Methods for treating autoimmune diseases

Abstract

Provided herein are methods for treating subjects having or suspected of having an autoimmune disease (e.g., systemic lupus erythematosus and / or lupus nephritis) using natural killer (NK) cells, as well as related compositions, uses, and products. In some embodiments, NK cells express recombinant receptors such as CD19-directed chimeric antigen receptors (CARs).

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Application No. 63 / 494902 filed on 7 April 2023, U.S. Provisional Application No. 63 / 523598 filed on 27 June 2023, U.S. Provisional Application No. 63 / 544111 filed on 13 October 2023, U.S. Provisional Application No. 63 / 618144 filed on 5 January 2024, and U.S. Provisional Application No. 63 / 567812 filed on 20 March 2024, the entire contents of each of these applications being incorporated herein by reference.

[0002] field This disclosure relates, in some embodiments, to methods for treating autoimmune diseases (e.g., lupus) using natural killer (NK) cells, as well as related compositions, uses, and products. NK cells generally express recombinant receptors, such as chimeric antigen receptors (CARs), for targeting antigens such as CD19. In some embodiments, subjects have or are suspected of having an autoimmune disease (e.g., B cell-mediated autoimmune disease). In some embodiments, subjects have or are suspected of having systemic lupus erythematosus (SLE) and / or lupus nephritis (LN). [Background technology]

[0003] Autoimmune diseases encompass a multitude of heterogeneous conditions in which the target immune system attacks target healthy cells, tissues, and / or organs. B cells contribute to the development of autoimmune diseases in various ways, including the production of autoantibodies, their role as antigen-presenting cells (APCs), and the production of cytokines. Current strategies for treating autoimmune diseases include the use of corticosteroids, immunosuppressants, and B-cell targeting agents. Such strategies are often limited in potency and / or persistence, unsuitable for chronic use, or both. Therefore, there is a need for effective treatments for patients with autoimmune diseases. Methods and uses that address these needs are provided.

[0004] Incorporating data by referencing sequence listing files This application incorporates by reference the data contained in the sequence listing XML file submitted concurrently with this application: file name: NKT104WO_ST26.xml; created on April 2, 2024, and size 39,468 bytes. [Overview of the project]

[0005] Provided herein are methods for treating autoimmune diseases, comprising administering to a subject having an autoimmune disease a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR). In some embodiments, the autoimmune disease is a B-cell mediated autoimmune disease.

[0006] Also provided herein is a method for reducing B cells in a subject, comprising administering to a subject with a B cell-mediated disease a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR). Also provided herein is a method for reducing autoantibody levels in a subject with a B cell-mediated disease, comprising administering to a subject with a B cell-mediated disease a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR).

[0007] Also provided herein is a method for reducing B cells in a subject having a B cell-mediated disease, comprising administering to the subject a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein (i) the composition comprising NK cells genetically engineered to express the CAR is administered to the subject in a dosing regimen comprising a dosing cycle, and (ii) the method reduces peripheral B cells in the subject by at least about 90%; peripheral B cells are significantly reduced in the subject for the duration of the dosing cycle; and / or at least about 75% of regrowing peripheral B cells are non-class-switched B cells. In some embodiments, the method reduces peripheral B cells in the subject by at least about 90%. In some embodiments, peripheral B cells are significantly reduced in the subject for the duration of the dosing cycle. In some embodiments, at least about 75% of regrowing peripheral B cells are non-class-switched B cells.

[0008] In some embodiments, B cell-mediated diseases are autoimmune diseases. In some embodiments, genetically modified NK cells are allogeneic to the target.

[0009] In some embodiments, the method reduces B cells in a subject by at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, or at least about 99%. In some embodiments, the method reduces B cells in a subject by at least about 70%. In some embodiments, the method reduces B cells in a subject by at least about 75%. In some embodiments, the method reduces B cells in a subject by at least about 80%. In some embodiments, the method reduces B cells in a subject by at least about 85%. In some embodiments, the method reduces B cells in a subject by at least about 90%.

[0010] In some embodiments, the method reduces B cells in the subject for at least about 30 days, at least about 45 days, at least about 60 days, at least about 75 days, or at least about 90 days. In some embodiments, the method reduces B cells in the subject for at least about 30 days. In some embodiments, the method reduces B cells in the subject for at least about 45 days. In some embodiments, the method reduces B cells in the subject for at least about 60 days. In some embodiments, the method reduces B cells in the subject for at least about 75 days. In some embodiments, the method reduces B cells in the subject for at least about 90 days. In some embodiments, the method reduces B cells in the subject for at least 30 days, at about 45 days, at about 60 days, at about 75 days, or at about 90 days. In some embodiments, the method reduces B cells in the subject for at least 30 days. In some embodiments, the method reduces B cells in the subject for at least 45 days. In some embodiments, the method reduces B cells in the subject for at least 60 days. In some embodiments, the method reduces B cells in the subject for at least 75 days. In some embodiments, this method reduces B cells in the subject for approximately 90 days. In some embodiments, the B cells are peripheral B cells.

[0011] In some embodiments, the autoimmune disease is a T-cell mediated autoimmune disease. In some embodiments, the autoimmune disease is a plasma cell mediated autoimmune disease.

[0012] In some embodiments, the subject is serologically positive for autoantibodies. In some embodiments, the autoantibody is associated with an autoimmune disease. In some embodiments, the autoantibody is associated with a B cell-mediated disease. In some embodiments, the autoantibody is an antinuclear antibody (ANA). In some embodiments, the autoantibody is an antithyroid antibody. In some embodiments, the autoantibody is an antineutrophil cytoplasmic antibody (ANCA). In some embodiments, the autoantibody is an antithrombin antibody. In some embodiments, the autoantibody is an anti-citrullinated peptide (CP) antibody. In some embodiments, the autoantibody is an anti-actin antibody. In some embodiments, the autoantibody is an antiphospholipid antibody. In some embodiments, the autoantibody is an anti-smooth muscle antibody. In some embodiments, the autoantibody is an anti-mitochondrial antibody. In some embodiments, the autoantibody is an anti-ganglioside antibody. In some embodiments, the autoantibody is an anti-signal recognition peptide (SRP) antibody. In some embodiments, the autoantibody is an anti-nicotinic acetylcholine receptor (AChR) antibody. In some embodiments, the autoantibody is an anti-muscle-specific kinase (MuSK) antibody. In some embodiments, the autoantibody is an anti-voltage-gated calcium channel (VGCC) antibody. In some embodiments, the autoantibody is an anti-bincrine antibody. In some embodiments, the autoantibody is an anti-Hu(ANNA-1) antibody. In some embodiments, the autoantibody is an anti-RF antibody. In some embodiments, this method reduces the level of autoantibodies in the subject.

[0013] In some embodiments, the subjects are serologically positive for anti-EBV antibodies.

[0014] In some embodiments, autoimmune diseases are selected from the group consisting of systemic lupus erythematosus (SLE), lupus nephritis (LN), scleroderma, rheumatoid arthritis (RA), myasthenia gravis (MG), multiple sclerosis (MS) NMDA / NMDAR encephalitis, transverse myelitis, neuromyelitis optica spectrum disorder (NMOSD), myelin oligodendrocyte glycoprotein antibody disease (MOGAD), myelin oligodendrocyte glycoprotein spectrum disorder (MOGSD), idiopathic inflammatory myopathy (IIM; also known as myositis), Sjögren's disease, pemphigus vulgaris, bullous pemphigoid (BP), membranous nephropathy (MN), immune thrombocytopenia (ITP), Hashimoto's disease, Grave's disease, insulin resistance, type 1 diabetes mellitus, antiphospholipid syndrome, vasculitis, antineutrophil cytoplasmic antibody (ANCA) vasculitis (AAV), and anti-synthetase syndrome (ASSD). In some embodiments, the autoimmune disease includes idiopathic inflammatory myopathy (IIM), multiple sclerosis (MS), myasthenia gravis (MG), rheumatoid arthritis (RA), scleroderma, thyroid disease, type 1 diabetes, vasculitis, or any combination thereof. In some embodiments, the autoimmune disease is selected from the group consisting of SLE, LN, scleroderma, MG, IIM, and vasculitis.

[0015] In some embodiments, the autoimmune disease includes scleroderma. In some embodiments, the autoimmune disease is scleroderma. In some embodiments, the autoimmune disease includes systemic sclerosis (also known as systemic scleroderma). In some embodiments, the autoimmune disease is systemic sclerosis (also known as systemic scleroderma). In some embodiments, the autoimmune disease includes focal scleroderma. In some embodiments, the autoimmune disease is focal scleroderma.

[0016] In some embodiments, the autoimmune disease includes myositis (also known as IIM). In some embodiments, the autoimmune disease is myositis (also known as IIM). In some embodiments, the autoimmune disease is selected from the group consisting of anti-synthetase syndrome (ASSD), overlap myopathy (OM), dermatomyositis (DM), clinical myopathic dermatomyositis, juvenile myositis (JM), necrotizing myositis (NM; e.g., necrotizing autoimmune myositis (or immune-mediated necrotizing myositis)), polymyositis (PM), and sporadic inclusion body myositis (sIBM). In some embodiments, the autoimmune disease is ASSD. In some embodiments, the autoimmune disease is OM. In some embodiments, the autoimmune disease is DM. In some embodiments, the autoimmune disease is JM. In some embodiments, the autoimmune disease is NM. In some embodiments, the autoimmune disease is PM. In some embodiments, the autoimmune disease is sIBM.

[0017] In some embodiments, the autoimmune disease includes vasculitis. In some embodiments, the autoimmune disease is vasculitis. In some embodiments, the vasculitis is large vasculitis. In some embodiments, the vasculitis is medium vasculitis. In some embodiments, the vasculitis is small vasculitis. In some embodiments, the vasculitis is anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis. In some embodiments, ANCA vasculitis is granulomatosis with polyangiitis (GPA). In some embodiments, ANCA vasculitis is microscopic polyangiitis (MPA). In some embodiments, ANCA vasculitis is eosinophilic granulomatosis with polyangiitis (EGPA).

[0018] In some embodiments, the autoimmune disease includes myasthenia gravis (MG). In some embodiments, the autoimmune disease is MG. In some embodiments, MG is ocular MG. In some embodiments, MG is early-onset systemic MG. In some embodiments, MG is late-onset MG.

[0019] In some embodiments, the autoimmune disease includes multiple sclerosis (MS). In some embodiments, the autoimmune disease is MS. In some embodiments, MS is primary progressive MS (PPMS). In some embodiments, MS is secondary progressive MS (SPMS). In some embodiments, MS is relapsing-remitting MS (RRMS).

[0020] In some embodiments, the autoimmune disease includes systemic lupus erythematosus (SLE). In some embodiments, the autoimmune disease is systemic lupus erythematosus (SLE). In some embodiments, the autoimmune disease is SLE without lupus nephritis (LN).

[0021] In some embodiments, the autoimmune disease includes lupus nephritis (LN). In some embodiments, the autoimmune disease is lupus nephritis (LN). In some embodiments, the autoimmune disease includes SLE and LN. In some embodiments, the autoimmune disease is SLE and LN.

[0022] Also provided herein is a method for reducing B cells in a subject, comprising administering to a subject having a B cell-mediated disease a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein the CAR comprises (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain. Also provided herein is a method for reducing autoantibody levels in a subject, comprising administering to a subject having a B cell-mediated disease a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein the CAR comprises (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain. In some embodiments, the B cell-mediated disease is an autoimmune disease. In some embodiments, the genetically engineered NK cells are allogeneic to the subject.

[0023] Also provided herein is a method for treating systemic lupus erythematosus (SLE), comprising administering to a subject having SLE a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein the CAR comprises (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain. Also provided herein is a method for preventing systemic lupus erythematosus (SLE), comprising administering to a subject determined to be at risk of SLE a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein the CAR comprises (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain.

[0024] Also provided herein is a method for treating lupus nephritis (LN), comprising administering to a subject having LN a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein the CAR comprises (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain. Also provided herein is a method for preventing lupus nephritis (LN), comprising administering to a subject determined to be at risk of LN a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein the CAR comprises (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain.

[0025] In some embodiments, the extracellular antigen-binding domain includes a heavy chain variable region (VH) having CDR-1, CDR-2, and CDR-3 containing the amino acid sequences described in SEQ ID NOs: 24, 25, and 26, respectively, and a light chain variable region (VL) having CDR-1, CDR-2, and CDR-3 containing the amino acid sequences described in SEQ ID NOs: 27, 28, and 29, respectively. In some embodiments, VH contains the amino acid sequence described in SEQ ID NO: 35. In some embodiments, VL contains the amino acid sequence described in SEQ ID NO: 36. In some embodiments, VH contains the amino acid sequence described in SEQ ID NO: 35, and VL contains the amino acid sequence described in SEQ ID NO: 36. In some embodiments, the extracellular antigen-binding domain is a single-chain variable fragment (scFv) containing the amino acid sequence described in SEQ ID NO: 37.

[0026] Also provided herein is a method for treating an autoimmune disease, comprising administering to a subject having an autoimmune disease a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein the CAR comprises (a) an extracellular antigen-binding domain comprising a heavy chain variable region (VH) comprising CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences described in SEQ ID NOs: 24, 25, and 26, respectively, and a light chain variable region (VL) comprising CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences described in SEQ ID NOs: 27, 28, and 29, respectively; (b) a transmembrane domain comprising a CD8 alpha transmembrane domain; and (c) an intracellular signaling domain comprising an intracellular signaling region of OX40 and a CD3 zeta domain.

[0027] Also provided herein is a method for preventing an autoimmune disease, comprising administering to a subject having an autoimmune disease a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein the CAR comprises (a) an extracellular antigen-binding domain comprising a heavy chain variable region (VH) containing CDR-1, CDR-2, and CDR-3 containing the amino acid sequences described in SEQ ID NOs: 24, 25, and 26, respectively, and a light chain variable region (VL) having CDR-1, CDR-2, and CDR-3 containing the amino acid sequences described in SEQ ID NOs: 27, 28, and 29, respectively; (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the genetically engineered NK cells are allogeneic to the subject.

[0028] Also provided herein is a method for preventing an autoimmune disease, comprising administering to a subject having an autoimmune disease a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein the genetically engineered NK cells are allogeneic to the subject. In some embodiments, the CAR comprises (a) an extracellular antigen-binding domain comprising a heavy chain variable region (VH) containing CDR-1, CDR-2, and CDR-3 containing the amino acid sequences described in SEQ ID NOs: 24, 25, and 26, respectively, and a light chain variable region (VL) having CDR-1, CDR-2, and CDR-3 containing the amino acid sequences described in SEQ ID NOs: 27, 28, and 29, respectively; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0029] Also provided herein are methods for treating autoimmune diseases, comprising administering to a subject having an autoimmune disease a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein the genetically engineered NK cells are allogeneic to the subject. In some embodiments, the CAR comprises (a) an extracellular antigen-binding domain comprising a heavy chain variable region (VH) containing CDR-1, CDR-2, and CDR-3 containing the amino acid sequences described in SEQ ID NOs: 24, 25, and 26, respectively, and a light chain variable region (VL) having CDR-1, CDR-2, and CDR-3 containing the amino acid sequences described in SEQ ID NOs: 27, 28, and 29, respectively; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0030] Also provided herein are methods for treating autoimmune diseases, comprising administering to a subject having an autoimmune disease a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein the CAR comprises (a) an extracellular antigen-binding domain comprising a heavy chain variable region (VH) containing CDR-1, CDR-2, and CDR-3 containing the amino acid sequences described in SEQ ID NOs: 24, 25, and 26, respectively, and a light chain variable region (VL) having CDR-1, CDR-2, and CDR-3 containing the amino acid sequences described in SEQ ID NOs: 27, 28, and 29, respectively; (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the genetically engineered NK cells are allogeneic to the subject.

[0031] Also provided herein are methods for treating autoimmune diseases, comprising administering to a subject having an autoimmune disease a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein (i) the subject has been administered lymphocytapheresis prior to administration of the composition comprising NK cells genetically engineered to express a CAR; (ii) the lymphocytapheresis comprises the administration of cyclophosphamide but not fludarabine; and (iii) the autoimmune disease is selected from the group consisting of scleroderma, myositis, and vasculitis.

[0032] Also provided herein is a method for treating an autoimmune disease, comprising administering to a subject having an autoimmune disease a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein (i) the composition comprising NK cells genetically engineered to express a CAR is administered to the subject in a dosing regimen comprising a dosing cycle, the dosing cycle comprising a first dose, a second dose, and a third dose of the composition; and (ii) each of the first, second, and third doses of the dosing cycle comprises approximately 1 × 10⁻¹⁶ 9 Individual CAR-expressing NK cells ~ approximately 2.5 × 10⁶ 9 The method comprises (iii) a CAR-expressing NK cell composition, (iii) a second dose administered to the subject about 2-4 days after the first dose was administered to the subject, and a third dose administered to the subject about 2-4 days after the second dose was administered to the subject; and (iv) about 3 days before administering the composition containing NK cells genetically engineered to express CAR, the subject is administered lymphocyte apheresis consisting of a single dose of about 1000 mg / m2 of cyclophosphamide. In some embodiments, each dose in the administration cycle is about 1 × 10 8 Individual CAR-expressing NK cells ~ approximately 1 × 10⁶ 10 A single CAR-expressing NK cell, or approximately 3 × 10⁶ cells. 8 Individual CAR-expressing NK cells ~ approximately 3 × 10⁶ 9 It contains CAR-expressing NK cells (including each end). In some embodiments, each dose in the administration cycle is approximately 1 × 10⁶ 8Individual CAR-expressing NK cells ~ about 1 × 10 10 Individual CAR-expressing NK cells (including both ends) are included. In some embodiments, each dose of the administration cycle is about 3 × 10 8 Individual CAR-expressing NK cells ~ about 3 × 10 9 Individual CAR-expressing NK cells (including both ends) are included.

