criteria that reflect the CRS level are those detailed in Table 2 below.

[0360] In some implementations, high-dose vasopressor therapy includes those described in Table 3 below.

[0361] 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 implementations, a subject is considered to have “severe CRS” (“sCRS”) in response to or secondary to administration of the cell therapy or a cellular dose thereof if, after administration, the subject exhibits any of the following: (1) fever at least 38 degrees Celsius for at least three days; (2) elevated cytokines, including (a) a maximum fold change of at least two of the following seven cytokines of at least 75 compared to levels immediately following administration: interferon-γ (IFNγ), GM-CSF, IL-6, IL-10, Flt-3L, fractal chemokine, and IL-5, and / or (b) a maximum fold change of at least one of the following seven cytokines of at least 250 compared to levels immediately following administration: interferon-γ (IFNγ), GM-CSF, IL-6, IL-10, Flt-3L, fractal chemokine, and IL-5; and (c) at least one clinical sign of toxicity, such as hypotension (requiring at least one intravenous vasopressor) or hypoxia (PO2 < 0.05). 90% or more of the following: or one or more neurological disorders (including altered mental status, lethargy, and / or epilepsy). In some implementations, severe CRS includes grade 3 or higher CRS, as shown in Tables 1 and 2.

[0362] In some embodiments, the level of toxic outcomes (e.g., CRS-related outcomes) is measured by ELISA (e.g., serum levels of CRS indicators). In some embodiments, fever and / or C-reactive protein (CRP) levels may be measured. In some embodiments, subjects with fever and CRP ≥ 15 mg / dL may be considered at high risk of developing severe CRS. In some embodiments, CRS-related serum factors or CRS-related outcomes include increased levels and / or concentrations of inflammatory cytokines and / or chemokines, including Flt-3L, fractal chemokines, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1β (IL-1β), IL-2, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, interferon-γ (IFN-γ), macrophage inflammatory protein (MIP)-1, MIP-1, sIL-2Rα, or tumor necrosis factor-α (TNFα). In some embodiments, the factor or outcome includes C-reactive protein (CRP). Besides being an early and easily measurable risk factor for CRS, CRP is also a marker of cell proliferation. In some embodiments, subjects measured to have high CRP levels (e.g., ≥ 15 mg / dL) have CRS. In some embodiments, subjects measured to have high CRP levels do not have CRS. In some embodiments, the measurement of CRS includes a measurement of CRP and another factor indicative of CRS.

[0363] In some implementations, outcomes associated with severe CRS or grade 3 or higher (e.g., grade 4 or higher) include one or more of the following: persistent fever, such as fever at a specified temperature (e.g., greater than or greater than about 38 degrees Celsius) for two or more days, such as three or more days, such as four or more days, or at least three consecutive days; fever greater than or greater than about 38 degrees Celsius; and elevated cytokines, such as at least two cytokines (e.g., at least two from the group consisting of: interferon-γ (IFNγ), GM-CSF, IL-6, IL-10, F...). The maximum fold change in IL-3L, fractal chemokines, and IL-5 and / or tumor necrosis factor α (TNFα) compared to pre-treatment levels is, for example, at least or at least about 75, or the maximum fold change in at least one of these cytokines is, for example, at least or at least about 250; and / or at least one clinical sign of toxicity, such as hypotension (e.g., as measured by at least one intravenous vasopressor); hypoxia (e.g., plasma oxygen (PO2) levels below or below about 90%); and / or one or more neurological disorders (including altered mental status, lethargy, and seizures). In some embodiments, severe CRS includes CRS requiring management or care in an intensive care unit (ICU).

[0364] In some embodiments, CRS (such as severe CRS) includes a combination of the following: (1) persistent fever (fever of at least 38 degrees Celsius for at least three days) and (2) serum CRP levels of at least or at least about 20 mg / dL. In some embodiments, CRS covers hypotension requiring the use of two or more vasopressors or respiratory failure requiring mechanical ventilation. In some embodiments, the dose of vasopressor is increased in a second or subsequent administration.

[0365] In some implementations, severe CRS or grade 3 CRS encompasses increased alanine aminotransferase, increased aspartate aminotransferase, chills, febrile neutropenia, headache, left ventricular dysfunction, encephalopathy, hydrocephalus, and / or tremor.

[0366] You can specify methods for measuring or detecting various outcomes.

[0367] In some embodiments, the toxic outcome is neurotoxicity or is associated with neurotoxicity. In some implementations, symptoms associated with the clinical risk of neurotoxicity include confusion, delirium, aphasia, expressive aphasia, lethargy, myoclonus, somnolence, altered mental status, seizures, seizure-like activity, seizures (optionally confirmed by electroencephalography (EEG)), elevated β-amyloid (Aβ) levels, elevated glutamate levels, and elevated oxygen free radical levels. In some implementations, neurotoxicity is graded based on severity (e.g., using a 1–5 scale) (see, for example, 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)).

[0368] In some cases, neurological symptoms may be the earliest symptom of sCRS. In some implementations, neurological symptoms have been observed to begin 5 to 7 days after cell therapy infusion. In some implementations, the duration of neurological changes may range from 3 to 19 days. In some cases, recovery of neurological changes occurs after the resolution of other symptoms of sCRS. In some implementations, treatment with anti-IL-6 and / or one or more steroids does not accelerate the time or extent of resolution of neurological changes.

[0369] In some implementations, a subject is considered to have “severe neurotoxicity” in response to or secondary to administration of the cell therapy or a dose thereof if, after administration, the subject exhibits symptoms of limited self-care (e.g., bathing, dressing and undressing, eating, toileting, taking medication) in the following ways: 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, sensory dulling (e.g., sensory perceptual distortion, resulting in abnormal and uncomfortable sensations), neuralgia (e.g., severe pain along a nerve or nerve group), and / or sensory abnormalities (e.g., dysfunction of sensory neurons, resulting in abnormal skin sensations of tingling, numbness, pressure, cold, and warmth in the absence of a stimulus). In some implementations, severe neurotoxicity includes grade 3 or higher neurotoxicity, as shown in Table 4.

[0370] In some embodiments, the method alleviates symptoms associated with CRS or neurotoxicity compared to other methods. In some aspects, the provided method alleviates symptoms, outcomes, or factors associated with CRS, including those associated with severe CRS or grade 3 or higher CRS, compared to other methods. For example, subjects treated according to the method of the invention may lack symptoms, outcomes, or factors of detectable CRS (e.g., severe CRS or grade 3 or higher CRS) and / or have reduced symptoms, outcomes, or factors, as described (e.g., those shown in Tables 1 and 2). In some embodiments, subjects treated according to the method of the invention may have reduced neurotoxicity symptoms, such as weakness or numbness in the limbs, memory, visual and / or intellectual impairment, uncontrollable compulsive and / or obsessive behaviors, delusions, headaches, cognitive and behavioral problems (including loss of motor control, cognitive decline, and autonomic dysfunction), and sexual dysfunction, compared to subjects treated by other methods. In some implementations, subjects treated according to the method of the present invention may experience reduced symptoms associated with peripheral motor neuropathy, peripheral sensory neuropathy, sensory dullness, neuralgia, or paresthesia.

[0371] In some embodiments, the method mitigates neurotoxicity-related outcomes, including damage to the nervous system and / or brain, such as neuronal death. In some aspects, the method reduces levels of neurotoxicity-related factors such as β-amyloid (Aβ), glutamate, and oxygen free radicals.