[0033] In some embodiments, the autoimmune disease is a B cell-mediated disease.

[0034] Also provided herein is a method for reducing B cells in a subject, comprising administering to a subject having a B cell-mediated disease a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein the CAR comprises (a) an extracellular antigen-binding domain comprising a heavy chain variable region (VH) comprising CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences described in SEQ ID NOs: 24, 25, and 26, respectively, and a light chain variable region (VL) comprising CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences described in SEQ ID NOs: 27, 28, and 29, respectively; (b) a transmembrane domain comprising a CD8 alpha transmembrane domain; and (c) an intracellular signaling domain comprising an intracellular signaling region of OX40 and a CD3 zeta domain. Also provided herein is a method for reducing the level of autoantibodies in a subject, comprising administering to a subject having a B cell-mediated disease a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein the CAR comprises (a) an extracellular antigen-binding domain comprising a heavy chain variable region (VH) comprising CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences described in SEQ ID NOs: 24, 25, and 26, respectively, and a light chain variable region (VL) comprising CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences described in SEQ ID NOs: 27, 28, and 29, respectively; (b) a transmembrane domain comprising a CD8 alpha transmembrane domain; and (c) an intracellular signaling domain comprising an intracellular signaling region of OX40 and a CD3 zeta domain. In some embodiments, the B cell-mediated disease is an autoimmune disease. In some embodiments, the genetically engineered NK cells are allogeneic to the subject.

[0035] In some embodiments, the autoimmune disease is a T-cell mediated disease. In some embodiments, the autoimmune disease is a plasma cell mediated disease. In some embodiments, the subject is seropositive for autoantibodies. In some embodiments, the autoantibodies are associated with the autoimmune disease.

[0036] In some embodiments, the autoimmune disease includes scleroderma. In some embodiments, the autoimmune disease is scleroderma. In some embodiments, the autoimmune disease includes systemic sclerosis (also known as systemic scleroderma). In some embodiments, the autoimmune disease is systemic sclerosis (also known as systemic scleroderma). In some embodiments, the autoimmune disease includes focal scleroderma. In some embodiments, the autoimmune disease is focal scleroderma.

[0037] In some embodiments, the autoimmune disease includes myositis (also known as IIM). In some embodiments, the autoimmune disease is myositis (also known as IIM). In some embodiments, the autoimmune disease is selected from the group consisting of anti-synthetase syndrome (ASSD), overlap myopathy (OM), dermatomyositis (DM), clinical myopathic dermatomyositis, juvenile myositis (JM), necrotizing myositis (NM), polymyositis (PM), and sporadic inclusion body myositis (sIBM). In some embodiments, the autoimmune disease is ASSD. In some embodiments, the autoimmune disease is OM. In some embodiments, the autoimmune disease is DM. In some embodiments, the autoimmune disease is JM. In some embodiments, the autoimmune disease is NM. In some embodiments, the autoimmune disease is PM. In some embodiments, the autoimmune disease is sIBM.

[0038] In some embodiments, the autoimmune disease includes vasculitis. In some embodiments, the autoimmune disease is vasculitis. In some embodiments, the vasculitis is large vasculitis. In some embodiments, the vasculitis is medium vasculitis. In some embodiments, the vasculitis is small vasculitis. In some embodiments, the vasculitis is anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis. In some embodiments, ANCA vasculitis is granulomatosis with polyangiitis (GPA). In some embodiments, ANCA vasculitis is microscopic polyangiitis (MPA). In some embodiments, ANCA vasculitis is eosinophilic granulomatosis with polyangiitis (EGPA).

[0039] In some embodiments, the autoimmune disease includes myasthenia gravis (MG). In some embodiments, the autoimmune disease is MG. In some embodiments, MG is ocular MG. In some embodiments, MG is early-onset systemic MG. In some embodiments, MG is late-onset MG.

[0040] In some embodiments, the autoimmune disease includes multiple sclerosis (MS). In some embodiments, the autoimmune disease is MS. In some embodiments, MS is primary progressive MS (PPMS). In some embodiments, MS is secondary progressive MS (SPMS). In some embodiments, MS is relapsing-remitting MS (RRMS).

[0041] In some embodiments, the autoimmune disease includes systemic lupus erythematosus (SLE). In some embodiments, the autoimmune disease is systemic lupus erythematosus (SLE). In some embodiments, SLE does not include lupus nephritis (LN). In some embodiments, the autoimmune disease includes lupus nephritis (LN). In some embodiments, the autoimmune disease is lupus nephritis (LN). In some embodiments, the autoimmune disease includes both SLE and LN. In some embodiments, the autoimmune disease is both SLE and LN.

[0042] Also provided herein is a method for treating systemic lupus erythematosus (SLE), comprising administering to a subject having SLE a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein the CAR comprises (a) an extracellular antigen-binding domain comprising a heavy chain variable region (VH) containing CDR-1, CDR-2, and CDR-3 containing the amino acid sequences described in SEQ ID NOs: 24, 25, and 26, respectively, and a light chain variable region (VL) containing CDR-1, CDR-2, and CDR-3 containing the amino acid sequences described in SEQ ID NOs: 27, 28, and 29, respectively; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0043] Also provided herein is a method for treating lupus nephritis (LN), comprising administering to a subject having LN a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein the CAR comprises (a) an extracellular antigen-binding domain comprising a heavy chain variable region (VH) containing CDR-1, CDR-2, and CDR-3 containing the amino acid sequences described in SEQ ID NOs: 24, 25, and 26, respectively, and a light chain variable region (VL) containing CDR-1, CDR-2, and CDR-3 containing the amino acid sequences described in SEQ ID NOs: 27, 28, and 29, respectively; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0044] In some embodiments, the transmembrane domain includes the CD8 alpha hinge. In some embodiments, the transmembrane domain includes the CD8 alpha transmembrane region. In some embodiments, the transmembrane domain includes the CD8 alpha hinge and the CD8 alpha transmembrane region. In some embodiments, the intracellular signaling domain includes the intracellular signaling region of OX40. In some embodiments, the intracellular signaling domain includes the CD3 zeta domain. In some embodiments, the intracellular signaling domain includes the intracellular signaling region and the CD3 zeta domain of OX40.

[0045] In some embodiments, VH includes the amino acid sequence described in SEQ ID NO: 35. In some embodiments, VL includes the amino acid sequence described in SEQ ID NO: 36. In some embodiments, VH includes the amino acid sequence described in SEQ ID NO: 35, and VL includes the amino acid sequence described in SEQ ID NO: 36. In some embodiments, the extracellular antigen-binding domain includes the amino acid sequence described in SEQ ID NO: 37. In some embodiments, the extracellular antigen-binding domain is scFv including the amino acid sequence described in SEQ ID NO: 37.

[0046] In some embodiments, the CD8 alpha hinge includes the amino acid sequence described in SEQ ID NO: 6. In some embodiments, the CD8 alpha transmembrane domain includes the amino acid sequence described in SEQ ID NO: 8. In some embodiments, the transmembrane domain includes the amino acid sequence described in SEQ ID NO: 6, SEQ ID NO: 8, and / or SEQ ID NO: 10. In some embodiments, the transmembrane domain includes the amino acid sequence described in SEQ ID NO: 8 or SEQ ID NO: 10. In some embodiments, the transmembrane domain includes the amino acid sequence described in SEQ ID NO: 8. In some embodiments, the transmembrane domain includes the amino acid sequences described in SEQ ID NO: 6 and SEQ ID NO: 8. In some embodiments, the transmembrane domain includes the amino acid sequence described in SEQ ID NO: 10. In some embodiments, the intracellular signaling region of OX40 includes the amino acid sequence described in SEQ ID NO: 14. In some embodiments, the CD3 zeta domain includes the amino acid sequence described in SEQ ID NO: 16. In some embodiments, the CAR includes the amino acid sequence described in SEQ ID NO: 38.

[0047] In some embodiments, NK cells genetically engineered to express CAR are also engineered to express interleukin-15 (IL15). In some embodiments, NK cells genetically engineered to express CAR also express membrane-bound interleukin-15 (mbIL15). In some embodiments, mbIL15 includes the amino acid sequence described in SEQ ID NO: 22. In some embodiments, mbIL15 includes the amino acid sequence described in SEQ ID NO: 23. In some embodiments, mbIL15 includes the amino acid sequence described in SEQ ID NO: 40. In some embodiments, CAR and mbIL15 are bisistronally encoded by the same nucleic acid molecule. In some embodiments, the nucleic acid sequences encoding CAR and mbIL15 are separated by the nucleic acid sequence encoding the T2A peptide. In some embodiments, the T2A peptide includes the amino acid sequence described in SEQ ID NO: 20.

[0048] In some embodiments, NK cells genetically engineered to express CAR are also gene-edited. In some embodiments, NK cells are gene-edited to increase IL15 signaling. In some embodiments, the method includes gene-editing NK cells to increase IL15 signaling. In some embodiments, NK cells are gene-edited to reduce the expression of the CISH gene. In some embodiments, the method includes gene-editing NK cells to reduce the expression of the CISH gene. In some embodiments, NK cells are gene-edited to reduce the expression of the Cis protein. In some embodiments, the method includes gene-editing NK cells to reduce the expression of the Cis protein. In some embodiments, NK cells involve the disruption of one or both alleles of the CISH gene. In some embodiments, NK cells involve the disruption of one allele of the CISH gene. In some embodiments, NK cells involve the disruption of both alleles of the CISH gene.

[0049] In some embodiments, a composition comprising NK cells genetically engineered to express CAR is administered to a subject in a dosing regimen comprising a dosing cycle. In some embodiments, the dosing cycle comprises a first dose, a second dose, and a third dose of the composition comprising NK cells genetically engineered to express CAR.

[0050] In some embodiments, the second dose is administered to the subject approximately 5 to 10 days after the first dose has been administered. In some embodiments, the third dose is administered to the subject approximately 5 to 10 days after the second dose has been administered. In some embodiments, the second dose is administered approximately 7 days after the first dose has been administered. In some embodiments, the third dose is administered approximately 7 days after the second dose has been administered. In some embodiments, the second dose is administered approximately 7 days after the first dose has been administered, and the third dose is administered approximately 7 days after the second dose has been administered.

[0051] In some embodiments, the second dose is administered to the subject approximately 2 to 4 days after the first dose has been administered. In some embodiments, the third dose is administered to the subject approximately 2 to 4 days after the second dose has been administered. In some embodiments, the second dose is administered approximately 3 days after the first dose has been administered. In some embodiments, the third dose is administered approximately 4 days after the second dose has been administered. In some embodiments, the second dose is administered approximately 3 days after the first dose has been administered, and the third dose is administered approximately 4 days after the second dose has been administered.

[0052] In some embodiments, the administration cycle is approximately 21 to 49 days (including both ends). In some embodiments, the administration cycle is approximately 14 to 35 days, or approximately 21 to 28 days (including both ends). In some embodiments, the administration cycle is approximately 14 days. In some embodiments, the administration cycle is approximately 21 days. In some embodiments, the administration cycle is approximately 28 days. In some embodiments, the administration cycle is approximately 35 days. In some embodiments, the administration cycle is approximately 42 days. In some embodiments, the administration cycle is approximately 42 days. In some embodiments, the administration cycle is approximately 49 days.

[0053] In some embodiments, the first dose is administered on approximately day 0 of the administration cycle. In some embodiments, the second dose is administered on approximately day 7 of the administration cycle. In some embodiments, the third dose is administered on approximately day 14 of the administration cycle. In some embodiments, the first dose is administered on approximately day 0 of the administration cycle, the second dose is administered on approximately day 7 of the administration cycle, and the third dose is administered on approximately day 14 of the administration cycle.

[0054] In some embodiments, the first dose is administered on approximately day 0 of the administration cycle. In some embodiments, the second dose is administered on approximately day 2 of the administration cycle. In some embodiments, the second dose is administered on approximately day 3 of the administration cycle. In some embodiments, the third dose is administered on approximately day 4 of the administration cycle. In some embodiments, the third dose is administered on approximately day 5 of the administration cycle. In some embodiments, the third dose is administered on approximately day 6 of the administration cycle. In some embodiments, the third dose is administered on approximately day 7 of the administration cycle. In some embodiments, each dose is administered at intervals of approximately 24 to 72 hours. In some embodiments, each dose is administered at least at approximately 24 hours apart. In some embodiments, each dose is administered at intervals of approximately 24 hours apart. In some embodiments, each dose is administered at least at approximately 48 hours apart. In some embodiments, each dose is administered at intervals of approximately 48 hours apart. In some embodiments, each dose is administered at least at approximately 72 hours apart. In some embodiments, each dose is administered at intervals of approximately 72 hours apart.

[0055] In some embodiments, the first dose is administered on approximately day 0 of the administration cycle, the second dose on approximately day 2 of the administration cycle, and the third dose on approximately day 4 of the administration cycle. In some embodiments, the first dose is administered on approximately day 0 of the administration cycle, the second dose on approximately day 2 of the administration cycle, and the third dose on approximately day 5 of the administration cycle. In some embodiments, the first dose is administered on approximately day 0 of the administration cycle, the second dose on approximately day 3 of the administration cycle, and the third dose on approximately day 5 of the administration cycle. In some embodiments, the first dose is administered on approximately day 0 of the administration cycle, the second dose on approximately day 3 of the administration cycle, and the third dose on approximately day 6 of the administration cycle. In some embodiments, the first dose is administered on approximately day 0 of the administration cycle, the second dose on approximately day 3 of the administration cycle, and the third dose on approximately day 7 of the administration cycle.

[0056] Also provided herein is a method for treating systemic lupus erythematosus (SLE), comprising administering to a subject having SLE a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein (i) the CAR comprises (a) an extracellular antigen-binding domain comprising a heavy chain variable region (VH) comprising the amino acid sequence described in SEQ ID NO: 35 and a light chain variable region (VL) comprising the amino acid sequence described in SEQ ID NO: 36; (b) a transmembrane domain comprising a CD8 alpha transmembrane domain; and (c) an intracellular signaling domain comprising an intracellular signaling region of OX40 and a CD3 zeta domain; (ii) the composition comprising NK cells genetically engineered to express the CAR is administered to the subject in a dosing regimen comprising a dosing cycle, the dosing cycle comprising a first dose, a second dose, and a third dose of the composition; and (iii) each of the first, second, and third doses of the dosing cycle comprises approximately 1 × 10⁻¹⁶ 8 Individual CAR-expressing NK cells ~ approximately 2 × 10⁶ 9 (iv) a method comprising CAR-expressing NK cells; (iv) a second dose administered to the subject approximately 7 days after the first dose was administered to the subject, and a third dose administered to the subject approximately 7 days after the second dose was administered to the subject. In some embodiments, each of the first, second, and third doses in the administration cycle is approximately 1 × 10⁶ 9 A single CAR-expressing NK cell, approximately 1.5 × 10⁶ 9 A single CAR-expressing NK cell, approximately 2 × 10⁶ 9 A single CAR-expressing NK cell, or approximately 2.5 × 10⁶ cells. 9 Contains CAR-expressing NK cells. In some embodiments, each of the first, second, and third doses of the administration cycle is approximately 1 × 10⁶ 9 Contains CAR-expressing NK cells. In some embodiments, each of the first, second, and third doses of the administration cycle is approximately 1.5 × 10⁶ 9 Contains CAR-expressing NK cells. In some embodiments, each of the first, second, and third doses of the administration cycle is approximately 2 × 10⁶ 9Contains CAR-expressing NK cells. In some embodiments, the first, second, and third doses of the administration cycle are approximately 2.5 × 10⁶ 9 Includes individual CAR-expressing NK cells.

[0057] Also provided herein is a method for treating systemic lupus erythematosus (SLE), comprising administering to a subject having SLE a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein (i) the subject has been administered lymphocyte apheresis prior to administration of the composition comprising NK cells genetically engineered to express a CAR; and (ii) the lymphocyte apheresis comprising the administration of cyclophosphamide but not the administration of fludarabine.

[0058] Also provided herein is a method for treating lupus nephritis (LN), comprising administering to a subject having LN a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein (i) the CAR comprises (a) an extracellular antigen-binding domain comprising a heavy chain variable region (VH) comprising the amino acid sequence described in SEQ ID NO: 35 and a light chain variable region (VL) comprising the amino acid sequence described in SEQ ID NO: 36; (b) a transmembrane domain comprising a CD8 alpha transmembrane domain; and (c) an intracellular signaling domain comprising an intracellular signaling region of OX40 and a CD3 zeta domain; (ii) the composition comprising NK cells genetically engineered to express the CAR is administered to the subject in a dosing regimen comprising a dosing cycle, the dosing cycle comprising a first dose, a second dose, and a third dose of the composition; and (iii) each of the first, second, and third doses of the dosing cycle comprises approximately 1 × 10⁻¹⁶ 8 CAR-expressing NK cells ~2 × 10⁶ 9(iv) a method comprising CAR-expressing NK cells; (iv) a second dose administered to the subject approximately 7 days after the first dose was administered to the subject, and a third dose administered to the subject approximately 7 days after the second dose was administered to the subject. In some embodiments, each of the first, second, and third doses in the administration cycle is approximately 1 × 10⁶ 9 A single CAR-expressing NK cell, approximately 1.5 × 10⁶ 9 A single CAR-expressing NK cell, approximately 2 × 10⁶ 9 A single CAR-expressing NK cell, or approximately 2.5 × 10⁶ cells. 9 Contains CAR-expressing NK cells. In some embodiments, each of the first, second, and third doses of the administration cycle is approximately 1 × 10⁶ 9 Contains CAR-expressing NK cells. In some embodiments, each of the first, second, and third doses of the administration cycle is approximately 1.5 × 10⁶ 9 Contains CAR-expressing NK cells. In some embodiments, each of the first, second, and third doses of the administration cycle is approximately 2 × 10⁶ 9 Contains CAR-expressing NK cells. In some embodiments, each of the first, second, and third doses of the administration cycle is approximately 2.5 × 10⁶ 9 Includes individual CAR-expressing NK cells.

[0059] Also provided herein is a method for treating lupus nephritis (LN), comprising administering to a subject having LN a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein (i) the subject has been administered lymphocyte apheresis prior to administration of the composition comprising NK cells genetically engineered to express a CAR; and (ii) the lymphocyte apheresis comprising the administration of cyclophosphamide but not the administration of fludarabine.

[0060] Also provided herein is a method for treating systemic lupus erythematosus (SLE), comprising administering to a subject having SLE a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein (i) the CAR comprises (a) an extracellular antigen-binding domain comprising a heavy chain variable region (VH) comprising the amino acid sequence described in SEQ ID NO: 35 and a light chain variable region (VL) comprising the amino acid sequence described in SEQ ID NO: 36; (b) a transmembrane domain comprising a CD8 alpha transmembrane domain; and (c) an intracellular signaling domain comprising an intracellular signaling region of OX40 and a CD3 zeta domain; (ii) the composition comprising NK cells genetically engineered to express the CAR is administered to the subject in a dosing regimen comprising a dosing cycle, the dosing cycle comprising a first dose, a second dose, and a third dose of the composition; and (iii) each of the first, second, and third doses of the dosing cycle comprises approximately 2 × 10⁻¹⁶ 9 Individual CAR-expressing NK cells or approximately 2.5 × 10⁶ 9 (iv) The method comprises 1 CAR-expressing NK cells; (iv) a second dose is administered to the subject approximately 3 days after the first dose is administered, and a third dose is administered to the subject approximately 4 days after the second dose is administered.

[0061] Also provided herein is a method for treating lupus nephritis (LN), comprising administering to a subject having LN a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein (i) the CAR comprises (a) an extracellular antigen-binding domain comprising a heavy chain variable region (VH) comprising the amino acid sequence described in SEQ ID NO: 35 and a light chain variable region (VL) comprising the amino acid sequence described in SEQ ID NO: 36; (b) a transmembrane domain comprising a CD8 alpha transmembrane domain; and (c) an intracellular signaling domain comprising an intracellular signaling region of OX40 and a CD3 zeta domain; (ii) the composition comprising NK cells genetically engineered to express the CAR is administered to the subject in a dosing regimen comprising a dosing cycle, the dosing cycle comprising a first dose, a second dose, and a third dose of the composition; and (iii) each of the first, second, and third doses of the dosing cycle comprises approximately 2 × 10⁻¹⁶ 9 Individual CAR-expressing NK cells or approximately 2.5 × 10⁶ 9 (iv) The method comprises 1 CAR-expressing NK cells; (iv) a second dose is administered to the subject approximately 3 days after the first dose is administered, and a third dose is administered to the subject approximately 4 days after the second dose is administered.

[0062] In some embodiments, NK cells genetically engineered to express CAR also express membrane-bound interleukin-15 (mbIL15). In some embodiments, mbIL15 comprises the amino acid sequence described in SEQ ID NO: 23. In some embodiments, mbIL15 comprises the amino acid sequence described in SEQ ID NO: 40. In some embodiments, CAR and mbIL15 are bisistronally encoded by the same nucleic acid molecule. In some embodiments, the nucleic acid sequences encoding CAR and mbIL15 are separated by the nucleic acid sequence encoding the T2A peptide. In some embodiments, the T2A peptide comprises the amino acid sequence described in SEQ ID NO: 20.

[0063] In some embodiments, the method reduces B cells in a subject. In some embodiments, the method reduces B cells in a subject by at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, or at least about 99%. In some embodiments, the method reduces B cells in a subject by at least about 70%. In some embodiments, the method reduces B cells in a subject by at least about 75%. In some embodiments, the method reduces B cells in a subject by at least about 80%. In some embodiments, the method reduces B cells in a subject by at least about 85%. In some embodiments, the method reduces B cells in a subject by at least about 90%. In some embodiments, among a plurality of subjects treated according to the method, the number of peripheral B cells in the subjects is reduced on average by at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%. In some embodiments, among multiple subjects treated according to this method, the number of peripheral B cells in the subjects is reduced by at least approximately 70% on average. In some embodiments, among multiple subjects treated according to this method, the number of peripheral B cells in the subjects is reduced by at least approximately 75% on average. In some embodiments, among multiple subjects treated according to this method, the number of peripheral B cells in the subjects is reduced by at least approximately 80% on average. In some embodiments, among multiple subjects treated according to this method, the number of peripheral B cells in the subjects is reduced by at least approximately 85% on average. In some embodiments, among multiple subjects treated according to this method, the number of peripheral B cells in the subjects is reduced by at least approximately 90% on average. In some embodiments, among multiple subjects treated according to this method, the number of peripheral B cells in the subjects is reduced by at least approximately 95% on average. In some embodiments, among multiple subjects treated according to this method, the number of peripheral B cells in the subjects is reduced by at least approximately 99% on average.

[0064] In some embodiments, the method reduces B cells in the subject for at least about 30 days, at least about 45 days, at least about 60 days, at least about 75 days, or at least about 90 days. In some embodiments, the method reduces B cells in the subject for at least about 30 days. In some embodiments, the method reduces B cells in the subject for at least about 45 days. In some embodiments, the method reduces B cells in the subject for at least about 60 days. In some embodiments, the method reduces B cells in the subject for at least about 75 days. In some embodiments, the method reduces B cells in the subject for at least about 90 days. In some embodiments, the method reduces B cells in the subject for at least 30 days, at about 45 days, at about 60 days, at about 75 days, or at about 90 days. In some embodiments, the method reduces B cells in the subject for at least 30 days. In some embodiments, the method reduces B cells in the subject for at least 45 days. In some embodiments, the method reduces B cells in the subject for at least 60 days. In some embodiments, the method reduces B cells in the subject for at least 75 days. In some embodiments, this method reduces B cells in the subject for approximately 90 days. In some embodiments, the B cells are peripheral B cells.

[0065] In some embodiments, among multiple subjects treated according to this method, the number of peripheral B cells in the subjects is significantly reduced for at least about 15 days, at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, or at least about 9 months after the final dose of the composition containing genetically engineered NK cells expressing CAR. In some embodiments, among multiple subjects treated according to this method, the number of peripheral B cells in the subjects is significantly reduced for at least about 15 days after the final dose of the composition containing genetically engineered NK cells expressing CAR. In some embodiments, among multiple subjects treated according to this method, the number of peripheral B cells in the subjects is significantly reduced for at least about 1 month after the final dose of the composition containing genetically engineered NK cells expressing CAR. In some embodiments, among multiple subjects treated according to this method, the number of peripheral B cells in the subjects is significantly reduced for at least about 2 months after the final dose of the composition containing genetically engineered NK cells expressing CAR. In some embodiments, among multiple subjects treated according to this method, the peripheral B cell count in the subjects is significantly reduced for at least about 3 months after the final dose of the composition containing genetically engineered NK cells expressing CAR. In some embodiments, among multiple subjects treated according to this method, the peripheral B cell count in the subjects is significantly reduced for at least about 6 months after the final dose of the composition containing genetically engineered NK cells expressing CAR. In some embodiments, among multiple subjects treated according to this method, the peripheral B cell count in the subjects is significantly reduced for at least about 9 months after the final dose of the composition containing genetically engineered NK cells expressing CAR. In some embodiments, the peripheral B cell count in the subjects is significantly reduced compared to subjects not treated according to this method. In some embodiments, the peripheral B cell count in the subjects is significantly reduced compared to subjects before administration of lymphocyte apheresis.

[0066] In some embodiments, among multiple subjects treated according to this method, the number of peripheral B cells in the subjects is significantly reduced for about 15 days, about 1 month, about 2 months, about 3 months, about 6 months, or about 9 months after the final dose of the composition containing NK cells genetically engineered to express CAR. In some embodiments, among multiple subjects treated according to this method, the number of peripheral B cells in the subjects is significantly reduced for about 15 days after the final dose of the composition containing NK cells genetically engineered to express CAR. In some embodiments, among multiple subjects treated according to this method, the number of peripheral B cells in the subjects is significantly reduced for about 1 month after the final dose of the composition containing NK cells genetically engineered to express CAR. In some embodiments, among multiple subjects treated according to this method, the number of peripheral B cells in the subjects is significantly reduced for about 2 months after the final dose of the composition containing NK cells genetically engineered to express CAR. In some embodiments, among multiple subjects treated according to this method, the number of peripheral B cells in the subjects is significantly reduced for about 3 months after the final dose of the composition containing NK cells genetically engineered to express CAR. In some embodiments, among multiple subjects treated according to this method, the number of peripheral B cells in the subjects is significantly reduced for about 6 months after the final dose of the composition containing NK cells genetically engineered to express CAR. In some embodiments, among multiple subjects treated according to this method, the number of peripheral B cells in the subjects is significantly reduced for about 9 months after the final dose of the composition containing NK cells genetically engineered to express CAR.

[0067] In some embodiments, the number of peripheral B cells in a subject is significantly reduced within approximately 10 days, approximately 15 days, or approximately 30 days after administration of a first dose of a composition containing NK cells genetically engineered to express CAR. In some embodiments, the number of peripheral B cells in a subject is significantly reduced within approximately 10 days after administration of a first dose of a composition containing NK cells genetically engineered to express CAR. In some embodiments, the number of peripheral B cells in a subject is significantly reduced within approximately 15 days after administration of a first dose of a composition containing NK cells genetically engineered to express CAR. In some embodiments, the number of peripheral B cells in a subject is significantly reduced within approximately 30 days after administration of a first dose of a composition containing NK cells genetically engineered to express CAR.

[0068] In some embodiments, approximately 3 months, approximately 6 months, approximately 9 months, and / or approximately 12 months after administration of the final dose of a composition containing NK cells genetically engineered to express CARs to the subject, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, or at least approximately 80% of the peripheral B cells in the subject are naive B cells.

[0069] In some embodiments, approximately 3 months after administration of a final dose of a composition containing NK cells genetically engineered to express CAR, at least approximately 30% of the peripheral B cells in the subjects are naive B cells. In some embodiments, approximately 3 months after administration of a final dose of a composition containing NK cells genetically engineered to express CAR, at least approximately 40% of the peripheral B cells in the subjects are naive B cells. In some embodiments, approximately 3 months after administration of a final dose of a composition containing NK cells genetically engineered to express CAR, at least approximately 50% of the peripheral B cells in the subjects are naive B cells. In some embodiments, approximately 3 months after administration of a final dose of a composition containing NK cells genetically engineered to express CAR, at least approximately 75% of the peripheral B cells in the subjects are naive B cells.

[0070] In some embodiments, approximately 6 months after administration of a final dose of a composition containing NK cells genetically engineered to express CAR, at least approximately 30% of the peripheral B cells in the subjects are naive B cells. In some embodiments, approximately 6 months after administration of a final dose of a composition containing NK cells genetically engineered to express CAR, at least approximately 40% of the peripheral B cells in the subjects are naive B cells. In some embodiments, approximately 6 months after administration of a final dose of a composition containing NK cells genetically engineered to express CAR, at least approximately 50% of the peripheral B cells in the subjects are naive B cells. In some embodiments, approximately 6 months after administration of a final dose of a composition containing NK cells genetically engineered to express CAR, at least approximately 75% of the peripheral B cells in the subjects are naive B cells.

[0071] In some embodiments, approximately 9 months after administration of a final dose of a composition containing NK cells genetically engineered to express CAR, at least about 30% of the peripheral B cells in the subjects are naive B cells. In some embodiments, approximately 9 months after administration of a final dose of a composition containing NK cells genetically engineered to express CAR, at least about 40% of the peripheral B cells in the subjects are naive B cells. In some embodiments, approximately 9 months after administration of a final dose of a composition containing NK cells genetically engineered to express CAR, at least about 50% of the peripheral B cells in the subjects are naive B cells. In some embodiments, approximately 9 months after administration of a final dose of a composition containing NK cells genetically engineered to express CAR, at least about 75% of the peripheral B cells in the subjects are naive B cells.

[0072] In some embodiments, approximately 12 months after administration of a final dose of a composition containing NK cells genetically engineered to express CAR, at least about 30% of the peripheral B cells in the subjects are naive B cells. In some embodiments, approximately 12 months after administration of a final dose of a composition containing NK cells genetically engineered to express CAR, at least about 40% of the peripheral B cells in the subjects are naive B cells. In some embodiments, approximately 12 months after administration of a final dose of a composition containing NK cells genetically engineered to express CAR, at least about 50% of the peripheral B cells in the subjects are naive B cells. In some embodiments, approximately 12 months after administration of a final dose of a composition containing NK cells genetically engineered to express CAR, at least about 75% of the peripheral B cells in the subjects are naive B cells.

[0073] In some embodiments, naive B cells are non-class-switched B cells. In some embodiments, non-class-switched B cells are IgM or IgD isotypes. In some embodiments, non-class-switched B cells are IgM isotypes. In some embodiments, non-class-switched cells are IgD isotypes.

[0074] In some embodiments, the method reduces the level of autoantibodies in a subject. In some embodiments, the method reduces the level of autoantibodies in a subject by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%. In some embodiments, among multiple subjects treated according to the method, the level of autoantibodies in the subjects is reduced on average by at least about 80%, at least about 90%, at least about 95%, or at least about 99%. In some embodiments, among multiple subjects treated according to the method, the level of autoantibodies in the subjects is reduced on average by at least about 50%. In some embodiments, among multiple subjects treated according to the method, the level of autoantibodies in the subjects is reduced on average by at least about 60%. In some embodiments, among multiple subjects treated according to the method, the level of autoantibodies in the subjects is reduced on average by at least about 70%. In some embodiments, among multiple subjects treated according to the method, the level of autoantibodies in the subjects is reduced on average by at least about 80%. In some embodiments, among multiple subjects treated according to this method, the level of autoantibodies in the subjects is reduced by at least about 90% on average. In some embodiments, among multiple subjects treated according to this method, the level of autoantibodies in the subjects is reduced by at least about 95% on average. In some embodiments, among multiple subjects treated according to this method, the level of autoantibodies in the subjects is reduced by at least about 99% on average. In some embodiments, the level of autoantibodies in the subjects is significantly reduced compared to subjects with disease or condition that have not been treated according to this method. In some embodiments, the level of autoantibodies in the subjects is significantly reduced compared to subjects before administration of a composition containing NK cells genetically engineered to express CAR.

[0075] Also provided herein is the use of a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR) for reducing B cells in subjects having a B cell-mediated disease, wherein the CAR comprises (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain. In some embodiments, the B cell-mediated disease is an autoimmune disease. In some embodiments, the subject is administered lymphocytapheresis before being administered a composition comprising NK cells genetically engineered to express a CAR. In some embodiments, the lymphocytapheresis does not involve the administration of fludarabine. In some embodiments, the lymphocytapheresis involves the administration of cyclophosphamide but does not involve the administration of fludarabine.

[0076] Also provided herein is the use of lymphocyte apheresis for the preparation of subjects with autoimmune diseases for treatment with a composition comprising natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds to CD19, wherein (i) the lymphocyte apheresis is administered to the subject before the composition is administered to the subject; and (ii) the lymphocyte apheresis is a use comprising cyclophosphamide but not fludarabine.

[0077] Also provided herein is the use of a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR) for treating subjects with autoimmune diseases, wherein the CAR comprises (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain. In some embodiments, the subject is administered lymphocytapheresis before being administered a composition comprising NK cells genetically engineered to express a CAR. In some embodiments, the lymphocytapheresis does not involve the administration of fludarabine. In some embodiments, the lymphocytapheresis involves the administration of cyclophosphamide but does not involve the administration of fludarabine.