[0372] In some embodiments, the toxic outcome is dose-limiting toxicity (DLT). In some embodiments, the toxic outcome is dose-limiting toxicity. In some embodiments, the toxic outcome is the absence of dose-limiting toxicity. In some embodiments, dose-limiting toxicity (DLT) is defined as any grade 3 or higher toxicity, as assessed by any known or published guidance for evaluating a particular toxicity, such as any of the guidelines mentioned above and including the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 4.0.

[0373] In some embodiments, a low rate, risk, or likelihood of toxicity (e.g., CRS or neurotoxicity, or severe CRS or neurotoxicity, such as grade 3 or higher CRS or neurotoxicity) observed when a dose of T cells is administered according to the provided method and / or with the provided product or composition allows for outpatient-based administration of cell therapy. In some embodiments, administration of cell therapy (e.g., T cells, such as CAR-T cells) according to the provided method and / or with the provided product or composition allows for outpatient-based administration of cell therapy. + The dosage of T cells is administered on an outpatient basis, or without the need for hospitalization, such as hospitalization requiring an overnight stay.

[0374] In some respects, cell therapy (e.g., T-cell therapy, e.g., CAR) is not administered before or concurrently with the administration of the cell dose, according to the provided method and / or with the provided product or composition. + Subjects receiving a dose of T-cell therapy (including those treated on an outpatient basis) shall be given an intervention to treat any toxicity unless or until the subject exhibits signs or symptoms of toxicity (such as neurotoxicity or CRS). Exemplary agents for treating, delaying, reducing, or improving toxicity are described in section IC.

[0375] In some implementations, if cell therapy (e.g., T-cell therapy, e.g., CAR) is administered... +If a subject (including subjects treated on an outpatient basis) exhibits fever after receiving a dose of T-cell therapy, the subject is given treatment or instructed to receive or administer treatment to reduce the fever. In some embodiments, the fever in a subject is characterized by the subject's body temperature being equal to or higher than a certain threshold temperature or level. In some aspects, the threshold temperature is a temperature associated with at least low-grade fever, at least moderate fever, and / or at least high-grade fever. In some embodiments, the threshold temperature is a specific temperature or range. For example, the threshold temperature may be 38, 39, 40, 41, or 42 degrees Celsius, and / or may be a range of 38 to 39 degrees Celsius, 39 to 40 degrees Celsius, 40 to 41 degrees Celsius, or 41 to 42 degrees Celsius.

[0376] In some embodiments, treatment designed to reduce fever includes treatment with an antipyretic. The antipyretic may include any agent that reduces fever, such as compounds, compositions, or ingredients, such as any number of agents known to have antipyretic effects, such as NSAIDs (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), salicylates (e.g., aspirin, salicylcholine, magnesium salicylate, and sodium salicylate), acetaminophen, antipyrine, methylaminophen, nabumetone, phenaxone, antipyrine, and antipyretics. In some embodiments, the antipyretic is acetaminophen. In some embodiments, acetaminophen may be administered orally or intravenously at a dose of up to 12.5 mg / kg every four hours. In some embodiments, the antipyretic is or includes ibuprofen or aspirin.

[0377] In some implementations, if the fever is persistent, the subject is given alternative treatment for toxicity. For subjects treated on an outpatient basis, if the subject has persistent fever and / or is determined to have or has persistent fever, the subject is instructed to return to the hospital. In some implementations, if a subject presents with a fever equal to or higher than a relative threshold temperature, and after prescribed treatment (such as treatment designed to reduce fever, such as treatment with an antipyretic (e.g., NSAID or salicylates, e.g., ibuprofen, acetaminophen, or aspirin)) the subject's fever or body temperature does not decrease, or does not decrease by a prescribed amount or decreases by no more than a prescribed amount (e.g., more than 1ºC, and generally without fluctuations of about or more than about 0.5ºC, 0.4ºC, 0.3ºC, or 0.2ºC), the subject has and / or is determined to have or is considered to have persistent fever. For example, if a subject exhibits or is determined to exhibit a fever of at least or at least about 38 or 39 degrees Celsius, said fever occurring over a 6-hour period, an 8-hour period, a 12-hour period, or a 24-hour period, and even after treatment with an antipyretic such as acetaminophen, does not decrease by about 0.5ºC, 0.4ºC, 0.3ºC, or 0.2ºC, or does not decrease by more than or equal to about 0.5ºC, 0.4ºC, 0.3ºC, or 0.2ºC, or decreases by about 1%, 2%, 3%, 4%, or 5%, then said subject is considered to have persistent fever. In some embodiments, the dose of the antipyretic is a dose that is typically effective in reducing fever or a specific type of fever (such as fever associated with bacterial or viral infections, e.g., local or systemic infections) in such subjects.

[0378] In some implementations, a subject is considered to have and / or is identified as having persistent fever if they exhibit a fever equal to or higher than a relative threshold temperature, and the subject's fever or body temperature does not fluctuate by about 1°C or more, and substantially does not fluctuate by about 0.5°C, 0.4°C, 0.3°C, or 0.2°C. The absence of such fluctuation above or equal to a certain amount is typically measured over a given time period (e.g., within a 24-hour, 12-hour, 8-hour, 6-hour, 3-hour, or 1-hour time period, which can be measured from the first sign of fever or the first time the body temperature exceeds the indicated threshold). For example, in some implementations, a subject is considered to have persistent fever if they exhibit a fever of at least or at least about 38 or 39 degrees Celsius, and the temperature of the fever does not fluctuate by more than or more than about 0.5°C, 0.4°C, 0.3°C, or 0.2°C over a 6-hour time period, an 8-hour time period, a 12-hour time period, or a 24-hour time period.

[0379] In some implementations, the fever is persistent; in some aspects, it is present in the initial therapy that may induce toxicity (such as cell therapy, such as T-cell therapy, e.g., CAR). + After administering a dose of T cells, the subject is treated if the subject has been diagnosed with persistent fever, such as within one, two, three, four, five, six hours or less after such diagnosis or the first diagnosis.

[0380] In some embodiments, one or more interventions or agents (such as toxicity-targeting therapies) are administered upon or immediately after a subject is identified or confirmed (e.g., upon initial identification or confirmation) of persistent fever (e.g., as measured according to any of the foregoing embodiments). In some embodiments, one or more toxicity-targeting therapies are administered within a specific time period of such identification or confirmation, such as within 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, or 8 hours of such identification or confirmation.

[0381] In some embodiments, the provided method does not result in a high rate or likelihood of toxicity or toxic outcomes (such as immune effector cell-associated neurotoxicity syndrome (ICANS)) compared to certain other cell therapies, or reduces the rate or likelihood of toxicity or toxic outcomes. In some embodiments, the method does not cause ICANS or does not increase its risk.

[0382] Exemplary ICANS-related outcomes include the ICANS grading scheme developed by the CAR T-cell therapy-related toxicities (CARTOX) consensus group, which consists of a 10-point scale (CARTOX-10) that incorporates key components of the Mini-Mental State Assessment to assess the level of encephalopathy through changes in attention, speech, handwriting, and orientation (see, for example, Neelapu et al., Nat Rev Clin Oncol., 2018, 15:47-62).

[0383] In some implementations, the outcome is related to a method of classifying the severity of ICANS using an immune effector cell encephalopathy (ICE) score. In some implementations, the ICE score includes an assessment of sensory aphasia. II. Cell Therapy and Engineered Cells

[0384] In some embodiments, the cell therapy (e.g., T-cell therapy) methods disclosed herein include administering engineered cells expressing a recombinant receptor (e.g., a CAR), said recombinant receptor being designed to recognize and / or specifically bind to an antigen associated with a disease or condition such as severe and refractory SLE. In a particular embodiment, the antigen bound to or recognized by the recombinant receptor (e.g., CAR) is CD19. In some embodiments, binding to the antigen leads to a response, such as an immune response against such an antigen. In some embodiments, the cells contain or are engineered to contain a recombinant receptor, such as a chimeric antigen receptor (CAR). Recombinant receptors such as CARs typically include an extracellular antigen (or ligand) binding domain that is specific to an antigen, said domain being linked to one or more intracellular signaling components, in some respects via a linker and / or one or more transmembrane domains. In some respects, the engineered cells are provided as pharmaceutical compositions and formulations suitable for administration to a subject (e.g., suitable for adoptive cell therapy). Treatment methods for administering cells and compositions to a subject (e.g., a patient) are also provided.

[0385] In some embodiments, the cells include one or more nucleic acids introduced through genetic engineering, and thereby express recombinant products or genetically engineered products of such nucleic acids. In some embodiments, gene transfer is accomplished by first stimulating the cells, such as by combining the cells with a stimulant that induces a response (e.g., proliferation, survival, and / or activation, as measured, for example, by the expression of cytokines or activation markers), then transducing the activated cells, and expanding them in culture to a quantity sufficient for clinical application. A. Chimeric antigen receptors (e.g., CARs targeting CD19)

[0386] In some embodiments of the provided methods and uses, the chimeric receptor (such as a chimeric antigen receptor) contains one or more domains that combine a ligand-binding domain (e.g., an antibody or antibody fragment) specific for a desired antigen (e.g., CD19) with an intracellular signaling domain. In some embodiments, the intracellular signaling domain is a portion of the intracellular domain that stimulates or activates, such as a T cell stimulation or activation domain, thereby providing a primary activation signal or primary signal. In some embodiments, the intracellular signaling domain contains or additionally contains a co-stimulatory signaling domain to promote effector function. In some embodiments, when the chimeric receptor is genetically engineered into immune cells, it can modulate T cell activity and, in some cases, T cell differentiation or homeostasis, thereby producing genetically engineered cells with improved lifespan, survival, and / or durability in vivo, such as for use in adoptive cell therapy methods.

[0387] Exemplary antigen receptors (including CARs) and methods for engineering and introducing such receptors into cells include, for example, those described in the following documents: International Patent Application Publications WO 200014257, WO 2013126726, WO2012 / 129514, WO 2014031687, WO 2013 / 166321, WO 2013 / 071154, WO 2013 / 123061; U.S. Patent Application Publications US 2002131960, US 2013287748, US 20130149337; US 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; and European Patent Application No. EP2537416; and / or those described in the following documents: Sadelain et al., Cancer Discov. April 2013; 3(4): 388-398; Davila et al. (2013) pLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., October 2012; 24(5): 633-39; Wu et al., Cancer, March 2012 18(2): 160-75. In some respects, antigen receptors include CARs as described in U.S. Patent No. 7,446,190, and those described in International Patent Application Publication No. WO / 2014055668A1. Examples of CARs include those disclosed in any of the aforementioned publications, such as WO2014031687, US 8,339,645, US 7,446,179, US 2013 / 0149337, US Patent No. 7,446,190, US 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 WO 2014031687, US 8,339,645, US 7,446,179, US2013 / 0149337, US Patent No. 7,446,190 and US Patent No. 8,389,282.

[0388] Chimeric receptors (such as CARs) typically include an extracellular antigen-binding domain, similar to a portion of an antibody molecule, and are often variable weights (V-weights) of the antibody. H ) chain region and / or variable light (V L (e.g., scFv antibody fragments). In some embodiments, the antibody or its antigen-binding portion is expressed on the cell as part of a recombinant receptor (such as a chimeric receptor, e.g., CAR), which binds to an antigen (e.g., CD19) (e.g., specific binding).

[0389] In some embodiments, the antigen targeted by the receptor is a peptide. In a particular embodiment, the antigen target is CD19. In some embodiments, the antigen is selectively expressed on B cells targeted for the treatment of autoimmune or inflammatory conditions such as lupus. In some embodiments, the CAR typically includes one or more antibody or antigen-binding fragments or portions targeting CD19 in its extracellular portion.

[0390] Chimeric receptors (such as CARs) typically include an extracellular antigen-binding domain, which is one or more antigen-binding portions of an antibody molecule. In some embodiments, the antigen-binding domain is part of the antibody molecule, typically a variable weight (V) region of the antibody. H ) chain region and / or variable light (V L ) chain regions, such as scFv antibody fragments. In some embodiments, the CAR includes one or more antigen-binding portions of the antibody molecule, such as variable heavy chains (V) derived from monoclonal antibodies (mAbs). H ) and variable light chains (V L The antigen-binding domain is a single-chain antibody fragment (scFv). In some embodiments, the antigen-binding domain is a single-domain antibody (sdAb), such as sdFv, nanobodies, V H H and V NAR In some implementations, the antigen-binding fragment includes an antibody variable region that binds via a flexible linker.

[0391] In some implementations, the 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 an antibody or antigen-binding fragment that specifically binds to CD19, or a variant of said antibody or antigen-binding fragment. 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 having six CDRs (CDRH1-3 and CDRL1-3) that confer binding to CD19.

[0392] The terms "complementarity-determining region" and "CDR," synonyms with "hypervariant region" or "HVR," are known to refer in some cases to a discontinuous amino acid sequence within the antibody variable region that confers antigen specificity and / or binding affinity. Typically, 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. The terms "frame region" and "FR" are known to refer in some cases to the non-CDR portions of the variable regions of both the heavy and light chains. Typically, 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.

[0393] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described in the following literature: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding sitetopography,” 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, January 2003; 27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, June 8, 2001; 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).

[0394] The boundaries of a given CDR or FR can vary depending on the scheme used for identification. For example, the Kabat scheme is based on structure alignment, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based on the most common antibody region sequence lengths, where insertions are provided by insert letters (e.g., "30a"), and deletions occur in some antibodies. These two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many ways. The AbM scheme is a compromise between the Kabat and Chothia definitions, based on the scheme used by the Oxford Molecular AbM antibody modeling software.

[0395] Table 5 below lists exemplary positional boundaries of CDR-L1, CDR-L2, CDR-L3, and CDR-H1, CDR-H2, and CDR-H3 identified by the Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbers are listed using both the Kabat and Chothia numbering schemes. FRs are located between CDRs; for example, FR-L1 precedes CDR-L1, FR-L2 is between CDR-L1 and CDR-L2, FR-L3 is between CDR-L2 and CDR-L3, and so on. Note that because the Kabat numbering scheme shown places insertions at H35A and H35B, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the loop length when numbering using the Kabat numbering convention shown. Table 5. CDR boundaries according to various numbering schemes. 1—- Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD 2—- Al-Lazikani et al., (1997) JMB 273, 927-948

[0396] Therefore, unless otherwise specified, the “CDR” or “complementation-determining region” or individually designated CDR (e.g., CDR-H1, CDR-H2, CDR-H3) of a given antibody or its region (such as its variable region) should be understood to encompass one (or a specific) complementation-determining region as defined by any of the foregoing protocols or other known protocols. For example, in stating that a particular CDR (e.g., CDR-H3) contains a given V H or V L In the case of the amino acid sequence corresponding to the CDR in the variable region amino acid sequence, it should be understood that this CDR has the sequence of the corresponding CDR (e.g., CDR-H3) within the variable region, as defined by any of the foregoing schemes or other known schemes. In some embodiments, a specific CDR sequence is specified. Exemplary CDR sequences of the provided antibody are described using various numbering schemes, but it should be understood that the provided antibody may include CDRs as described according to any other numbering scheme described above or other numbering schemes known to a skilled person.