[0078] In some embodiments, the genetically modified NK cells are allogeneic to the target. In some embodiments, the autoimmune disease is a B cell-mediated autoimmune disease. In some embodiments, autoimmune diseases are selected from the group consisting of systemic lupus erythematosus (SLE), lupus nephritis (LN), scleroderma, rheumatoid arthritis (RA), myasthenia gravis (MG), multiple sclerosis (MS), NMDA / NMDAR encephalitis, transverse myelitis, neuromyelitis optica spectrum disorder (NMOSD), myelin oligodendrocyte glycoprotein antibody disease (MOGAD), myelin oligodendrocyte glycoprotein spectrum disorder (MOGSD), idiopathic inflammatory myopathy (IIM; also called myositis), Sjögren's disease, pemphigus vulgaris, bullous pemphigoid (BP), membranous nephropathy (MN), immune thrombocytopenia (ITP), Hashimoto's disease, Grave's disease, insulin resistance, type 1 diabetes mellitus, antiphospholipid syndrome, vasculitis, antineutrophil cytoplasmic antibody (ANCA) vasculitis (AAV), and anti-synthetase syndrome (ASSD). In some embodiments, the autoimmune disease is selected from the group consisting of SLE, LN, scleroderma, MG, myositis (also known as IIM), and vasculitis. In some embodiments, the autoimmune disease is SLE. In some embodiments, the autoimmune disease is LN. In some embodiments, the autoimmune disease is scleroderma. In some embodiments, the autoimmune disease is MG. In some embodiments, the autoimmune disease is IIM. In some embodiments, the autoimmune disease is vasculitis. In some embodiments, the autoimmune disease is MS. In some embodiments, the autoimmune disease is NMDA / NMDAR encephalitis. In some embodiments, the autoimmune disease is transverse myelitis. In some embodiments, the autoimmune disease is NMOSD. In some embodiments, the autoimmune disease is MOGAD. In some embodiments, the autoimmune disease is MOGSD. In some embodiments, the autoimmune disease is Sjögren's disease. In some embodiments, the autoimmune disease is pemphigus vulgaris. In some embodiments, the autoimmune disease is BP. In some embodiments, the autoimmune disease is MN. In some embodiments, the autoimmune disease is ITP. In some embodiments, the autoimmune disease is Hasmimoto's disease. In some embodiments, the autoimmune disease is Grave's disease.In some embodiments, the autoimmune disease is type 1 diabetes. In some embodiments, the autoimmune disease is antiphospholipid syndrome.

[0079] Also provided herein is the use of a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR) for treating subjects having systemic lupus erythematosus (SLE), wherein the CAR comprises (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain.

[0080] Also provided herein is the use of a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR) for treating subjects having lupus nephritis (LN), wherein the CAR comprises (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain.

[0081] Also provided herein is the use of a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR) for treating subjects with autoimmune diseases, wherein the CAR comprises (a) an extracellular antigen-binding domain comprising a heavy chain variable region (VH) containing CDR-1, CDR-2, and CDR-3 containing the amino acid sequences described in SEQ ID NOs: 24, 25, and 26, respectively, and a light chain variable region (VL) containing CDR-1, CDR-2, and CDR-3 containing the amino acid sequences described in SEQ ID NOs: 27, 28, and 29, respectively; (b) a transmembrane domain comprising a CD8 alpha transmembrane domain; and (c) an intracellular signaling domain comprising an intracellular signaling region of OX40 and a CD3 zeta domain.

[0082] Also provided herein is the use of a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR) for treating subjects having systemic lupus erythematosus (SLE), wherein the CAR comprises (a) an extracellular antigen-binding domain comprising a heavy chain variable region (VH) containing CDR-1, CDR-2, and CDR-3 containing the amino acid sequences described in SEQ ID NOs: 24, 25, and 26, respectively, and a light chain variable region (VL) containing CDR-1, CDR-2, and CDR-3 containing the amino acid sequences described in SEQ ID NOs: 27, 28, and 29, respectively; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0083] Also provided herein is the use of a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR) for treating subjects having lupus nephritis (LN), wherein the CAR comprises (a) an extracellular antigen-binding domain comprising a heavy chain variable region (VH) containing CDR-1, CDR-2, and CDR-3 containing the amino acid sequences described in SEQ ID NOs: 24, 25, and 26, respectively, and a light chain variable region (VL) containing CDR-1, CDR-2, and CDR-3 containing the amino acid sequences described in SEQ ID NOs: 27, 28, and 29, respectively; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0084] Also provided herein is the use of a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR) for treating subjects having systemic lupus erythematosus (SLE), wherein (i) the CAR comprises (a) an extracellular antigen-binding domain comprising a heavy chain variable region (VH) comprising the amino acid sequence described in SEQ ID NO: 35 and a light chain variable region (VL) comprising the amino acid sequence described in SEQ ID NO: 36; (b) a transmembrane domain comprising a CD8 alpha transmembrane domain; and (c) an intracellular signaling domain comprising an intracellular signaling region of OX40 and a CD3 zeta domain; (ii) the composition comprising NK cells genetically engineered to express the CAR is formulated for administration in a dosing regimen comprising a dosing cycle, the dosing cycle comprising a first dose, a second dose, and a third dose of the composition; and (iii) each of the first, second, and third doses of the dosing cycle comprises approximately 1 × 10⁻¹⁶ 8 CAR-expressing NK cells ~2 × 10⁶ 9 (iv) The second dose is to be administered to the subject approximately 7 days after the first dose has been administered, and the third dose is to be administered to the subject approximately 7 days after the second dose has been administered.

[0085] Also provided herein is the use of a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR) for treating subjects having lupus nephritis (LN), wherein (i) the CAR comprises an extracellular antigen-binding domain comprising (a) a heavy chain variable region (VH) comprising the amino acid sequence described in SEQ ID NO: 35 and a light chain variable region (VL) comprising the amino acid sequence described in SEQ ID NO: 36; (b) a transmembrane domain comprising a CD8 alpha transmembrane domain; and (c) an intracellular signaling domain comprising an intracellular signaling region of OX40 and a CD3 zeta domain; (ii) the composition comprising NK cells genetically engineered to express the CAR is formulated for administration in a dosing regimen comprising a dosing cycle, the dosing cycle comprising a first dose, a second dose and a third dose of the composition; and (iii) each of the first dose, second dose and third dose of the dosing cycle is approximately 1 × 10⁻¹⁶ 8 Individual CAR-expressing NK cells ~3 × 10⁶ 9 (iv) The second dose is to be administered to the subject approximately 7 days after the first dose has been administered, and the third dose is to be administered to the subject approximately 7 days after the second dose has been administered.

[0086] Also provided herein is the use of a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR) for treating subjects having systemic lupus erythematosus (SLE), wherein (i) the CAR comprises (a) an extracellular antigen-binding domain comprising a heavy chain variable region (VH) comprising the amino acid sequence described in SEQ ID NO: 35 and a light chain variable region (VL) comprising the amino acid sequence described in SEQ ID NO: 36; (b) a transmembrane domain comprising a CD8 alpha transmembrane domain; and (c) an intracellular signaling domain comprising an intracellular signaling region of OX40 and a CD3 zeta domain; (ii) the composition comprising NK cells genetically engineered to express the CAR is formulated for administration in a dosing regimen comprising a dosing cycle, the dosing cycle comprising a first dose, a second dose, and a third dose of the composition; and (iii) each of the first, second, and third doses of the dosing cycle comprises approximately 2 × 10⁻¹⁶ 9 Individual CAR-expressing NK cells or approximately 2.5 × 10⁶ 9 (iv) The use is such that the second dose is administered to the subject approximately 3 days after the first dose has been administered, and the third dose is administered to the subject approximately 4 days after the second dose has been administered.

[0087] Also provided herein is the use of a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR) for treating subjects having lupus nephritis (LN), wherein (i) the CAR comprises an extracellular antigen-binding domain comprising (a) a heavy chain variable region (VH) comprising the amino acid sequence described in SEQ ID NO: 35 and a light chain variable region (VL) comprising the amino acid sequence described in SEQ ID NO: 36; (b) a transmembrane domain comprising a CD8 alpha transmembrane domain; and (c) an intracellular signaling domain comprising an intracellular signaling region of OX40 and a CD3 zeta domain; (ii) the composition comprising NK cells genetically engineered to express the CAR is formulated for administration in a dosing regimen comprising a dosing cycle, the dosing cycle comprising a first dose, a second dose, and a third dose of the composition; and (iii) each of the first, second, and third doses of the dosing cycle comprises approximately 2 × 10⁻¹⁶ 9 A single CAR-expressing NK cell or approximately 2 × 10⁶ cells 9 (iv) The use is such that the second dose is administered to the subject approximately 3 days after the first dose has been administered, and the third dose is administered to the subject approximately 4 days after the second dose has been administered.

[0088] Also provided herein is the use of a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR) for reducing B cells in subjects having B cell-mediated diseases, wherein (i) the composition comprising NK cells genetically engineered to express a CAR is for administration to a subject in a dosing regimen comprising a dosing cycle; and (ii) the composition reduces peripheral B cells in the subject by at least about 90%; peripheral B cells are significantly reduced in the subject for the duration of the dosing cycle; and / or at least about 75% of regrowing peripheral B cells are non-class-switched B cells. In some embodiments, the composition reduces peripheral B cells in the subject by at least about 90%. In some embodiments, peripheral B cells are significantly reduced in the subject for the duration of the dosing cycle. In some embodiments, at least about 75% of regrowing peripheral B cells are non-class-switched B cells.

[0089] Also provided herein are (i) compositions comprising natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds to CD19; and (ii) kits comprising instructions for administering the compositions to subjects having a B cell-mediated disease. In some embodiments, the B cell-mediated disease is an autoimmune disease.

[0090] Also provided herein are (i) a composition comprising natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds to CD19; and (ii) a kit comprising instructions for administering the composition to a subject having an autoimmune disease.

[0091] In some embodiments, the genetically engineered NK cells are allogeneic to the subject. In some embodiments, the CAR comprises (a) an extracellular antigen-binding domain comprising a heavy chain variable region (VH) containing CDR-1, CDR-2, and CDR-3 containing the amino acid sequences described in SEQ ID NOs: 24, 25, and 26, respectively, and a light chain variable region (VL) having CDR-1, CDR-2, and CDR-3 containing the amino acid sequences described in SEQ ID NOs: 27, 28, and 29, respectively; (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, administration of the composition to the subject comprises administration of the composition to the subject in a dosing regimen comprising a dosing cycle. In some embodiments, the dosing cycle comprises a first dose, a second dose, and a third dose of the composition.

[0092] In some embodiments, each of the first, second, and third doses of the administration cycle is approximately 2 × 10 9 Contains CAR-expressing NK cells. In some embodiments, each of the first, second, and third doses of the administration cycle is approximately 2.5 × 10⁶ 9 Includes individual CAR-expressing NK cells.

[0093] In some embodiments, each dose in the administration cycle is approximately 1 × 10⁻⁶ 8 Individual CAR-expressing NK cells ~ approximately 1 × 10⁶ 10 Contains CAR-expressing NK cells. In some embodiments, each dose in the administration cycle is approximately 3 × 10⁶ 8 Individual CAR-expressing NK cells ~ approximately 3 × 10⁶ 10 Contains CAR-expressing NK cells. In some embodiments, each dose in the administration cycle is approximately 1 × 10⁶ 8 Contains CAR-expressing NK cells. In some embodiments, each dose in the administration cycle is approximately 3 × 10⁶ 8 Contains CAR-expressing NK cells. In some embodiments, each dose in the administration cycle is approximately 5 × 10⁶ 8 Contains CAR-expressing NK cells. In some embodiments, each dose in the administration cycle is approximately 1 × 10⁶ 9 Contains CAR-expressing NK cells. In some embodiments, each dose in the administration cycle is approximately 1.25 × 10⁶ 9Contains CAR-expressing NK cells. In some embodiments, each dose in the administration cycle is approximately 1.5 × 10⁶ 9 Contains CAR-expressing NK cells. In some embodiments, each dose in the administration cycle is approximately 1.75 × 10⁶ 9 Contains CAR-expressing NK cells. In some embodiments, each dose in the administration cycle is approximately 2 × 10⁶ 9 Contains CAR-expressing NK cells. In some embodiments, each dose in the administration cycle is approximately 2.5 × 10⁶ 9 Contains CAR-expressing NK cells. In some embodiments, each dose in the administration cycle is approximately 3 × 10⁶ 9 Includes individual CAR-expressing NK cells.

[0094] In some embodiments, each dose in the administration cycle is approximately 1 × 10⁻⁶ 6 CAR-expressing NK cells per kilogram (kg) ~ approximately 1 × 10⁻⁶ 8 Contains CAR-expressing NK cells / kg. In some embodiments, if the subject's body weight is less than 50 kg, each dose in the administration cycle is approximately 1 × 10⁶ 6 CAR-expressing NK cells / kg ~ approximately 1 × 10⁶ 8 Contains CAR-expressing NK cells / kg. In some embodiments, each dose in the administration cycle is approximately 1 × 10⁶ 6 Contains CAR-expressing NK cells / kg. In some embodiments, each dose in the administration cycle is approximately 2 × 10⁶ 6 Contains CAR-expressing NK cells / kg. In some embodiments, each dose in the administration cycle is approximately 3 × 10⁶ 6 Contains CAR-expressing NK cells / kg. In some embodiments, each dose in the administration cycle is approximately 4 × 10⁶ 6 Contains CAR-expressing NK cells / kg. In some embodiments, each dose in the administration cycle is approximately 5 × 10⁶ 6 Contains CAR-expressing NK cells / kg. In some embodiments, each dose in the administration cycle is approximately 6 × 10⁶ 6 Contains CAR-expressing NK cells / kg. In some embodiments, each dose in the administration cycle is approximately 7 × 10⁶ 6 Contains CAR-expressing NK cells / kg. In some embodiments, each dose in the administration cycle is approximately 8 × 10⁶ 6Contains CAR-expressing NK cells / kg. In some embodiments, each dose in the administration cycle is approximately 9 × 10⁶ 6 Contains CAR-expressing NK cells / kg. In some embodiments, each dose in the administration cycle is approximately 1 × 10⁶ 7 Contains CAR-expressing NK cells / kg. In some embodiments, each dose in the administration cycle is approximately 2 × 10⁶ 7 Contains CAR-expressing NK cells / kg. In some embodiments, each dose in the administration cycle is approximately 3 × 10⁶ 7 Contains CAR-expressing NK cells / kg. In some embodiments, each dose in the administration cycle is approximately 4 × 10⁶ 7 Contains CAR-expressing NK cells / kg. In some embodiments, each dose in the administration cycle is approximately 5 × 10⁶ 7 Contains CAR-expressing NK cells / kg. In some embodiments, each dose in the administration cycle is approximately 6 × 10⁶ 7 Contains CAR-expressing NK cells / kg. In some embodiments, each dose in the administration cycle is approximately 7 × 10⁶ 7 Contains CAR-expressing NK cells / kg. In some embodiments, each dose in the administration cycle is approximately 8 × 10⁶ 7 Contains CAR-expressing NK cells / kg. In some embodiments, each dose in the administration cycle is approximately 9 × 10⁶ 7 Contains CAR-expressing NK cells / kg. In some embodiments, each dose in the administration cycle is approximately 1 × 10⁶ 8 Contains individual CAR-expressing NK cells / kg.

[0095] In some embodiments, NK cells genetically engineered to express CAR are allogeneic to the subject. In some embodiments, NK cells are obtained from donors without B cell-mediated disease. In some embodiments, NK cells are obtained from donors without autoimmune disease. In some embodiments, NK cells are obtained from donors without SLE. In some embodiments, NK cells are obtained from donors without LN.

[0096] In some embodiments, NK cells genetically engineered to express the CD19 CAR also express a CAR that binds to an antigen associated with an autoimmune disease. In some embodiments, the composition further comprises immune cells genetically engineered to express a CAR that binds to an antigen associated with an autoimmune disease. In some embodiments, the antigen is selected from the group consisting of BAFF-R, BCMA, CD20, CD22, CD27, CD28, CD33, CD38, CD45, CD47, CD54, CD56, CD81, CD117, CD138, CD200, FcRH5, GPRC5D, and SLAMF7. In some embodiments, the antigen is BAFF-R. In some embodiments, the antigen is BCMA. In some embodiments, the antigen is CD20. In some embodiments, the antigen is CD22. In some embodiments, the antigen is CD27. In some embodiments, the antigen is CD28. In some embodiments, the antigen is CD38. In some embodiments, the antigen is CD45. In some embodiments, the antigen is CD47. In some embodiments, the antigen is CD54. In some embodiments, the antigen is CD56. In some embodiments, the antigen is CD81. In some embodiments, the antigen is CD117. In some embodiments, the antigen is CD138. In some embodiments, the antigen is CD200. In some embodiments, the antigen is FcRH5. In some embodiments, the antigen is GPRC5D. In some embodiments, the antigen is SLAMF7. In some embodiments, the immune cells include NK cells. In some embodiments, the immune cells include T cells. In some embodiments, the immune cells include NK cells and T cells.

[0097] In some embodiments, the method further includes administering lymphocyte apheresis to a subject before administering a composition comprising NK cells genetically engineered to express CAR. In some embodiments, the subject has been administered lymphocyte apheresis before administering a composition comprising NK cells genetically engineered to express CAR.

[0098] In some embodiments, lymphocyte apheresis includes the administration of cyclophosphamide. In some embodiments, lymphocyte apheresis does not include the administration of fludarabine. In some embodiments, lymphocyte apheresis includes the administration of cyclophosphamide but does not include the administration of fludarabine.

[0099] Also provided herein is a method for preparing a subject having an autoimmune disease for treatment with a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein the method comprises administering a lymphocyte apheresis to the subject before administering the composition, the lymphocyte apheresis comprising cyclophosphamide.