[0397] Similarly, unless otherwise specified, a FR of a given antibody or its region (such as its variable region) or one or more individually designated FRs (e.g., FR-H1, FR-H2, FR-H3, FR-H4) should be understood to encompass a (or specific) frame region as defined by any known protocol. In some cases, a protocol for identifying one or more specific CDRs or FRs is specified, such as a CDR defined by the Kabat, Chothia, AbM, or Contact methods or other known protocols. In other cases, the specific amino acid sequence of the CDR or FR is given.

[0398] The term "variable region" or "variable domain" refers to the structural domain in the heavy or light chain of an antibody that participates in the binding of the antibody to the antigen. The variable regions of the heavy and light chains of natural antibodies (V1 and V2, respectively) H and V L These typically have similar structures, with each domain containing four conserved frame regions (FRs) and three core regions (CDRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). Single V H or V L The structural domain may be sufficient to confer antigen-binding specificity. Furthermore, V-terminal molecules derived from antibodies that bind to specific antigens can be used. H or V L The structural domain is used to separate antibodies that bind to the antigen, in order to screen complementary V antibodies respectively. L or V HLibrary of structural domains. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0399] The antibodies provided include antibody fragments. An "antibody fragment" refers to a molecule other than a complete antibody, containing the portion of the complete antibody that binds to the antigen bound by the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; and variable heavy chains (V...). H ) region, single-chain antibody molecules (such as scFv) and single-domain V H Single antibodies; and multispecific antibodies formed from antibody fragments. In a particular embodiment, the antibody is a single-chain antibody fragment containing a variable heavy chain region and / or a variable light chain region, such as scFv.

[0400] A single-domain antibody (sdAb) is an antibody fragment comprising all or part of the antibody's heavy chain variable domain or light chain variable domain. In some 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 B cells, like CD19. Exemplary single-domain antibodies include sdFv, nanobodies, and V... H H or V NAR .

[0401] Antibody fragments can be prepared using various techniques, including but not limited to the proteolytic digestion of intact antibodies and production via recombinant host cells. In some embodiments, the antibody is a recombinantly generated fragment, such as a fragment containing an arrangement that is not naturally occurring, such as those having two or more antibody regions or chains bound by synthetic linkers (e.g., peptide linkers). In some embodiments, the antibody fragment is a fragment not generated by enzymatic digestion of a naturally occurring intact antibody. In some embodiments, the antibody fragment is an scFv.

[0402] A “humanized” antibody is an antibody in which all or substantially all of the CDR amino acid residues are derived from a non-human CDR, and all or substantially all of the FR amino acid residues are derived from a human FR. A humanized antibody may optionally include at least a portion of the antibody constant region derived from a human antibody. A “humanized form” of a non-human antibody refers to a variant of a non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in the humanized antibody are replaced by corresponding residues from a non-human antibody (e.g., an antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0403] In some implementations, the scFv contains V derived from an antibody or antibody fragment specific to CD19. H and V L . In some implementations, the extracellular binding domain of the CD19 CAR is derived from antibodies specific to CD19, including, 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 may contain the V of any antibody. H V LAnd / or one or more CDRs, or composed of them. In some embodiments, the antibody or antibody fragment binding 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 U.S. Patent Publication No. US 2016 / 0152723.

[0404] In some implementations, the antigen-binding domain includes a V derived from FMC63. H and / or V L In some respects, it can be scFv. FMC63 generally refers to a mouse monoclonal IgG1 antibody against human-derived Nalm-1 and Nalm-16 cells expressing CD19 (Ling, NR et al. (1987). Leucocyte typing III. 302). In some embodiments, the FMC63 antibody comprises the sequences of CDR-H1 and CDR-H2 shown in SEQ ID NO: 38 and 39, and CDR-H3 shown in SEQ ID NO: 40 or 54, respectively; and CDR-L1 shown in SEQ ID NO: 35, and CDR-L2 shown in SEQ ID NO: 36 or 55 and CDR-L3 shown in SEQ ID NO: 37 or 56. In some embodiments, the FMC63 antibody comprises a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 41. H ) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 42. L ).

[0405] 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 thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with any of the foregoing. In some embodiments, scFv comprises the FMC63 variable heavy chain region shown in SEQ ID NO: 41 and the FMC63 variable light chain region shown in SEQ ID NO: 42, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the foregoing.

[0406] In some embodiments, the FMC63 antibody comprises the CDR-H1 and CDR-H2 shown in SEQ ID NO: 38 and 39, and the CDR-H3 shown in SEQ ID NO: 40 or 54, respectively; and the CDR-L1 shown in SEQ ID NO: 35, and the CDR-L2 shown in SEQ ID NO: 36 or 55 and the CDR-L3 shown in SEQ ID NO: 37 or 56, respectively. In some embodiments, the FMC63 antibody comprises a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 41. H ) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 42. LIn 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 thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with any of the foregoing. In some embodiments, scFv comprises the FMC63 variable heavy chain region shown in SEQ ID NO: 41 and the FMC63 variable light chain region shown in SEQ ID NO: 42, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the foregoing.

[0407] In some embodiments, the variable heavy chain and the variable light chain are connected by a joint. In some embodiments, the joint is shown in SEQ ID NO: 24. In some embodiments, scFv contains V in sequence. H , connectors and V L In some implementations, scFv contains V in sequence. L , connectors and V H In some embodiments, the scFv is encoded by the nucleotide sequence shown in SEQ ID NO: 25 or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 25. In some embodiments, the scFv comprises the amino acid sequence shown in SEQ ID NO: 43 or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 43.

[0408] In some implementations, the antigen-binding domain includes a V derived from SJ25C1. H and / or V LIn some respects, it can be scFv. SJ25C1 is a mouse monoclonal IgG1 antibody against human-derived Nalm-1 and Nalm-16 cells expressing CD19 (Ling, NR et al. (1987). Leucocyte typing III. 302). In some embodiments, the SJ25C1 antibody comprises the sequences CDR-H1, CDR-H2, and CDR-H3 shown in SEQ ID NO: 47-49, and CDR-L1, CDR-L2, and CDR-L3 shown in SEQ ID NO: 44-46, respectively. In some embodiments, the SJ25C1 antibody comprises a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 50. H ) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 51. L In some embodiments, 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 thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with any of the foregoing. In some embodiments, the scFv comprises the SJ25C1 variable heavy chain region shown in SEQ ID NO: 50 and the SJ25C1 variable light chain region shown in SEQ ID NO: 51, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with any of the foregoing. In some embodiments, the variable heavy chain and the variable light chain are connected by a connector. In some embodiments, the connector is shown in SEQ ID NO: 52. In some embodiments, the scFv comprises V in sequence. H , connectors and V L In some implementations, scFv contains V in sequence. L , connectors and V HIn some embodiments, scFv comprises the amino acid sequence shown in SEQ ID NO: 53 or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 53.

[0409] In some embodiments, the adapter is shown in SEQ ID NO: 23. In some embodiments, the adapter has the formula -PGGG-(SGGGG)5-P-, where P is proline, G is glycine and S is serine, and the adapter is shown in SEQ ID NO: 22.

[0410] In some embodiments, the anti-CD19 CAR includes the antigen-binding domain described in PCT Publication No. WO 2015187528. In some embodiments, the anti-CD19 CAR is the CAR described in PCT Publication No. WO 2015187528.

[0411] In some embodiments, the anti-CD19 CAR includes an antigen-binding domain, which 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 the following publications: PCT Publication No. WO 2016033570, PCT Publication No. WO 2020233589, US Publication No. US2010 / 0104509, and US Publication No. US 20220220200.

[0412] Exemplary antigen receptors (e.g., CARs) also include CARs of the following FDA-approved products: BREYANZI® (lisocabtagene maraleucel), TECARTUS™ (brexucabtagene autoleucel), KYMRIAH™ (tisagenlecleucel), and YESCARTA™ (axicabtagene ciloleucel). In some embodiments of any of the provided implementations, the CAR is one of the following: BREYANZI® (lisocabtagene maraleucel), TECARTUS™ (brexucabtagene maraleucel), KYMRIAH™ (tisagenlecleucel), or YESCARTA™ (axicabtagene maraleucel). In some embodiments of any of the provided implementations, the CAR is BREYANZI® (lisocabtagene maraleucel), see Sehgal et al., 2020, Journal of Clinical Oncology 38:15_Supplement, 8040; Teoh et al., 2019, Blood 134(Supplement_1):593; and Abramson et al., 2020, The Lancet 396(10254): 839-852). In some embodiments of any of the provided embodiments, the CAR is the CAR of TECARTUS™ (Brekiolenzae, 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 embodiments of any of the provided embodiments, the CAR is the CAR of KYMRIAH™ (Texalenzae, 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., CAR (2021, Blood Adv.5(23):4980-4991; and Fowler et al., 2022, Nature Medicine 28:325-332).In some implementations of any of the provided implementations, the CAR is the CAR of YESCARTA™ (Akirunsai, 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).

[0413] In some aspects, recombinant receptors (e.g., chimeric antigen receptors) include an extracellular portion containing one or more antigen-binding domains (such as antibodies or fragments thereof), and one or more intracellular signaling regions or domains (which may also be interchangeably referred to as cytoplasmic signaling domains or regions). In some aspects, recombinant receptors (e.g., CARs) also include spacers and / or transmembrane domains or portions. In some aspects, spacers and / or transmembrane domains may connect the extracellular portion containing the antigen-binding domain to one or more intracellular signaling regions or domains.

[0414] In some embodiments, the recombinant receptor (such as a CAR) further comprises a spacer, which may contain a hinge domain. In some embodiments, the spacer is in 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 shown in SEQ ID NO: 93. In some embodiments, the hinge domain comprises a CD28 hinge domain, such as a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 94. In some embodiments, the CD28 hinge domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 95. In some embodiments, the hinge domain has an amino acid sequence having at least 80% sequence identity with any of the foregoing embodiments (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).

[0415] In some embodiments, the spacer may be or include at least a portion of an immunoglobulin constant region or a variant or modification thereof, such as a hinge region (e.g., an IgG4 hinge region) and / or C H 1 / C Land / or the Fc region. In some embodiments, the recombinant receptor further comprises a spacer and / or a hinge region. In some embodiments, the constant region or portion is a constant region or portion of human IgG (such as IgG4 or IgG1). In some aspects, a portion of the constant region serves as a spacer between an antigen recognition component (e.g., scFv) and a transmembrane domain. The spacer may have a length that can provide enhanced cellular reactivity after antigen binding compared to the absence of a spacer. In some examples, the length of the spacer is one or about 12 amino acids, or no more than 12 amino acids. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and include any integer between the endpoints of any of the listed ranges. In some embodiments, the spacer region has about 12 or fewer amino acids, about 119 or fewer amino acids, or about 229 or fewer amino acids. Exemplary spacers include only IgG4 hinges, IgG4 hinges connected to both CH2 and CH3 domains, or IgG4 hinges connected to the CH3 domain. Exemplary spacers include, but are not limited to, those described in the following literature: 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. WO 2014031687.

[0416] In some embodiments, the spacer contains only the hinge region of IgG, such as a hinge containing only IgG4 or IgG1, as shown in SEQ ID NO: 1 and encoded by the sequence shown in SEQ ID NO: 2, which is a hinge-only spacer. In some embodiments, the spacer is associated with C. H 2 and / or C H 3. Ig hinges (e.g., IgG4 hinges) connected by structural domains. In some embodiments, the spacer is connected to C... H 2 and C H 3. Ig hinges (e.g., IgG4 hinges) connected by a structural domain, as shown in SEQ ID NO: 4. In some embodiments, the spacer is only connected to C. HA 3-domain connected Ig hinge (e.g., an IgG4 hinge), as shown in SEQ ID NO: 3. In some embodiments, the spacer is or contains a glycine-serine-rich sequence or other flexible linker, such as known flexible linkers. In some embodiments, the constant region or portion is a constant region or portion of IgD. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 5. In some embodiments, the spacer has an amino acid sequence exhibiting at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with any one of SEQ ID NO: 1, 3, 4 and 5.

[0417] In some aspects, the spacer is a polypeptide spacer, said polypeptide spacer: (a) comprising all or part of an immunoglobulin hinge or a modified form thereof, or comprising about 15 or fewer amino acids and not comprising a CD28 extracellular region or a CD8 extracellular region; (b) comprising all or part of an immunoglobulin hinge (optionally an IgG4 hinge) or a modified form thereof, and / or comprising about 15 or fewer amino acids and not comprising a CD28 extracellular region or a CD8 extracellular region; or (c) having a length of about 12 amino acids and / or comprising all or part of an immunoglobulin hinge (optionally an IgG4 hinge) or a modified form thereof; or (d) comprising or containing the following: SEQ ID NO: 1. The amino acid sequence shown in 3-5, 27-34 or 24, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with any of the preceding items, or (e) the inclusion formula X1PPX2P or consisting thereof, wherein X1 is glycine, cysteine ​​or arginine, and X2 is cysteine ​​or threonine, as shown in SEQ ID NO: 26.

[0418] In some embodiments, the antigen receptor includes an intracellular domain directly or indirectly connected to an extracellular domain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain connecting the extracellular domain and the intracellular signaling domain. In some embodiments, the intracellular signaling domain includes an ITAM. For example, in some aspects, the antigen recognition domain (e.g., the extracellular domain) is typically linked to one or more intracellular signaling components (such as signaling components that mimic activation via an antigen receptor complex (e.g., a TCR complex, in the case of CAR) and / or signal transduction via another cell surface receptor). In some embodiments, the chimeric receptor includes a transmembrane domain that is linked or fused between an extracellular domain (e.g., scFv) and an intracellular signaling domain. Thus, in some embodiments, the antigen-binding component (e.g., an antibody) is linked to one or more transmembrane domains and intracellular signaling domains.