[0100] Also provided herein are methods for treating or preventing autoimmune diseases, comprising administering a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR) to a subject who has or is suspected of having an autoimmune disease, has been determined to be at risk of an autoimmune disease, or has been determined to be at risk of recurrence of an autoimmune disease, wherein (i) the CAR comprises (a) an extracellular antigen-binding domain; (b) a transmembrane domain; and (c) an intracellular signaling domain; (ii) the subject has been administered lymphocyte apheresis prior to administration of the composition comprising NK cells genetically engineered to express the CAR; and (iii) the lymphocyte apheresis comprising the administration of cyclophosphamide but not the administration of fludarabine.

[0101] In some embodiments, NK cells genetically engineered to express CAR also express mbIL15. In some embodiments, the method is a method of treating an autoimmune disease. In some embodiments, the method is a method of preventing an autoimmune disease. In some embodiments, the subject has an autoimmune disease. In some embodiments, the subject has been determined to be at risk of an autoimmune disease. In some embodiments, the subject has been determined to be at risk of recurrence of an autoimmune disease. In some embodiments, the genetically engineered NK cells are allogeneic to the subject.

[0102] In some embodiments, the lymphodepletion therapy comprises administering cyclophosphamide at about 200 mg / m 2 ~ about 600 mg / m 2 per day. In some embodiments, the lymphodepletion therapy comprises administering cyclophosphamide at about 200 mg / m 2 per day. In some embodiments, the lymphodepletion therapy comprises administering cyclophosphamide at about 300 mg / m 2 per day. In some embodiments, the lymphodepletion therapy comprises administering cyclophosphamide at about 400 mg / m 2 per day. In some embodiments, the lymphodepletion therapy comprises administering cyclophosphamide at about 500 mg / m 2 per day. In some embodiments, the lymphodepletion therapy comprises administering cyclophosphamide at about 600 mg / m 2 per day. In some embodiments, the lymphodepletion therapy comprises administering cyclophosphamide daily for 2 - 4 days. In some embodiments, the lymphodepletion therapy comprises administering cyclophosphamide daily for 3 days. In some embodiments, the lymphodepletion therapy comprises administering cyclophosphamide at about 500 mg / m 2 daily for 3 days. In some embodiments, the lymphodepletion therapy comprises administering cyclophosphamide at about 500 mg / m 2 per day on each of days -5, -4, and -3.

[0103] In some embodiments, the lymphodepletion therapy includes administration of a single dose of cyclophosphamide. In some embodiments, the single dose of cyclophosphamide comprises cyclophosphamide at about 500 mg / m 2 to about 1500 mg / m 2 of cyclophosphamide. In some embodiments, the single dose of cyclophosphamide comprises cyclophosphamide at about 500 mg / m 2 of cyclophosphamide. In some embodiments, the single dose of cyclophosphamide comprises cyclophosphamide at about 750 mg / m 2 of cyclophosphamide. In some embodiments, the single dose of cyclophosphamide comprises cyclophosphamide at about 1000 mg / m 2 2 to about 1500 mg / m 2 of cyclophosphamide. In some embodiments, the single dose of cyclophosphamide comprises cyclophosphamide at about 500 mg / m 2 of cyclophosphamide. In some embodiments, the single dose of cyclophosphamide comprises cyclophosphamide at about 750 mg / m 2 of cyclophosphamide. In some embodiments, the single dose of cyclophosphamide comprises cyclophosphamide at about 1000 mg / m 2 of cyclophosphamide. In some embodiments, the single dose of cyclophosphamide comprises cyclophosphamide at about 1250 mg / m 2 of cyclophosphamide. In some embodiments, the single dose of cyclophosphamide comprises cyclophosphamide at about 1500 mg / m 2 of cyclophosphamide. In some embodiments, the single dose of cyclophosphamide is administered about 3 days prior to administration of a composition comprising NK cells genetically engineered to express a CAR. In some embodiments, about 1000 mg / m 2 of the single dose of cyclophosphamide is administered about 3 days prior to administration of a composition comprising NK cells genetically engineered to express a CAR. In some embodiments, a single dose of about 1000 mg / m 2 of cyclophosphamide is administered on day - 3.

[0104] [[ID=The]] In some embodiments, the lymphodepletion therapy includes administration of fludarabine. In some embodiments, the lymphodepletion therapy includes administering about 20 mg / m 2 to about 40 mg / m 2 of fludarabine daily. In some embodiments, the lymphodepletion therapy includes administering about 20 mg / m 2 of fludarabine daily. In some embodiments, the lymphodepletion therapy includes administering about 25 mg / m 2 of fludarabine daily. In some embodiments, the lymphodepletion therapy includes administering about 30 mg / m 2This includes administering fludarabine daily. In some embodiments, lymphocyte apheresis therapy is administered at approximately 40 mg / m². 2 This includes administering fludarabine daily. In some embodiments, the lymphocyte apheresis therapy includes administering fludarabine daily for 2 to 4 days. In some embodiments, the lymphocyte apheresis therapy includes administering fludarabine daily for 3 days. In some embodiments, the lymphocyte apheresis therapy includes administering 25 mg / m². 2 The procedure includes administering fludarabine daily for three days. In some embodiments, the lymphocyte apheresis involves administering 25 mg / m² of a composition containing NK cells genetically engineered to express CAR, each on days 5, 4, and 3, prior to the administration of the composition. 2 This includes administering fludarabine daily. In some embodiments, lymphocyte apheresis is administered at 25 mg / m² on days -5, -4, and -3, respectively. 2 This includes administering fludarabine daily.

[0105] In some embodiments, lymphocyte apheresis therapy includes the administration of cyclophosphamide and fludarabine. In some embodiments, lymphocyte apheresis therapy is administered at approximately 300 mg / m². 2 Cyclophosphamide daily, and approximately 30 mg / m² 2 This includes administering fludarabine daily for three days. In some embodiments, the lymphocyte apheresis therapy is approximately 500 mg / m². 2 Cyclophosphamide daily, and approximately 30 mg / m² 2 The regimen includes administering fludarabine daily for three days each day. In some embodiments, lymphocyte apheresis is administered at approximately 300 mg / m² on days -5, -4, and -3, respectively. 2 Cyclophosphamide and approximately 30 mg / m² 2 This includes administering fludarabine. In some embodiments, lymphocyte apheresis therapy is administered at approximately 500 mg / m² on days -5, -4, and -3, respectively. 2 Cyclophosphamide and approximately 30 mg / m² 2 This includes administering fludarabine. In some embodiments, lymphocyte apheresis therapy is administered at approximately 1000 mg / m². 2A single dose of cyclophosphamide and a daily dose of approximately 25 mg / m². 2 The procedure includes administering fludarabine. In some embodiments, the lymphocyte apheresis involves administering a single dose of approximately 1000 mg / m² of a composition containing NK cells genetically engineered to express CAR, approximately 3 days prior to administration. 2 Administer cyclophosphamide and a composition containing genetically engineered NK cells expressing CAR, at approximately 25 mg / m² each day, 5, 4, and 3 days prior to administration. 2 This includes administering a dose of fludarabine. In some embodiments, lymphocyte apheresis therapy involves a single dose of approximately 1000 mg / m² on day 3. 2 Cyclophosphamide, and approximately 25 mg / m² each on days -5, -4, and -3. 2 This includes administering a dose of fludarabine.

[0106] In some embodiments, the method includes administering a corticosteroid to a subject before, during, and / or after the administration of lymphocyte apheresis. In some embodiments, the subject is administered a corticosteroid before, during, and / or after the administration of lymphocyte apheresis. In some embodiments, the subject is administered a corticosteroid before the administration of lymphocyte apheresis. In some embodiments, the subject is administered a corticosteroid during the administration of lymphocyte apheresis. In some embodiments, the subject is administered a corticosteroid after the administration of lymphocyte apheresis. In some embodiments, the subject is administered a corticosteroid before, during, and after the administration of lymphocyte apheresis. In some embodiments, the corticosteroid comprises a glucocorticoid. In some embodiments, the corticosteroid is prednisone or comprises prednisone.

[0107] In some embodiments, the method includes administering a corticosteroid to a subject before, during, and / or after administration of the composition. In some embodiments, the subject is administered a corticosteroid before, during, and / or after administration of the composition. In some embodiments, the subject is administered a corticosteroid before administration of the composition. In some embodiments, the subject is administered a corticosteroid during administration of the composition. In some embodiments, the subject is administered a corticosteroid after administration of the composition. In some embodiments, the subject is administered a corticosteroid before, during, and after administration of the composition. In some embodiments, the corticosteroid comprises a glucocorticoid. In some embodiments, the corticosteroid is prednisone or comprises prednisone.

[0108] In some embodiments, the method includes administering an immunosuppressant to a subject before, during, and / or after the administration of lymphocyte apheresis. In some embodiments, the subject is administered an immunosuppressant before, during, and / or after the administration of lymphocyte apheresis. In some embodiments, the subject is administered an immunosuppressant before the administration of lymphocyte apheresis. In some embodiments, the subject is administered an immunosuppressant during the administration of lymphocyte apheresis. In some embodiments, the subject is administered an immunosuppressant after the administration of lymphocyte apheresis. In some embodiments, the subject is administered an immunosuppressant before, during, and after the administration of lymphocyte apheresis.

[0109] In some embodiments, the method includes administering an immunosuppressant to a subject before, during, and / or after administration of the composition. In some embodiments, the subject is administered an immunosuppressant before, during, and / or after administration of the composition. In some embodiments, the subject is administered an immunosuppressant before administration of the composition. In some embodiments, the subject is administered an immunosuppressant during administration of the composition. In some embodiments, the subject is administered an immunosuppressant after administration of the composition. In some embodiments, the subject is administered an immunosuppressant before, during, and after administration of the composition.

[0110] In some embodiments, the immunosuppressant includes anti-thymocyte globulin (ATG), mammalian targeted rapamycin inhibitor (mTOR), calcineurin inhibitor, or any combination thereof. In some embodiments, the immunosuppressant is anti-thymocyte globulin (ATG). In some embodiments, the immunosuppressant is a mammalian targeted rapamycin inhibitor (mTOR). In some embodiments, the immunosuppressant is a calcineurin inhibitor (e.g., voclosporine).

[0111] In some embodiments, the subject was diagnosed with an autoimmune disease (e.g., SLE or LN) at least about 18 weeks to at least about 30 weeks before administering the composition. In some embodiments, the subject was diagnosed with an autoimmune disease (e.g., SLE or LN) at least about 18 weeks to at least about 30 weeks before administering the composition. In some embodiments, the subject was diagnosed with an autoimmune disease at least about 18 weeks, at least about 20 weeks, at least about 22 weeks, at least about 24 weeks, at least about 26 weeks, at least about 28 weeks, or at least about 30 weeks before administering the composition. In some embodiments, the subject was diagnosed with an autoimmune disease at least about 20 weeks before administering the composition. In some embodiments, the subject was diagnosed with an autoimmune disease at least about 21 weeks before administering the composition. In some embodiments, the subject was diagnosed with an autoimmune disease at least about 22 weeks before administering the composition. In some embodiments, the subject was diagnosed with an autoimmune disease at least about 23 weeks before administering the composition. In some embodiments, the subjects were diagnosed with an autoimmune disease at least about 24 weeks prior to administration of the composition. In some embodiments, the subjects were diagnosed with an autoimmune disease at least about 25 weeks prior to administration of the composition. In some embodiments, the subjects were diagnosed with an autoimmune disease at least about 26 weeks prior to administration of the composition. In some embodiments, the subjects were diagnosed with an autoimmune disease at least about 27 weeks prior to administration of the composition. In some embodiments, the subjects were diagnosed with an autoimmune disease at least about 28 weeks prior to administration of the composition. In some embodiments, the subjects were diagnosed with an autoimmune disease at least about 29 weeks prior to administration of the composition. In some embodiments, the subjects were diagnosed with an autoimmune disease at least about 30 weeks prior to administration of the composition.

[0112] In some embodiments, the subjects are human. In some embodiments, the subjects are under 18 years of age. In some embodiments, the subjects are approximately 12 to 18 years of age. In some embodiments, the subjects are at least 12 years of age. In some embodiments, the subjects are approximately 14 to 18 years of age. In some embodiments, the subjects are at least 14 years of age. In some embodiments, the subjects are approximately 16 to 18 years of age. In some embodiments, the subjects are at least 16 years of age. In some embodiments, the subjects are adults. In some embodiments, the subjects are at least 18 years of age. In some embodiments, the subjects are approximately 18 to 65 years of age.

[0113] In some embodiments, the autoimmune disease is relapsing / refractory. In some embodiments, the subject has relapsed after treatment with a prior line of treatment for the autoimmune disease and / or is refractory to a prior line of treatment for the autoimmune disease. In some embodiments, the subject has relapsed after treatment with a prior line of treatment for the autoimmune disease. In some embodiments, the subject is refractory to a prior line of treatment for the autoimmune disease.

[0114] In some embodiments, the subject does not have lupus nephritis (LN). In some embodiments, the subject has lupus nephritis (LN). In some embodiments, at the time of administration of the composition to the subject, the subject has SLE with active LN. In some embodiments, the LN is refractory LN. In some embodiments, the refractory LN is LN that has not responded to at least two prior lines of treatment. In some embodiments, at the time of administration of the composition to the subject, the subject has active LN.

[0115] In some embodiments, the subjects do not have neuropsychiatric systemic lupus erythematosus (NPSLE). In some embodiments, the subjects have neuropsychiatric systemic lupus erythematosus (NPSLE). In some embodiments, the subjects have systemic lupus erythematosus (SLE) without renal lesions.

[0116] In some embodiments, the SLE is relapsing / refractory SLE. In some embodiments, the subject has relapsed after treatment with a previous treatment line for SLE and / or is refractory to a previous treatment line for SLE. In some embodiments, the subject has relapsed after treatment with a previous treatment line for SLE. In some embodiments, the subject is refractory to a previous treatment line for SLE. In some embodiments, the subject has relapsed after treatment with a previous treatment line for SLE and is refractory to a previous treatment line for SLE.

[0117] In some embodiments, the previous treatment line includes two, three, or four previous treatment lines. In some embodiments, the previous treatment line includes two previous treatment lines. In some embodiments, the previous treatment line includes three previous treatment lines. In some embodiments, the previous treatment line includes four previous treatment lines. In some embodiments, the subject has previously received at least two previous treatment lines for LN. In some embodiments, the subject has previously received at least three previous treatment lines for LN.

[0118] In some embodiments, at least two previous treatment lines for LN include immunosuppressive agents and / or immunomodulatory agents. In some embodiments, at least two previous treatment lines for LN include immunosuppressive agents. In some embodiments, at least two previous treatment lines for LN include immunomodulatory agents. In some embodiments, at least two previous treatment lines for LN include immunosuppressive agents and immunomodulatory agents. In some embodiments, at least three previous treatment lines for LN include immunosuppressive agents and / or immunomodulatory agents. In some embodiments, at least three previous treatment lines for LN include immunosuppressive agents. In some embodiments, at least three previous treatment lines for LN include immunomodulatory agents. In some embodiments, at least three previous treatment lines for LN include immunosuppressive agents and immunomodulatory agents.

[0119] In some embodiments, the subject was treated with a prior line of therapy for at least about 2 months. In some embodiments, the subject was treated with a prior line of therapy for about 3 months to about 24 months. In some embodiments, the subject did not achieve a partial response to the prior line of therapy. In some embodiments, the subject did not achieve a complete response to the prior line of therapy.

[0120] In some embodiments, the prior line of therapy includes corticosteroids, immunosuppressive agents, antimalarial agents, B cell targeting agents, hematopoietic stem cell transplantation (HSCT), or any combination thereof. In some embodiments, the prior line of therapy includes corticosteroids. In some embodiments, the prior line of therapy includes glucocorticoids. In some embodiments, the prior line of therapy includes antimalarial agents. In some embodiments, the prior line of therapy includes immunosuppressive agents. In some embodiments, the prior line of therapy includes B cell targeting agents. In some embodiments, the prior line of therapy includes hematopoietic stem cell transplantation (HSCT). In some embodiments, the prior line of therapy does not include HSCT.

[0121] In some embodiments, the subject achieves a clinical response after a cycle of administration. In some embodiments, the subject achieves a complete response (CR) after a cycle of administration. In some embodiments, the subject achieves a complete renal response (CRR) after a cycle of administration. In some embodiments, the subject achieves a clinical remission after a cycle of administration. In some embodiments, the subject achieves a partial response (PR) after a cycle of administration. In some embodiments, the subject achieves a partial renal response (PRR) after a cycle of administration. In some embodiments, the subject achieves a reduction in disease activity after a cycle of administration. In some embodiments, the subject achieves a reduction in the level of autoantibodies after a cycle of administration. In some embodiments, the autoantibodies are associated with an autoimmune disease.

[0122] In some embodiments, if the subject shows a clinical response to the treatment, the administration regimen includes an additional administration cycle. In some embodiments, if the subject shows a partial response to the treatment, the administration regimen includes an additional administration cycle. In some embodiments, if the subject shows a complete response to the treatment, the administration regimen includes an additional administration cycle. In some embodiments, if the subject shows an initial clinical response to the treatment and then relapses, the administration regimen includes an additional administration cycle. In some embodiments, if the subject shows an initial clinical response to the treatment and then the disease progresses, the administration regimen includes an additional administration cycle. In some embodiments, the administration regimen includes two, three, four, or five administration cycles. In some embodiments, the administration regimen consists of two administration cycles. In some embodiments, the administration regimen consists of three administration cycles. In some embodiments, the administration regimen consists of four administration cycles. In some embodiments, the administration regimen consists of five administration cycles. In some embodiments, the administration regimen includes five or fewer administration cycles.

[0123] In some embodiments, the method treats autoimmune diseases. In some embodiments, the method prevents autoimmune diseases. In some embodiments, among a group of subjects treated according to the method, the mean time between disease relapses is reduced compared to a group of subjects with autoimmune diseases who are not treated according to the method. In some embodiments, among a group of subjects treated according to the method, the mean severity of disease relapses is reduced compared to a group of subjects with autoimmune diseases who are not treated according to the method.

[0124] In some embodiments, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% of subjects treated according to this method show a clinical response. In some embodiments, at least about 50% of subjects treated according to this method show a clinical response. In some embodiments, at least about 60% of subjects treated according to this method show a clinical response. In some embodiments, at least about 70% of subjects treated according to this method show a clinical response. In some embodiments, at least about 80% of subjects treated according to this method show a clinical response. In some embodiments, at least about 90% of subjects treated according to this method show a clinical response. In some embodiments, at least about 95% of subjects treated according to this method show a clinical response. In some embodiments, the clinical response includes partial response (PR). In some embodiments, the clinical response includes complete response (CR). In some embodiments, the clinical response includes a partial renal response (PRR). In some embodiments, the clinical response includes a complete renal response (CRR). In some embodiments, the clinical response includes a reduction in disease activity. In some embodiments, the reduction in disease activity is assessed by disease indicators.

[0125] In some embodiments, the subject is human. In some embodiments, the subject is at least about 12 years old. In some embodiments, the subject is at least about 14 years old. In some embodiments, the subject is at least about 16 years old. In some embodiments, the subject is an adult. In some embodiments, the subject is at least about 18 years old. [Brief explanation of the drawing]

[0126] [Figure 1] Figure 1 shows a non-restrictive schematic diagram of a CD19-targeted chimeric antigen receptor (CAR). [Figure 2]Figures 2A–2D show non-limiting schematic diagrams of administration cycles for treating autoimmune diseases (e.g., SLE) with CD19 CAR-expressing NK cells ("CD19 CAR NK cells"). [Figure 3] Figure 3A shows the in vitro cytotoxicity of untransduced ("control") NK cells and CD19 CAR NK cells against CD19+, CD14+, and CD3+ subpopulations of PBMCs. Figure 3B shows the ex vivo cytotoxicity of untransduced ("control") NK cells and CD19 CAR NK cells against CD19+ B cells derived from donors with systemic lupus erythematosus (SLE; n=3), scleroderma (n=3), myositis (n=3), and myasthenia gravis (MG; n=1). [Figure 4] Figure 4 shows the in vitro cytotoxicity of control NK cells, CD19 CAR NK cells, and CD19 CAR-expressing T cells ("CD19 CAR T cells") against CD19+ target cells (Nalm6 and REH cell lines) during 24-hour or 72-hour co-culture. [Figure 5] Figure 5 shows cytokine levels from control NK cells, CD19 CAR NK cells, and CD19 CAR T cells after co-culture with Nalm-6 or REH target cells at a 1:1 E:T ratio for 24 hours (from left to right: control NK cells, CD19 CAR NK cells, CD19 CAR T cells). [Figure 6] Figure 6 shows tumor load (left) and body weight (right) in a mouse CD19-positive xenograft tumor model after treatment with control NK cells or CD19 CAR NK cells. [Figure 7]Figure 7A shows the Cmax of interleukin-15 (IL15) relative to the peak concentration (Cmax) of CD19 CAR NK cells in subjects with CD19+ B-cell malignancies treated with CD19 CAR NK cells according to a non-restrictive dosing regimen. Figure 7B shows the mean IL15 Cmax in subjects with CD19+ B-cell malignancies treated with CD19 CAR NK cells according to a non-restrictive dosing regimen who achieved complete response (CR), partial response (PR), or stable or progressive disease (SD / PD). Figure 7C shows the number of CD19+ cells per microliter (uL) of whole blood in subjects with CD19+ B-cell malignancies treated with CD19 CAR NK cells on days 0, 7, and 14 according to a non-restrictive dosing regimen (each row represents a different subject). Figure 7D shows the absolute number of B cells per 800 μL of whole blood in NHL subjects at baseline (C1D-5) and on the indicated days after one or more administration cycles with CD19 CAR NK cells (C1: 1st administration cycle; C2: 2nd administration cycle; C3: 3rd administration cycle; C4: 4th administration cycle; EOT: End of treatment; FUP1: Follow-up 1; FUP2: Follow-up 2; FUP3: Follow-up 3; FUP4: Follow-up 4). [Figure 8] Figure 8A shows the B cell receptor (BCR) heavy chain isotypes of representative NHL subjects before lymphocyte removal (pre-LD) and the B cell receptor (BCR) heavy chain isotypes of FUP1, FUP2, FUP3, and FUP4 evaluated by mRNA sequencing (n=1). Figure 8B shows the B cell receptor (BCR) heavy chain isotypes of FUP1 in NHL subjects evaluated by mRNA sequencing (n=5). Figure 8C shows the percentage of B cell subtypes of FUP1 in NHL subjects evaluated by transcriptome analysis (n=5). [Figure 9]Figure 9 shows the concentration of CD19 CAR NK cells in two subjects with CD19+ B-cell malignancies. These subjects received lymphocyte apheresis with cyclophosphamide and fludarabine (cy / flu) before the first administration cycle, and lymphocyte apheresis with cyclophosphamide alone (cy) before the second administration cycle. [Modes for carrying out the invention]

[0127] Detailed explanation Provided are methods and uses of genetically modified immune cells and / or compositions thereof for the treatment of subjects having autoimmune diseases (e.g., lupus). Also provided are methods and uses of genetically modified immune cells and / or compositions thereof for the prevention of autoimmune diseases in subjects suspected of having an autoimmune disease or determined to be at risk of autoimmune disease. In any particular embodiment of the methods provided, natural killer (NK) cells are genetically modified to express a CD19-targeted chimeric antigen receptor (CAR).

[0128] In some embodiments, autoimmune diseases are B-cell mediated diseases, T-cell mediated diseases, and / or plasma cell mediated diseases. In some embodiments, autoimmune diseases are B-cell mediated diseases. For example, in some embodiments, autoimmune diseases are systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and / or multiple sclerosis (MS). In some aspects, autoimmune diseases include acquired immunodeficiency syndrome (AIDS), Addison's disease, alopecia areata, vasculitis (e.g., anti-neutrophil cytoplasmic antibody (ANCA) vasculitis), antiphospholipid syndrome, anti-synthetase syndrome, atherosclerosis, bullous pemphigoid (BP), celiac disease, chronic inflammatory demyelinating polyneuropathy (CIDP), Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, immune thrombocytopenia (ITP), inflammatory bowel disease (IBD) such as Crohn's disease or ulcerative colitis, insulin resistance, membranous nephropathy (MN), myasthenia gravis (MG), and myelin These include oligodendrocyte glycoprotein antibody disease (MOGAD), myelin oligodendrocyte glycoprotein spectrum disorder (MOGSD), myocardial aneurysm, myocardial infarction, IIM (including anti-synthetase syndrome, dermatomyositis, juvenile myositis, necrotizing myositis, polymyositis, and sporadic inclusion body myositis), neuromyelitis optica spectrum disorder (NMOSD), NMDA / NMDAR encephalitis, pemphigus vulgaris, pernicious anemia, psoriasis, psoriatic arthritis, reactive arthritis, scleroderma (e.g., focal or systemic scleroderma), Sjögren's disease, transverse myelitis, and / or type 1 diabetes.

[0129] In some embodiments, the method and use involve administering genetically engineered NK cells to a subject having a B cell-mediated disease (e.g., autoimmune disease) that expresses a recombinant receptor (e.g., CAR) expressed by B cells, associated with B cells, and / or specific to B cells (e.g., CD19). Thus, in some embodiments, the method and use involve administering genetically engineered NK cells to a subject having an autoimmune disease (e.g., SLE) that expresses a recombinant receptor (e.g., CAR) expressed by cells involved in the pathogenesis of the autoimmune disease (e.g., B cells), associated with B cells, and / or specific to B cells (e.g., CD19). NK cells are generally administered in a formulation for administration; the method generally involves administering CAR-expressing NK cells as part of an administration cycle. In some embodiments, the administration cycle comprises multiple (e.g., three) doses of genetically engineered NK cells. In some embodiments, the subject is administered lymphocyte apheresis prior to the administration of genetically engineered NK cells. If the patient is to receive two or more administration cycles, the patient may receive lymphocyte apheresis before each administration cycle.

[0130] While we do not wish to be bound by theory, the methods and uses described herein are intended to result in or achieve improved and / or more persistent responses or efficacy, and / or reduced risks of toxicity or other side effects, compared to alternative methods for treating such autoimmune diseases. For example, as further described below, the methods provided herein are intended to be advantageous by producing increased and / or more persistent responses compared to other methods such as B-cell targeting agents (e.g., anti-BAFF antibodies, anti-CD19 antibodies, anti-CD20 antibodies, and / or anti-CD22 antibodies). Also, while we do not wish to be bound by theory, the methods provided may be advantageous by reducing the risk of toxicity and / or increasing the ability to re-treat compared to alternative methods such as CAR T-cell therapy.

[0131] CD19 is a glycoprotein that is highly expressed by B cells throughout all stages of B cell differentiation (Jin et al., Cell Mol Immunol (2020) 18:1896-1093). Furthermore, CD19 is not expressed in normal tissues that are not derived from hematopoietic stem cells or B cell lineages. B cells are thought to play a central role in the development of autoimmune diseases such as rheumatoid arthritis (RA), multiple sclerosis (MS), and systemic lupus (SLE).

[0132] SLE is considered an incurable disease characterized by loss of self-tolerance with autoantibody production, cell and tissue infiltration, and peripheral organ damage that can lead to severe organ complications and even death (Doglio et al., J Allergy Clin Immunol (2022) 150(6):1289-1301). In particular, hyperactivation of autoreactive B cells is observed in the onset of SLE, inducing plasma cells to produce large amounts of autoantibodies, which then circulate and encounter autoantigens and complement to form immune complexes. These immune complexes may then be deposited in small blood vessels or distal sites, which can ultimately lead to organ destruction or dysfunction. See Jin et al. (2020). The most important sign of SLE is lupus nephritis (LN). Multiple mechanisms are involved in renal injury in lnN, and nephrotic proteinuria is observed in up to 50% of cases (Parikh et al., Am J Kidney Dis. (2020) 76(2):265-81). LN is a major risk factor for morbidity and mortality, with more than half of patients with SLE developing it within 10 years. LN causes end-stage renal disease (ESRD) in 10% of patients and accounts for a 12% mortality rate (Almaani et al., Clin J Am Soc Nephrol (2017) 12(5):825-35; Hahn et al., Arthritis Care Res (2012) 64(6):797-808).

[0133] In the treatment of SLE, in addition to non-steroidal anti-inflammatory drugs (NSAIDs), antimalarial drugs, glucocorticoids, and immunosuppression, B cell depletion strategies are being explored. B cell targeting agents such as anti-BAFF antibodies (e.g., belimumab) have been found to only partially deplete B cells in SLE patients. Other BAFF blockers, and the anti-CD20 antibody rituximab, have yielded negative or mixed results in clinical trials. See Jin et al. (2020). For example, memory B cells can escape depletion by rituximab, while some studies have shown that rituximab-treated patients with complete B cell depletion have a better response than those with only partial depletion (Schett et al., Lancet (2023) S0140-6736(23)01126-1).

[0134] Options for patients with treatment-refractory SLE are limited. Hematopoietic cell transplantation (HCT) has also been explored. Unfortunately, this approach has not been consistently successful and has the potential for significant toxicity (de Silva et al., Allergy Asthma Clin Immunol (2019) 15:59). Autologous HCT can induce remission in some patients, but there are also studies with treatment-related mortality exceeding 10% due to complications such as bleeding and infection (Jayne et al., Lupus. (2004) 13(3):168-76). Since autologous HCT can be beneficial for selected patients but not for others, allogeneic HCT has also been evaluated. Allogeneic HCT may enhance disease management, but it also has the potential for even higher treatment-related mortality, with a reported 2-year mortality of 20% (Daikeler et al., Bone Marrow Transplant (2009) 44(1):27-33). In summary, there is an urgent need for new therapies that can limit toxicity for patients with systemic autoimmune diseases such as SLE, especially those with severe disease and LN.

[0135] While T cells are widely considered a major contributor to inflammatory demyelination in multiple sclerosis (MS), there is growing evidence suggesting a crucial role for B cells in disease pathogenesis. Both antibody-dependent and antibody-independent mechanisms are thought to underlie B cell-mediated central nervous system (CNS) injury in MS. B cell activity can contribute to both MS relapses and disease progression. Primary progressive MS (PPMS), affecting 10–15% of MS patients, is a particularly difficult form of MS to recognize and treat. Rituximab was tested in PPMS patients in a phase 2 / 3 trial but failed to meet the primary endpoint (Comi et al., Annal. Neurol. (2021) 89(1):13-23). Another anti-CD20 antibody, ocrelizumab, is approved as the first and only treatment for PPMS and is recommended as first-line therapy according to the ECTRIMS-EAN (European Committee for Treatment and Research in Multiple Sclerosis-European Academy of Neurology; Montalban et al., Eur J Neurol. (2018) 25(2):215-37) guidelines. Nevertheless, the need for more effective treatments remains in MS, including PPMS.

[0136] Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease characterized by persistent, symmetrical polyarthritis (synovitis) primarily affecting small joints. Significant extra-articular lesions can also occur in organs such as the skin, heart, lungs, and eyes (Muzes and Sipos, Cells (2023) 12(11):1534). The prominent involvement of B cells in RA has been recognized since the discovery of rheumatoid factor (RF), but has recently received renewed attention; RF and anti-cyclic citryl peptide (anti-CCP) autoantibodies are well-established indicators of the disease and its severity. Initially, the hypothesis was that B cell depletion would have beneficial effects in patients with RA, based on the idea that RF-producing B cells self-replicate and induce TNF production. Transient B-cell depletion induced by rituximab, approved for TNF-refractory RA, can improve the disease over the long term, but it is typically not indefinite (Marston et al., Curr. Opin. Rheumatol. (2010) 22(2):307-15). Therefore, since some patients do not respond well to rituximab treatment, new therapeutic strategies are needed to treat RA, including TNF-refractory RA.

[0137] B cells are involved in several other autoimmune diseases, including scleroderma (Kraaij and van Laar, Biologics (2008) 2(3):389-95), myositis (Oddis and Aggarwal, Nat. Rev. Rheumatol. (2018) 14:279-89), myasthenia gravis (MG; Wu et al., Front. Neurol. (2020) 11: 593431), and vasculitis (Merino-Vico et al., Int J Mol Sci. (2022) 23(1): 387).

[0138] Scleroderma is a rare, chronic autoimmune connective tissue disorder characterized primarily by thickening and hardening of the skin and other tissues. The two main types of scleroderma are systemic scleroderma (also known as SSc) and focal scleroderma. In systemic scleroderma, internal organs such as the gastrointestinal tract, heart, lungs, and kidneys can be affected. Depending on how systemic scleroderma manifests, treatment may include immunosuppressants, cyclophosphamide, mycophenolate mofetil, calcium channel blockers (for Raynaud's phenomenon), gastrointestinal motility enhancers and proton pump inhibitors (for esophageal lesions), ACE inhibitors (for renal lesions), and corticosteroids. Rituximab, a CD20 monoclonal antibody (mAb), has been evaluated in several clinical trials and is used in practice to manage the cutaneous and pulmonary manifestations of SSc, but it is not approved for this indication. Limitations of rituximab include its effect of inducing the secretion of B-cell activator (BAFF), which perpetuates autoreactive B cells, and its inability to target autoreactive long-lived plasma cells (Benfaremo & Gabrielli, 2019; Ehrenstein & Wing, 2016). Autologous CD19-CAR T-cell therapy was evaluated in four patients with severe refractory SSc (Muller et al., N Engl J Med (2024) 390(8):687-700). The treatment was generally well-tolerated, and in three patients with follow-up data of more than 6 months, EUSTAR and MRSS scores decreased. In focal scleroderma, the skin is the main organ system involved, while muscles and bones may or may not be affected. There are two main forms of focal scleroderma: morphea and linear scleroderma. Morphea is the most common form, presenting as one or more patches, while linear scleroderma presents as thickened, hardened bands of skin, often appearing on the face or limbs. Different clinical forms of focal scleroderma can coexist in the same patient. Treatment for focal scleroderma may include systemic or topical steroids (e.g., corticosteroids), methotrexate, and phototherapy. There is no cure for scleroderma; therefore, treatment is designed to alleviate symptoms and slow disease progression.The use of various immunosuppressants remains disappointing. (Odonwodo A, Badri T, Hariz A. Scleroderma. [Updated 2022 Aug 1]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan. Available from ncbi.nlm.nih.gov / books / NBK537335 / ).

[0139] Idiopathic inflammatory myopathy (IIM, also known as myositis) is heterogeneous and rare, with few large-scale treatment trial results available to guide clinicians. The common feature of myositis is chronic inflammation of skeletal muscle, leading to muscle weakness, but other organs such as the skin, joints, lungs, gastrointestinal tract, and heart are also frequently affected. Myositis can be subdivided into anti-synthetase syndrome, polymyositis, dermatomyositis, inclusion body myositis, and immune-mediated necrotizing myositis (Lundberg et al., Nat. Rev. Rheumatol. (2018) 14:269-78). Conventional treatments for myositis include glucocorticoids and immunosuppressants, but biological therapies are increasingly being used. Treatment of myositis with rituximab has yielded mixed results; in the largest clinical trial, the primary endpoint was not met despite most patients meeting the definition of improvement (DOI) by the end of the trial (Oddis and Aggarwal 2018).