[0419] In one implementation, a transmembrane domain that naturally associates with one of the domains in the receptor (e.g., CAR) is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0420] In some embodiments, the transmembrane domain is derived from a natural or synthetic source. When the source is natural, in some aspects, the domain is derived from any membrane-binding or transmembrane protein. Transmembrane regions include those derived from (i.e., containing at least one or more of) the following: the α, β, or ζ chain of the T cell receptor; CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 (4-1BB), or CD154. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain primarily contains hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain. In some embodiments, the connection is achieved through a linker, a spacer, and / or one or more transmembrane domains. In some aspects, the transmembrane domain contains the transmembrane portion of CD28 or a variant thereof. The extracellular domain and the transmembrane domain may be directly or indirectly connected. In some embodiments, the extracellular domain and the transmembrane domain are connected by spacers (as described herein).

[0421] In some embodiments, the transmembrane domain is a transmembrane domain of human CD28 or a variant thereof, such as the 27-amino acid transmembrane domain of human CD28 (accession number: P10747.1). In some embodiments, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence exhibiting at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 8. In some embodiments, the transmembrane domain-containing portion of the recombinant receptor comprises the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with it.

[0422] 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 amino acid sequence shown in SEQ ID NO: 96, or an amino acid sequence exhibiting at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 96.

[0423] In some embodiments, the recombinant receptor (e.g., CAR) includes at least one or more intracellular signaling components, such as intracellular signaling regions or domains. In some aspects, T cell activation is described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and those that act in a non-antigen-dependent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of these signaling components. The intracellular signaling region includes those that mimic or approximate signals via: signals via a native antigen receptor, signals via a combination of such receptor and a co-stimulatory receptor, and / or signals via the co-stimulatory receptor alone. In some embodiments, short oligopeptide or polypeptide linkers (e.g., linkers of 2 to 10 amino acids in length, such as linkers containing glycine and serine, e.g., glycine-serine dinucleotides) exist between and form a connection between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[0424] In some embodiments, upon CAR conjugation, the cytoplasmic domains or intracellular signaling regions of the CAR activate 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 cases, the CAR induces T cell functions such as cytolytic activity or T helper cell activity such as the secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling region of an antigen receptor component or co-stimulatory molecule (e.g., if it transduces effector function signals) is used instead of the complete immune stimulation chain. In some embodiments, the intracellular signaling region (e.g., comprising one or more intracellular domains) includes the cytoplasmic sequence of a T cell receptor (TCR) and, in some respects, also includes those cytoplasmic sequences of co-receptors (which, in their natural context, synergize with such receptors to initiate signal transduction upon antigen receptor conjugation) and / or any derivatives or variants of such molecules, and / or any synthetic sequences having the same functional capabilities. In some embodiments, the intracellular signaling region (e.g., comprising one or more intracellular domains) includes the cytoplasmic sequence of a region or domain involved in providing co-stimulatory signals.

[0425] In some aspects, the CAR contains a primary cytoplasmic signaling sequence that regulates the primary activation of the TCR complex. The primary cytoplasmic signaling sequence that functions in a stimulatory manner may contain a signaling motif called an immune receptor tyrosine activation motif or ITAM. Examples of primary cytoplasmic signaling sequences containing ITAMs include those derived from the CD3ζ chain, FcRγ, CD3γ, CD3δ, and CD3ε. In some embodiments, one or more cytoplasmic signaling molecules in the CAR contain a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3ζ.

[0426] In some embodiments, the receptor includes an intracellular component of the TCR complex, such as the TCR CD3 chain, for example, the CD3ζ chain, which mediates T cell activation and cytotoxicity. Therefore, in some aspects, the antigen-binding portion is linked to one or more cell signaling modules. In some embodiments, the cell signaling module includes a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. In some embodiments, the receptor further includes, for example, a portion of one or more additional molecules, such as Fc receptor γ, CD8α, CD8β, CD4, CD25, or CD16. For example, in some aspects, CAR or other chimeric receptors include a chimeric molecule between CD3-ζ (CD3ζ) or Fc receptor γ and CD8α, CD8β, CD4, CD25, or CD16.

[0427] In some embodiments, the intracellular (or cytoplasmic) signaling region comprises the human CD3 chain, optionally the CD3ζ stimulatory signaling domain, or a functional variant thereof, such as the cytoplasmic domain of 112 AAs of an allotype 3 of human CD3ζ (accession number: P20963.2), or the CD3ζ signaling domain as described in U.S. Patent Nos. 7,446,190 or 8,911,993. In some embodiments, the intracellular signaling region comprises the amino acid sequence shown in SEQ ID NO: 13, 14, or 15, or an amino acid sequence exhibiting at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with SEQ ID NO: 13, 14, or 15. In some embodiments, the CD3ζ signaling domain comprises or is composed of the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the CD3ζ signaling domain comprises or is composed of the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the CD3ζ signaling domain comprises or is composed of the amino acid sequence shown in SEQ ID NO: 15.

[0428] In the case of a natural TCR, full activation typically requires not only signal transduction via the TCR but also co-stimulatory signals. Therefore, in some embodiments, components for generating secondary or co-stimulatory signals are also included in the CAR to promote full activation. In other embodiments, the CAR does not contain components for generating co-stimulatory signals. In some aspects, an additional CAR is expressed in the same cell and provides components for generating secondary or co-stimulatory signals.

[0429] In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T-cell co-stimulatory molecule. In some embodiments, the CAR includes a signal transduction domain and / or transmembrane portion of a co-stimulatory receptor (such as CD28, 4-1BB, OX40 (CD134), CD27, DAP10, DAP12, ICOS, and / or other co-stimulatory receptors). In some embodiments, the CAR includes a co-stimulatory region or domain of CD28 or 4-1BB (such as human CD28 or human 4-1BB).

[0430] In some embodiments, the intracellular signaling region or domain comprises the intracellular co-stimulatory signaling domain of human CD28 or a functional variant or portion thereof, such as its 41-amino acid domain and / or such domain having LL to GG substitutions at positions 186-187 of the native CD28 protein. In some embodiments, the intracellular signaling domain may comprise the amino acid sequence shown in SEQ ID NO: 10 or 11 or an amino acid sequence exhibiting at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 10 or 11. In some embodiments, the intracellular region includes an intracellular costimulatory signaling domain of 4-1BB or a functional variant or portion thereof, such as the cytoplasmic domain of 42 amino acids of human 4-1BB (accession number Q07011.1) or a functional variant or portion thereof, such as the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence exhibiting at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 12.

[0431] In some respects, the same CAR includes both the primary (or activating) cytoplasmic signal transduction region and the co-stimulatory signal transduction component.

[0432] In some embodiments, the activation domain is contained within a CAR, while the co-stimulatory component is provided by another CAR that recognizes a different antigen. In some embodiments, the CAR includes an activating or stimulating CAR and a co-stimulatory CAR, both expressed on the same cell (see WO2014 / 055668). In some aspects, the cell includes one or more stimulating or activating CARs and / or co-stimulatory CARs. In some embodiments, the cell also includes an inhibitory CAR (iCAR, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December 2013)), such as a CAR that recognizes antigens other than those associated with and / or specific to a disease or condition, thereby reducing or inhibiting activation signals delivered via a disease-targeting CAR through binding of the inhibitory CAR to its ligand, for example, to reduce off-target effects.