[0140] Myasthenia gravis (MG) is a T-cell-dependent, B-cell-mediated chronic autoimmune disease caused by antibodies against AChR, MuSK, or low-density LRP4 expressed in postsynaptic muscle cells, resulting in muscle weakness and fatigue. Approximately 80% of patients with MG are positive for anti-AChR antibodies, and approximately 40% of patients who are negative for anti-AChR antibodies are positive for anti-MuSK antibodies. The presence of anti-LRP4 autoantibodies can also be detected in patients outside the aforementioned groups (Muzes and Sipos Cells (2023) 12(11):1534). While conventional treatment options, including symptomatic treatment and general immunosuppression, may be helpful, sustained remission remains impossible, and chronic treatment with high doses of nonspecific immunosuppressants is usually required to maintain disease remission (Wu 2020). Recent treatment approaches include B-cell targeted therapies such as monoclonal antibodies and proteasome-targeted inhibitors. However, there is still an unmet need for effective treatments, particularly for patients with intractable diseases (Huda, Front. Immunol. (2020) 11:240).

[0141] Vasculitis is primarily classified according to the size of the major blood vessels involved, and the 2012 Chapel Hill Consensus Conference (CHCC) is widely used as the nomenclature and classification system for vasculitis (Jennette et al., Arthritis Rheum. (2013) 65:1-11). Specifically, vasculitis is classified into large vasculitis, medium vasculitis, and small vasculitis. Large vasculitis includes Takayasu's arteritis (TAK) and giant cell arteritis (GCA), which primarily affect the aorta and its major branches. Medium vasculitis includes polyarteritis nodosa (PAN) and Kawasaki disease, which typically affect medium-sized and small arteries. Small vasculitis includes ANCA-associated vasculitis (AAV), which affects small blood vessels and is a systemic autoimmune disease that can cause serious complications in the lungs and kidneys (Merino-Vico 2022). AAV encompasses three major types of vasculitis with distinct clinical characteristics: granulomatosis with polyangiitis (GPA, formerly known as Wegener's granulomatosis), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA) (Jennette and Falk, Semin. Immunopathol. (2014) 36:327-38). The prominent presence of ANCA autoantibodies in this disease suggests B cell involvement in its pathogenesis, given that B cells are precursors to plasma cells (PCs) that produce ANCA. Further evidence supporting the potential role of B cell lineages in vasculitis includes elevated B cell cytokine levels and dysregulated B cell populations in patients. The beneficial effects of anti-CD20 therapy (i.e., rituximab) in AAV patients confirm the pathological B cell contribution. These anti-CD20 antibodies deplete circulating B cells, resulting in disease improvement. However, not all patients respond completely, and this treatment does not target PCs that can maintain ANCA production (Merino-Vico 2022).

[0142] Recently, anti-CD19 autologous CAR T cells have been studied for the treatment of autoimmune diseases such as SLE (Muller et al., N Engl J Med (2024) 390(8):687-700), MG (NCT05828225), myositis (Pecher et al., JAMA (2023) 329(24):2154-62), anti-synthetase syndrome (Mueller et al., Lancet (2023) 401(10379):815-18), systemic sclerosis (Bergmann et al., Ann Rheum Dis. (2023) 82(8):1117-20), and vasculitis (NCT05263817). Treatment with autologous CD19 CAR T cells achieved sustained circulating B cell depletion, disappearance of serum autoantibodies, and clinical remission in patients with severe, refractory SLE who had not responded to treatment with anti-BAFF and anti-CD20 antibodies (Mougiakakos et al., N Engl J Med (2021) 385:567-59). Following administration to patients, CD19 CAR T cells rapidly expanded and remained detectable for 7 weeks thereafter, with the expansion of CAR T cells preceding complete and sustained depletion of circulating B cells. See Mougiakaos et al. (2021); and Jin et al., Cell Mol Immunol (2021) 18:2581-82. In long-term follow-up with additional patients, B cells reappeared an average of 110 days after CAR T cell treatment (Mackensen et al., Nat Med (2022) 28(10):2124-32), and B cell aplasia persisted for a median of 120 days (Taubmann et al. (2023) Ann Rheum Dis 82(Suppl. 1):93-4).

[0143] Nevertheless, autologous CAR T cells have been plagued by concerns regarding associated toxicities such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Furthermore, because autologous cell therapy is manufactured after cells are harvested from the subject, it cannot be used for several weeks and sometimes fails to expand. Additionally, because autologous CAR T cells originate from unhealthy subjects, they generally cannot be retreated (including further lymphocyte apheresis) if manufacturing or treatment is unsuccessful or if the disease relapses. The limited ability of autologous CAR T cells to retreat patients may be particularly important in the context of autoimmune diseases that tend to become chronic and require long-term treatment or retreatment. In addition, while CD19 CAR T cells have been observed to completely and persistently deplete circulating B cells in patients with SLE, complete elimination of B cells (including all subpopulations) may increase the patient's risk of infection.

[0144] Unlike T cells, allogeneic NK cells have not been observed to induce graft-versus-host disease (GvHD) and can kill cells in an antigen-independent manner. Furthermore, because allogeneic cell therapy (e.g., allogeneic NK cell therapy) is derived from a healthy donor, the patient does not need to undergo leukocyte apheresis. Therefore, allogeneic therapy can be provided on demand. Thus, allogeneic CAR NK cell therapy can produce allogeneic cells from a healthy subject as needed and has a low risk of toxicity such as CRS and ICANS, thus potentially offering further opportunities for retreatment of subjects with chronic autoimmune diseases. For example, in some embodiments, if the subject shows an initial clinical benefit (e.g., PR or CR), an additional dosing cycle is provided to enhance or deepen the response. In some embodiments, if the subject shows a CR, an additional dosing cycle is provided to enhance the response. In some embodiments, if the subject shows a PR, an additional dosing cycle is provided to deepen the response. In some embodiments, if the subject shows an initial clinical benefit (e.g., PR or CR) and subsequent disease progression, an additional dosing cycle is provided as retreatment.

[0145] Although the in vivo persistence of CAR NK cells has been observed to be shorter than that of CAR T cells, this property of CAR NK cells is thought to enable long-term reduction of B cell suppression and infection risk. In fact, findings described herein have shown that treatment with NK cells expressing CD19 CAR achieves deep suppression of CD19+ B cells within approximately 30 days, and B cell counts recover significantly within a few months of the final administration. Furthermore, case data reveal that the recovery of B cells after CD19 CAR NK treatment results in a population of primarily naive (e.g., non-class-switched) B cells. Without being constrained by theory, and given that the generation of pathogenic autoreactive antibodies in SLE appears to require class switching to mature B cell isotypes (Liu et al., Autoimmunity (2004) 37(6-7):431-43), this data suggests that treatment with CD19 CAR-expressing NK cells may reset the B cell compartment, thereby reducing or eliminating the production of autoreactive antibodies by mature B cells. Therefore, there is a need for alternative treatments that offer improved persistence, potency, and / or availability compared to current strategies, while minimizing potential toxicity and side effects.

[0146] Furthermore, given the relatively limited persistence of CAR NK cells in vivo, this specification suggests that standard lymphocyte depletion regimens used in CAR T cell therapy may be modified to mitigate the potential risk of toxicity. Multiple studies have shown that adequate suppression of the host immune system correlates with responses in cell therapy clinical trials (Miller et al., Blood (2005) 105(8):3051-57; Turtle et al., J. Clin. Oncol. (2017) 35(26):3010-20). Thus, lymphocyte depletion therapy has become an essential part of CAR T cell clinical trials. Similarly, the use of high-dose lymphocyte depletion regimens before adoptive transfer of NK cells resulted in increased and sustained NK cell activity in vivo, which was not achieved with low-dose lymphocyte depletion regimens (Kilgour et al., Front. Immunol. (2023) 14:1166038). In particular, lymphocyte depletion regimens of fludarabine and cyclophosphamide have been associated with the ability to detect adoptively transferred immune cells. The benefits of lymphocyte elimination with fludarabine-containing lymphocyte depletion therapy can be realized not only by reducing rejection of adoptively transferred immune cells but also by improving the availability of cytokines such as interleukin-15 (IL-15) (Gauthier et al., Blood (2020) 136(Supp. 1):37-38).

[0147] However, since the CD19 CAR-expressing NK cells provided herein are expected to exhibit much of their activity soon after administration to the subject, the primary benefit of lymphocyte removal against NK cells may be obtained from cyclophosphamide, which has activity against lymphocytes earlier than fludarabine. For example, the lowest point in white blood cell count has been reported to be approximately 13 days after fludarabine treatment (FLUDARA® USPI 2010) compared to approximately 9 days after cyclophosphamide treatment (Buckner et al., Cancer (1972) 29(2):357-65). In this regard, if multiple doses of CD19 CAR-expressing NK cells are provided in a single administration cycle, it may be particularly advantageous to provide the entire dose within the peak activity window of cyclophosphamide (e.g., within approximately 7-10 days after cyclophosphamide administration). Therefore, the inventors envision a combination of cyclophosphamide-only lymphocyte apheresis and a dosing cycle that provides the full dose of CD19 CAR-expressing NK cells within approximately 7-10 days after administration of cyclophosphamide-only lymphocyte apheresis. For example, a single dose of cyclophosphamide can be provided approximately 3 days before the administration of the first dose of CD19 CAR NK cells (-3 days), and doses of CD19 CAR NK cells can be provided on days 0, 3, and 7. Similarly, a single dose of cyclophosphamide can be provided approximately 3 days before the administration of the first dose of CD19 CAR NK cells (-3 days), and doses of CD19 CAR NK cells can be provided on days 0, 2, and 4, or on days 0, 2, and 5. For this purpose, the inventors believe that a dosing cycle including the administration of a single dose of cyclophosphamide on day 3 and the administration of CD19 CAR NK cells on days 0, 2, and 4 may be particularly convenient for outpatients, where the cyclophosphamide may be provided, for example, on Friday, and the dose of CD19 CAR NK cells may be provided, for example, on the following Monday, Wednesday, and Friday.While we do not wish to be bound by theory, delivering each dose of CD19 CAR-expressing NK cells within approximately 7–10 days of cyclophosphamide administration may allow for improved peak concentration and / or persistence of NK cells compared to administration regimens where one or more doses in the dosing cycle are delivered later in time.

[0148] Furthermore, increased bioavailability of cytokines (e.g., IL-15) may not be necessary in CD19 CAR-expressing NK cells expressing membrane-bound interleukin 15 (mbIL15), including those provided herein. Similar considerations can be applied to NK cells genetically engineered to increase IL-15 signaling (e.g., by knockout of the CISH gene). Since fludarabine can increase not only short-term toxicity (Hay et al., Blood (2017) 130(21):2295-2306) but also the likelihood of secondary malignancies, excluding fludarabine from lymphocyte apheresis may improve the risk-benefit profile. Therefore, when using CD19 CAR-expressing NK cells provided herein to treat or prevent autoimmune diseases, it is intended that fludarabine-containing lymphocyte apheresis regimens, as commonly used for hematological malignancies, may not be necessary. Rather, a cyclophosphamide-only lymphocyte apheresis regimen may be sufficient to achieve efficacy and mitigate potential LD-related toxicity.

[0149] All publications, including patent documents, scientific articles, and databases, referenced in this application are incorporated by reference in whole for any purpose to the same extent as individual publications are incorporated by reference individually. If any definition contained herein contradicts or is inconsistent with any definition contained herein in a patent, application, published application, or other publication incorporated herein by reference, the definition contained herein shall prevail over the definition incorporated herein by reference.

[0150] The section headings used herein are for organizational purposes only and should not be construed as limiting the subjects described.

[0151] I. Cell Therapy and Cell Engineering In some embodiments, a composition for use in accordance with the provided method (e.g., a composition of genetically engineered NK cells) involves administering engineered NK cells expressing recombinant receptors (e.g., CARs) designed to recognize and / or specifically bind to antigens associated with autoimmune diseases. In certain embodiments, the antigen bound to or recognized by the recombinant receptor (e.g., CAR) is CD19. In some embodiments, binding to the antigen results in a response such as an immune response to such an antigen. In some embodiments, binding to the antigen results in a reduction or depletion of antigen-expressing cells (e.g., CD19-expressing B cells, or a subset thereof). For example, binding to the antigen may reduce or deplete peripheral B cells in the subject being treated. The reduction or depletion of B cells may, correspondingly, reduce the levels and / or activity of autoantibodies in the subject.

[0152] In some embodiments, genetically engineered cells contain or are engineered to contain recombinant receptors, such as chimeric antigen receptors (CARs). Recombinant receptors such as CARs generally contain an antigen-specific extracellular antigen-binding domain (e.g., CD19), which, in some embodiments, is linked to one or more intracellular signaling components via a linker and / or transmembrane domain. In some embodiments, genetically engineered NK cells are provided as pharmaceutical compositions and formulations suitable for administration to a subject, such as for cell therapy. Also provided are therapeutic methods for administering the cells and compositions to a subject, the use of the compositions for treating the subject, and the use of the compositions in the manufacture of pharmaceuticals for treating the subject.

[0153] A. Chimeric antigen receptor Among the recombinant receptors provided, e.g., CD19-targeted CARs, are chimeric receptors that specifically bind to CD19, e.g., receptors containing an anti-CD19 antibody, e.g., an antibody fragment. Among antigen receptors are chimeric antigen receptors (CARs). Also provided are immune cells (e.g., NK cells) that express recombinant receptors, and their use in the treatment of diseases and conditions such as autoimmune diseases (e.g., SLE). Chimeric receptors such as CARs generally contain an extracellular antigen-binding domain containing an anti-CD19 antibody. Such recombinant receptors include antibodies (including their antigen-binding fragments) that specifically bind to CD19 proteins, such as human CD19 protein (e.g., SEQ ID NO: 39). In some embodiments, the antibody is V H and V L This includes multi-domain antibodies, such as those containing domains. In some embodiments, the antibody includes a variable heavy chain and a variable light chain, such as scFv. Among the anti-CD19 antibodies offered are human antibodies and humanized antibodies.

[0154] In this specification, the term “antibody” is used in its broadest sense and includes intact antibodies and polyclonal and monoclonal antibodies containing functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, and variable heavy chains (V) capable of specifically binding to antigens. HThis term includes polyclonal and monoclonal antibodies containing ) regions, single-chain antibody fragments containing single-chain variable fragments (scFv), and single-domain antibody fragments (e.g., sdAb, sdFv, nanobody). This term encompasses intrabody, peptide-body, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific antibodies such as bispecific antibodies, diabody, triabody, and tetrabody, genetically engineered immunoglobulins and / or otherwise modified forms of immunoglobulins such as tandem di-scFv, tandem tri-scFv. Unless otherwise specified, the term “antibody” should be understood to include its functional antibody fragment. This term also encompasses intact antibodies or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

[0155] The terms "complementarity-determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art to refer to discontinuous sequences of amino acids within the antibody variable region that confer antigen specificity and / or binding affinity. Generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, CDR-H3), and each light chain variable region has three CDRs (CDR-L1, CDR-L2, CDR-L3). The terms "framework region" and "FR" are known in the art to refer to the non-CDR portions of the heavy and light chain variable regions. Generally, each full-length heavy chain variable region has four FRs (FR-H1, FR-H2, FR-H3, and FR-H4), and each full-length light chain variable region has four FRs (FR-L1, FR-L2, FR-L3, and FR-L4).

[0156] The precise amino acid sequence boundaries of a given CDR or FR can be determined using the Kabat numbering scheme (Sequences of Proteins of Immunological Interest, 1987 and 1991, NIH, Bethesda, MD), the Chothia numbering scheme (Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883), the Contact numbering scheme (MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745), and the AbM numbering scheme (Martin et al., Proc. Natl. Acad. Sci., This can be easily determined using several well-known schemes, including the IMGT numbering scheme (the international ImMunoGeneTics information system; Lefranc et al, Dev. Comp. Immunol. 29:185-203; 2005) and the Aho numbering scheme (Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001).

[0157] The boundaries of a given CDR or FR may differ depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering in both the Kabat and Chothia schemes is based on the sequence length of the most common antibody regions, with insertions corresponding to insertion letters, e.g., "30a," and deletions appearing in some antibodies. The numbering differs between these two schemes because they place certain insertions and deletions ("indels") in different positions. The Contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme.

[0158] Table 1 below lists the non-restrictive positional boundaries of CDR-L1, CDR-L2, CDR-L3, and CDR-H1, CDR-H2, CDR-H3 as 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 is located between CDR-L1 and CDR-L2. Note that, because insertions are located at H35A and H35B in the shown Kabat numbering scheme, the end of the Chothia CDR-H1 loop, when numbered using the shown Kabat numbering rules, will vary between H32 and H34 depending on the loop length.