[0433] In some implementations, the two receptors induce activation and inhibition signals to the cell, respectively, such that the binding of one receptor to its antigen activates the cell or induces a response, while the binding of the second inhibitory receptor to its antigen induces signals that inhibit or attenuate the response. An example is a combination of an activating CAR and an inhibitory CAR (iCAR). For instance, this strategy can be used to reduce the likelihood of off-target effects, for example, in a context where the activating CAR binds to an antigen expressed in a disease or symptom but also on normal cells, and the inhibitory receptor binds to a separate antigen expressed on normal cells but not on cells with the disease or symptom.

[0434] In some aspects, chimeric receptors are or include inhibitory CARs (e.g., iCARs) and include intracellular components that attenuate or suppress immune responses, such as those induced by ITAM and / or co-stimulatory responses in cells. Examples 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 aspects, engineered cells contain inhibitory CARs that contain or are derived from signaling domains of such inhibitory molecules, such that they attenuate cellular responses, for example, induced by activating and / or co-stimulating CARs.

[0435] In some contexts, CARs are referred to as first-generation, second-generation, and / or third-generation CARs. In some respects, first-generation CARs are CARs that provide only CD3 chain-induced signaling upon antigen binding; in some respects, second-generation CARs are CARs that provide both this signal and co-stimulatory signals, such as CARs that include intracellular signaling domains from co-stimulatory receptors (e.g., CD28 or CD137); and in some respects, third-generation CARs are CARs that contain multiple co-stimulatory domains from different co-stimulatory receptors.

[0436] In some implementations, the CAR includes one or more (e.g., two or more) co-stimulatory domains and activation domains (e.g., primary activation domains) in the cytoplasm. Exemplary CARs include intracellular components of CD3-ζ, CD28, and 4-1BB.

[0437] In some embodiments, the antigen receptor further includes a marker, and / or cells expressing CAR or other antigen receptors also include alternative markers (such as cell surface markers) that can be used to confirm that the cells have been transduced or engineered to express the receptor. In some aspects, the marker includes all or part of CD34, NGFR, or epidermal growth factor receptors (e.g., truncated forms), such as truncated forms of cell surface receptors (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operatively linked to a polynucleotide encoding an adapter sequence (such as a cleavable adapter sequence, e.g., T2A). For example, the marker and optionally the adapter sequence can be any of those disclosed in published patent application number WO 2014031687. For example, the marker can be a truncated EGFR (tEGFR) optionally linked to an adapter sequence, such as the T2A cleavable adapter sequence.

[0438] Exemplary polypeptides of truncated EGFR (e.g., tEGFR) comprise the amino acid sequence shown in SEQ ID NO: 7 or 16 or exhibit amino acid sequence identity with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the sequence in SEQ ID NO: 7 or 16. Exemplary T2A adapter sequences comprise the amino acid sequence shown in SEQ ID NO: 6 or 17 or exhibit amino acid sequence identity with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the sequence in SEQ ID NO: 6 or 17.

[0439] In some embodiments, the marker is a molecule (e.g., a cell surface protein) or a portion thereof that is not naturally found on T cells or on the surface of T cells. In some embodiments, the molecule is a non-self molecule, such as a non-self protein, i.e., a molecule that is not recognized as "self" by the immune system of the host to which the cell is to be adopted.

[0440] In some embodiments, the marker does not provide therapeutic function and / or produces any effect other than serving as a marker for genetic engineering (e.g., for selecting successfully engineered cells). In other embodiments, the marker may be a therapeutic molecule or a molecule that originally performs a certain desired function, such as a ligand of cells that will be encountered in vivo, such as a co-stimulatory or immune checkpoint molecule, to enhance and / or weaken the cell response after adoptive transfer and encounter with the ligand.

[0441] In some embodiments, the chimeric antigen receptor includes an extracellular portion containing an antibody or fragment described herein. In some aspects, the chimeric antigen receptor includes an extracellular portion containing an antibody or fragment described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment includes scFv or a single-domain V. H The antibody, and the intracellular domain contains ITAM. In some aspects, the intracellular signaling domain includes a signaling domain of the ζ chain of the CD3-ζ (CD3ζ) chain. In some embodiments, the CD3-ζ chain is the human CD3-ζ chain. In some embodiments, the intracellular signaling region further includes a CD28 and CD137 (4-1BB, TNFRSF9) co-stimulatory domain linked to the CD3ζ intracellular domain. In some embodiments, CD28 is human CD28. In some embodiments, 4-1BB is human 4-1BB. In some embodiments, the chimeric antigen receptor includes a transmembrane domain disposed between the extracellular domain and the intracellular signaling region. In some aspects, the transmembrane domain contains a transmembrane portion of CD28. The extracellular domain and the transmembrane domain may be directly or indirectly connected. In some embodiments, the extracellular domain and the transmembrane domain are connected by a spacer (as described herein).

[0442] In some embodiments, the CAR contains an antibody (e.g., an antibody fragment), a transmembrane domain (which is or contains a transmembrane portion of CD28 or a functional variant thereof), and an intracellular signaling domain containing a signaling portion of CD28 or a functional variant thereof and a signaling portion of CD3ζ or a functional variant thereof. For example, in some embodiments, the CAR includes an antibody (such as an antibody fragment, including scFv, for example, specific to CD19, as any of the above), a spacer (such as a spacer containing a portion of an immunoglobulin molecule (such as a hinge region and / or one or more constant regions of a heavy chain molecule), such as a spacer containing an Ig hinge), a transmembrane domain containing all or part of a transmembrane domain derived from CD28, an intracellular signaling domain derived from CD28, and a CD3ζ signaling domain.

[0443] In some embodiments, the CAR contains an antibody (e.g., an antibody fragment), a transmembrane domain (which is or contains a transmembrane portion of CD28 or a functional variant thereof), and an intracellular signaling domain containing a 4-1BB signaling portion or a functional variant thereof and a CD3ζ signaling portion or a functional variant thereof. In some such embodiments, the receptor further comprises a spacer containing a portion of an Ig molecule (such as a human Ig molecule) (such as an Ig hinge, e.g., an IgG4 hinge), such as a hinge-only spacer. In some embodiments, the CAR comprises an antibody or fragment (such as scFv, e.g., specific for CD19, as any of the above), a spacer (such as any spacer containing an Ig hinge), a CD28-derived transmembrane domain, a 4-1BB-derived intracellular signaling domain, and a CD3ζ-derived signaling domain.

[0444] In a particular embodiment, 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 signal transduction domain containing a co-stimulatory signal transduction region (which is a 4-1BB signal transduction domain and a CD3-ζ (CD3ζ) chain signal transduction domain). In some embodiments, the scFv contains the sequence shown in SEQ ID NO: 43. In some embodiments, the scFv has a VL and a VH, wherein the VL has a CDR containing the amino acid sequences RASQDISKYLN (SEQ ID NO: 35), SRLHSGV (SEQ ID NO: 36), and GNTLPYTFG (SEQ ID NO: 37); and the VH has a CDR containing the amino acid sequences DYGVS (SEQ ID NO: 38), VIWGSETTYYNSALKS (SEQ ID NO: 39), and YAMDYWG (SEQ ID NO: 40). In some embodiments, the transmembrane domain has the sequence shown in SEQ ID NO: 8. In some embodiments, the transmembrane domain has a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO: 8. In some embodiments, the 4-1BB co-stimulatory signal transduction domain has the sequence shown in SEQ ID NO: 12. In some embodiments, the 4-1BB co-stimulatory signal transduction domain has a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO: 12. In some embodiments, the CD3-ζ domain has the sequence shown in SEQ ID NO: 13. In some embodiments, the CD3ζ signaling domain has a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO: 13. In some embodiments, the CAR contains a hinged immunoglobulin spacer between the scFv and the transmembrane domain. In some embodiments, the spacer is shown in SEQ ID NO: 1.

[0445] In any particular embodiment of the provided method, the CAR contains, in sequence from the N-terminus to the C-terminus: an extracellular antigen-binding domain of the 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 co-stimulatory signal transduction domain shown in SEQ ID NO: 12, and a signal transduction domain of the CD3-ζ (CD3ζ) chain shown in SEQ ID NO: 13.

[0446] 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 higher sequence identity with SEQ ID NO: 91. In some embodiments, the CAR comprises the sequence shown in SEQ ID NO: 91. In some embodiments, the CAR is shown in SEQ ID NO: 91. In some embodiments, the CAR is a CD19 CAR present in a leek-my-ren.

[0447] In some embodiments, the CAR is encoded by a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 92. In some embodiments, the CAR is encoded by the nucleotide sequence shown in SEQ ID NO: 92.

[0448] In some embodiments, the CAR contains, sequentially from the N-terminus to the C-terminus: an extracellular antigen-binding domain of an scFv (such as the scFv shown in SEQ ID NO: 43) comprising the variable heavy chain region of FMC63 shown in SEQ ID NO: 41 and the variable light chain region of FMC63 shown in SEQ ID NO: 42; a CD8α hinge domain of SEQ ID NO: 93; a CD8α transmembrane domain of SEQ ID NO: 96; a 4-1BB co-stimulatory 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 higher sequence identity with any of the aforementioned sequences. In some embodiments, the CAR has an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 97. In some embodiments, the CAR has the sequence shown in SEQ ID NO: 97. In some embodiments, the CAR is a CD19 CAR present in tesalenproxetine.

[0449] In some embodiments, the CAR contains, sequentially from the N-terminus to the C-terminus: an extracellular antigen-binding domain as an scFv (such as the scFv shown in SEQ ID NO: 43) comprising the variable heavy chain region of FMC63 shown in SEQ ID NO: 41 and the variable light chain region of FMC63 shown in SEQ ID NO: 42; a CD28 hinge domain of SEQ ID NO: 94; a CD28 transmembrane domain of SEQ ID NO: 8 or 9; a CD28 co-stimulatory domain of SEQ ID NO: 10; and a CD3ζ signaling domain of SEQ ID NO: 13. In some embodiments, the CAR has an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 98. In some embodiments, the CAR has the sequence shown in SEQ ID NO: 98. In some embodiments, the CAR is a CD19 CAR present in acerencin.

[0450] In some embodiments, the CAR contains an extracellular binding domain consisting of an scFv derived from an anti-CD19 antibody called 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 co-stimulatory domain (e.g., SEQ ID NO: 10), and a CD3ζ signaling domain (e.g., SEQ ID NO: 13). In some embodiments, the scFv named 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 contain a human CD8α leader sequence (SEQ ID NO: 115). In some embodiments, the CAR has the sequence shown in SEQ ID NO: 116. In some embodiments, the CAR has the sequence shown in SEQ ID NO: 117. In some embodiments, the CAR contains a Hu19-derived scFv, a CD8a hinge and transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain. In some embodiments, the CAR has the sequence shown in SEQ ID NO: 118. In some embodiments, the CAR does not contain a signaling sequence.

[0451] In some embodiments, the CAR comprises an extracellular binding domain consisting of an scFv derived from a fully human antibody and an intracellular signal transduction domain comprising a 4-1BB co-stimulatory domain and a CD3ζ signaling domain. In some embodiments, the light chain variable region of the scFv comprises the amino acid sequence shown in SEQ ID NO: 106, and the heavy chain variable region of the scFv comprises the amino acid sequence shown in SEQ ID NO: 107. In some embodiments, the light chain variable region of the scFv comprises the amino acid sequence shown in SEQ ID NO: 109, and the heavy chain variable region of the scFv comprises the amino acid sequence shown in SEQ ID NO: 110. In some embodiments, the scFv has the sequence shown in SEQ ID NO: 105. In some embodiments, the scFv has the sequence shown in SEQ ID NO: 108.

[0452] In some embodiments, the CAR contains a fully human anti-CD19 antibody, a CD8α hinge and transmembrane domain, a CD28 co-stimulatory domain, and a CD3ζ activation domain. In some embodiments, the CAR has the sequence shown in SEQ ID NO: 119 or a sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO: 119. In some embodiments, the CAR has the sequence shown in SEQ ID NO: 120 or a sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO: 120. In some embodiments, the CAR has the sequence shown in SEQ ID NO: 121 or a sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO: 121. In some embodiments, the CAR has the sequence shown in SEQ ID NO: 122 or a sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO: 122. In some embodiments, the CAR has the sequence shown in SEQ ID NO: 123 or a sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO: 123. In some embodiments, the CAR has the sequence shown in SEQ ID NO: 124 or a sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO: 124. In some embodiments, the CAR has the sequence shown in SEQ ID NO: 125 or a sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO: 125. In some embodiments, the CAR has the sequence shown in SEQ ID NO: 126 or a sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO: 126. In some embodiments, the CAR has the sequence shown in SEQ ID NO: 127 or a sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO: ...

Claims

1. A method of treating a subject with a systemic autoimmune disease, the method comprising administering to a subject with or suspected of having a severe systemic autoimmune disease a dose of CD19-directed genetically modified T cells derived from a composition comprising engineered T cells expressing a chimeric antigen receptor (CAR), wherein the dose of the T cells is positive for expression of a CD19-binding CAR, and the dose is from 1 × 10⁻⁶. 6 Up to 50 × 10 6 One CAR-positive live T cell.

2. A method of treating a subject with a systemic autoimmune disease, the method comprising administering to a subject with or suspected of having a moderate systemic autoimmune disease a dose of CD19-directed genetically modified T cells, wherein the dose of the T cells is positive for expression of a CD19-binding chimeric antigen receptor (CAR), and the dose is from 1 × 10⁻⁶. 6 Up to 50 × 10 6 One CAR-positive live T cell.

3. The method according to claim 1 or claim 2, wherein the systemic autoimmune disease is selected from systemic lupus erythematosus (SLE), Sjögren'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, Behçet's disease, rheumatoid arthritis, IgA nephropathy, pemphigus vulgaris, myasthenia gravis, autoimmune hemolytic anemia, immune thrombocytopenic purpura, IgG...

4. Related diseases, 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 motor neuropathy, necrotizing myopathy, neuromyelitis optica spectrum disorder, mixed connective tissue disorder, POEMS syndrome, primary biliary cholangitis, psoriasis, Rh hemolytic disease, Still's disease, type 1 diabetes mellitus, urticaria, capillary leak syndrome, cytokine release syndrome, erythema multiforme, pyoderma gangrenosa, X-linked agammaglobulinemia, antiphospholipid syndrome, and chronic inflammatory demyelinating polyneuropathy.

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

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

6. The method according to any one of claims 1-3, wherein the systemic autoimmune disease is myasthenia gravis.

7. The method according to any one of claims 1-3, wherein the systemic autoimmune disease is bullous pemphigoid.

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

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

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

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