[0159] [Table 1]

[0160] Therefore, unless otherwise specified, a given antibody or its region, for example, its variable region's "CDR" or "complementary determination region," or individual specific CDRs (e.g., CDR-H1, CDR-H2), should be understood to encompass the complementary determination region defined (or specific) by any of the schemes described above. For example, a particular CDR (e.g., CDR-H3) is a given V Hor V L When it is stated that an amino acid sequence contains the amino acid sequence of the corresponding CDR, such a CDR is understood to have the sequence of the corresponding CDR (e.g., CDR-H3) within a variable region defined by one of the schemes described above. In some embodiments, a specific CDR sequence is specified.

[0161] Similarly, unless otherwise specified, a given antibody or its region, for example, its variable region FR or individual specific FRs (e.g., FR-H1, FR-H2), should be understood to encompass a framework region defined by one of the known schemes (or a specific one). In some cases, a scheme is specified for identifying a specific CDR, FR, or multiple FRs or CDRs, such as a CDR defined by the IMGT, Kabat, Chothia, or Contact method. In other cases, a specific amino acid sequence of the CDR or FR is given.

[0162] The term "variable region" or "variable domain" refers to the domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to the antigen. The variable domains of the heavy and light chains of a native antibody (V H and V L ) generally have a similar structure, and each domain contains four conserved framework regions (FRs) and three CDRs. A single V H or V L The domain may be sufficient to confer antigen-binding specificity.

[0163] Some of the antibodies provided may contain antibody fragments. An "antibody fragment" refers to a molecule other than the intact antibody, containing a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2, diabody; linear antibodies, variable heavy chains (V H ) domain, single-chain antibody molecules such as scFv; single domain V HExamples include, but are not limited to, single antibodies and multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single-chain antibody fragment containing a variable heavy chain region and a variable light chain region, such as an scFv.

[0164] In some embodiments, the antibody is a recombinantly produced fragment, such as one having two or more antibody regions or antibody chains joined by a synthetic linker, such as a peptide linker, and containing an arrangement that does not exist in nature and / or may not be produced by the enzymatic digestion of naturally occurring intact antibodies. In some embodiments, the antibody fragment is an scFv.

[0165] A single-domain antibody is an antibody fragment that contains all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human or humanized single-domain antibody.

[0166] A “humanized” antibody is one in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. Humanized antibodies may optionally contain at least a portion of the constant region of a human antibody. A “humanized form” of a non-human antibody refers to a variant of a non-human antibody that has been humanized, typically to reduce its immunogenicity against humans, while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some of the FR residues of a humanized antibody are replaced with corresponding residues of a non-human antibody (e.g., an antibody from which CDR residues are derived) to restore or improve the specificity or affinity of the antibody, for example.

[0167] Among the anti-CD19 antibodies offered are human antibodies. “Human antibody” refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, human cell, or non-human source utilizing a human antibody repertoire, including a human antibody library, or other human antibody coding sequences. This term excludes humanized forms of non-human antibodies containing non-human antigen-binding regions, such as those where all or substantially all CDRs are non-human. This term includes antigen-binding fragments of human antibodies.

[0168] The antibodies offered include monoclonal antibodies (including monoclonal antibody fragments). As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, or an antibody within that population; that is, the individual antibodies comprising the population are identical, but excluding variants that may contain naturally occurring mutations or variants that may occur during the production of the monoclonal antibody preparation (such variants are generally present in trace amounts). In contrast to polyclonal antibody preparations, which generally contain different antibodies targeting different epitopes, each monoclonal antibody in a monoclonal antibody preparation targets a single epitope on an antigen. This term should not be construed as requiring the production of antibodies by any particular method. Monoclonal antibodies can be produced by a variety of techniques, including but not limited to production from hybridomas, recombinant DNA methods, phage display methods, and other antibody display methods.

[0169] The terms “polypeptide” and “protein” are used interchangeably to refer to polymers of amino acid residues and are not limited to the minimum length. The antibodies and antibody chains provided, as well as other peptides, such as linkers and CD19-binding peptides, may contain amino acid residues including native and / or non-native amino acid residues. This term also includes post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, and phosphorylation. In some embodiments, polypeptides may contain modifications to their native or natural sequence, as long as the protein maintains the desired activity.

[0170] The antigen-binding domain may be any antibody described herein (e.g., an anti-CD19 antibody), or may contain such an antibody.

[0171] In some embodiments, the extracellular antigen-binding domain includes a heavy chain variable region (VH) having CDR-1, CDR-2, and CDR-3, respectively, containing the amino acid sequences described in SEQ ID NOs: 24, 25, and 26. In some embodiments, the extracellular antigen-binding domain includes a light chain variable region (VL) having CDR-1, CDR-2, and CDR-3, respectively, containing the amino acid sequences described in SEQ ID NOs: 27, 28, and 29. In some embodiments, the extracellular antigen-binding domain includes a VH having CDR-1, CDR-2, and CDR-3, respectively, containing the amino acid sequences described in SEQ ID NOs: 24, 25, and 26; and a VL having CDR-1, CDR-2, and CDR-3, respectively, containing the amino acid sequences described in SEQ ID NOs: 27, 28, and 29.

[0172] In some embodiments, the extracellular antigen-binding domain includes a heavy chain variable region (VH) having CDR-1, CDR-2, and CDR-3, respectively, containing the amino acid sequences described in SEQ ID NOs: 30, 31, and 32. In some embodiments, the extracellular antigen-binding domain includes a light chain variable region (VL) having CDR-1, CDR-2, and CDR-3, respectively, containing the amino acid sequences described in SEQ ID NOs: 33, 34, and 29. In some embodiments, the extracellular antigen-binding domain includes a VH having CDR-1, CDR-2, and CDR-3, respectively, containing the amino acid sequences described in SEQ ID NOs: 30, 31, and 32; and a VL having CDR-1, CDR-2, and CDR-3, respectively, containing the amino acid sequences described in SEQ ID NOs: 33, 34, and 29.

[0173] In some embodiments, VH includes an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with respect to the amino acid sequence described in SEQ ID NO: 35. In some embodiments, VH includes the amino acid sequence described in SEQ ID NO: 35. In some embodiments, VL includes an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with respect to the amino acid sequence described in SEQ ID NO: 36. In some embodiments, VL includes the amino acid sequence described in SEQ ID NO: 36. In some embodiments, VH comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with the amino acid sequence described in SEQ ID NO: 35; VL comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with the amino acid sequence described in SEQ ID NO: 36. In some embodiments, VH comprises the amino acid sequence described in SEQ ID NO: 35, and VL comprises the amino acid sequence described in SEQ ID NO: 36.

[0174] In some embodiments, the antigen-binding domain is an scFv containing VH and VL linked by a linker (e.g., a linker containing any of SEQ ID NOs: 1-3). In some embodiments, the linker contains the amino acid sequence described in SEQ ID NOs: 1 or 3. In some embodiments, the extracellular antigen-binding domain is an scFv containing the linker described in SEQ ID NOs: 1. In some embodiments, the antigen-binding domain is an scFv containing an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with respect to the amino acid sequence described in SEQ ID NOs: 37. In some embodiments, the antigen-binding domain is an scFv containing the amino acid sequence described in SEQ ID NOs: 37. In some embodiments, the extracellular antigen-binding domain is an scFv containing the linker described in SEQ ID NOs: 2. In some embodiments, the extracellular antigen-binding domain is an scFv containing the linker described in SEQ ID NOs: 3.

[0175] Additional CD19 binding domains are known and described in the art, including any of those described in PCT applications PCT / US2015 / 024671, PCT / US2018 / 029107, PCT / US2020 / 020824, PCT / US2020 / 033559, PCT / IB2021 / 060213, and PCT / CN2021 / 106892 (each of which is incorporated herein by reference in its entirety).

[0176] Provided herein are recombinant receptors (e.g., CARs) comprising either a CD19 antibody or a binding domain as described herein. The extracellular antigen-binding domain is generally ligated to an intracellular signaling domain that includes an intracellular signaling component, for example, in the case of a CAR, a signaling component that mimics activation mediated by an antigen-receptor complex such as the TCR complex. In some embodiments, the extracellular antigen-binding domain of a CAR is ligated to the intracellular signaling domain by a transmembrane domain. Thus, in some embodiments, a CD19-binding molecule (e.g., an antibody) is ligated to both a transmembrane domain and an intracellular signaling domain. In some embodiments, a CAR comprises an extracellular antigen-binding domain that binds to CD19, a transmembrane domain, and an intracellular signaling domain that includes a co-stimulatory signaling region and a primary signaling domain (e.g., CD3 zeta).

[0177] In some embodiments, the transmembrane domain is fused to the extracellular domain. In some embodiments, the transmembrane domain is derived from either a natural or synthetic source. If the source is natural, the domain, in some embodiments, is derived from any membrane-bound or transmembrane protein. The transmembrane region includes those derived from CD3, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or combinations thereof (e.g., including at least the transmembrane region). Alternatively, in some embodiments, the transmembrane domain is synthetic.

[0178] In some embodiments, the transmembrane domain comprises at least a portion of CD8, a transmembrane glycoprotein normally expressed in both T cells and NK cells. In some embodiments, the transmembrane domain comprises CD8 alpha (CD8a). In some embodiments, the transmembrane domain comprises a CD8 (e.g., CD8a) hinge and a CD8 (e.g., CD8a) transmembrane region.

[0179] In some embodiments, the transmembrane domain includes a hinge, such as a CD8a hinge. In some embodiments, the sequence encoding the CD8a hinge is cleaved or modified. In some embodiments, the CD8a hinge is encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the CD8a hinge includes the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the CD8a hinge is cleaved or modified. In some embodiments, the CD8a hinge has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the CD8a hinge includes the amino acid sequence of SEQ ID NO: 6.

[0180] In some embodiments, the transmembrane domain includes a CD8a transmembrane region. In some embodiments, the CD8a transmembrane region is cleaved or modified. In some embodiments, the CD8a transmembrane region is encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments, the CD8a transmembrane region is encoded by the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the CD8a transmembrane region is cleaved or modified. In some embodiments, the CD8a transmembrane region has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments, the CD8a transmembrane region includes the amino acid sequence of SEQ ID NO: 8.

[0181] Therefore, in some embodiments, the CD8 transmembrane domain is cleaved or modified and encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments, the CD8 transmembrane domain is encoded by the nucleic acid sequence of SEQ ID NO: 9. In some embodiments, the CD8 transmembrane domain is cleaved or modified and contains an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments, the transmembrane domain contains the amino acid sequence of SEQ ID NO: 10.

[0182] In some embodiments, the transmembrane domain comprises the CD28 transmembrane domain or a fragment thereof. In some embodiments, the CD28 transmembrane domain is cleaved or modified. In some embodiments, the CD28 transmembrane domain has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 11.

[0183] Receptors, such as CARs, generally include an intracellular signaling domain containing intracellular signaling components. In some embodiments, the receptor includes intracellular components of the TCR complex, such as the TCR CD3 chain, such as the CD3 zeta chain, which mediates T cell activation and cytotoxicity. Thus, in some embodiments, the antigen-binding moiety is linked to one or more cellular signaling modules. In some embodiments, upon ligation of a CAR or other chimeric receptor, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of immune cells, such as NK cells engineered to express the CAR. For example, in some contexts, the CAR induces functions of immune cells (e.g., NK cells) such as cytolytic activity and / or secretion of cytokines or other factors. In some embodiments, a cleaved portion of the intracellular signaling domain of an antigen receptor component or co-stimulatory molecule is used instead of an intact immunostimulatory chain, for example, to transmit effector function signals. In some embodiments, the intracellular signaling domain includes a cytoplasmic sequence of a T cell receptor (TCR), and in some embodiments, also includes a sequence of a co-receptor that acts in cooperation with such receptors in a natural context to initiate signaling following engagement with an antigen receptor.

[0184] In the context of natural TCRs, complete activation generally requires not only TCR-mediated signaling but also co-stimulatory signals. Therefore, in some embodiments, the receptor also includes components for generating secondary or co-stimulatory signals to facilitate complete activation. T cell activation is described in some embodiments 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 an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences). In some embodiments, the receptor includes one or both of these signaling components.

[0185] In some embodiments, the receptor includes a primary cytoplasmic signaling sequence that modulates the primary activation of the TCR complex. The stimulative primary cytoplasmic signaling sequence may contain an immunoreceptor tyrosine-based activation motif or a signaling motif known as an ITAM. Examples of ITAMs containing primary cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d. In some embodiments, the cytoplasmic signaling molecule in the CAR contains a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3 zeta.

[0186] For example, immune cells operated according to some embodiments disclosed herein may include at least one subunit (or fragment thereof) of the CD3 T cell receptor complex. In some embodiments, the signaling domain includes a CD3 zeta subunit. In some embodiments, the CD3 zeta may be cleaved or modified. In some embodiments, the CD3 zeta is encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 17. In some embodiments, the CD3 zeta is encoded by the nucleic acid sequence of SEQ ID NO: 17. In some embodiments, the CD3 zeta is cleaved or modified. In some embodiments, the CD3 zeta includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the CD3 zeta includes the amino acid sequence of SEQ ID NO: 18.

[0187] In some embodiments, the intracellular signaling domain includes a co-stimulatory signaling region such as CD28, 4-1BB, OX40, DAP10, ICOS, or any combination thereof. In some embodiments, the intracellular signaling domain includes the intracellular signaling region of CD28. In some embodiments, the intracellular signaling domain includes the intracellular signaling region of 4-1BB. In some embodiments, the intracellular signaling domain includes the intracellular signaling region of OX40. In some embodiments, the intracellular signaling domain includes the intracellular signaling region of DAP10. In some embodiments, the intracellular signaling domain includes the intracellular signaling region of ICOS. In some embodiments, the intracellular signaling domain does not include DAP10 and / or DAP12. In some embodiments, the intracellular signaling domain does not include DAP10. In some embodiments, the intracellular signaling domain does not include DAP12. In some embodiments, the same receptor includes both the CD3 zeta and a co-stimulatory signaling region. Therefore, in some embodiments, the intracellular signaling domain of recombinant receptors such as CAR includes both the CD3 zeta intracellular domain and a co-stimulatory signaling region.

[0188] In some embodiments, the intracellular signaling domain includes the intracellular signaling region of OX40. In some embodiments, the OX40 intracellular signaling region is encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the OX40 intracellular signaling region is encoded by the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the OX40 intracellular signaling region includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the OX40 intracellular signaling region includes the amino acid sequence of SEQ ID NO: 14. In some embodiments, OX40 is used as the sole intracellular signaling component in the construct, but in some embodiments, OX40 may be used in combination with one or more other components. For example, in some embodiments, a combination of OX40 and CD3 zeta is used. In some embodiments, the intracellular signaling domain includes an OX40 co-stimulatory signaling region linked to the CD3 zeta.

[0189] In some embodiments, the CAR includes an extracellular antigen-binding domain containing the sequence described in SEQ ID NO: 37, a CD8 alpha transmembrane domain containing the amino acid sequence described in SEQ ID NO: 8, an OX40 intracellular signaling region containing the amino acid sequence described in SEQ ID NO: 14, and a CD3 zeta domain containing the amino acid sequence described in SEQ ID NO: 18. In some embodiments, the CAR includes an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with respect to the amino acid sequence described in SEQ ID NO: 38. In some embodiments, the CAR includes the amino acid sequence described in SEQ ID NO: 38.

[0190] As a further example, combinations of CD28, OX40, 4-1BB, and / or CD3 zeta are used in some embodiments.

[0191] In some embodiments, the intracellular signaling domain includes the intracellular signaling region of 4-1BB. In some embodiments, the 4-1BB intracellular signaling region is encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the 4-1BB intracellular signaling region is encoded by the nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the 4-1BB intracellular signaling region includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the 4-1BB intracellular signaling region includes the amino acid sequence of SEQ ID NO: 16. In some embodiments, 4-1BB is used as the sole intracellular signaling component in the construct, but in some embodiments, 4-1BB may be used in combination with one or more other components. For example, in some embodiments, a combination of 4-1BB and CD3 zeta is used. In some embodiments, the intracellular signaling domain includes a 4-1BB co-stimulatory signaling region linked to the CD3 zeta. As a further example, a combination of CD28, OX40, 4-1BB, and / or CD3 zeta is used in some embodiments.

[0192] In some embodiments, the intracellular signaling domain includes an intracellular signaling region of CD28. In some embodiments, the CD28 intracellular signaling region includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the CD28 intracellular signaling region includes the amino acid sequence of SEQ ID NO: 12. In some embodiments, CD28 is used as the sole intracellular signaling component in the construct, but in some embodiments, CD28 may be used in conjunction with one or more other components. For example, in some embodiments, a combination of CD28 and CD3 zeta is used. In some embodiments, the intracellular signaling domain includes a CD28 co-stimulatory signaling region linked to CD3 zeta. As a further example, a combination of CD28, OX40, 4-1BB, and / or CD3 zeta is used in some embodiments.

[0193] Additional CD19 directional CARs are known and described in the art, including any of those described in Kalos et al., Sci Transl Med 3:95ra73 (2011); Porter et al., NEJM 365:725-733 (2011); Grupp et al., NEJM 368: 1509-1518 (2013); and PCT applications PCT / US2015 / 024671, PCT / US2018 / 029107, PCT / US2020 / 020824, and PCT / CN2021 / 106892.

[0194] In any of the embodiments provided, the nucleic acid or portion thereof encoding the chimeric receptor is codon-optimized. In some embodiments, the polynucleotide is optimized to reduce RNA heterogeneity and / or modify the expression of the encoded receptor, e.g., surface expression, e.g., to increase or make it more consistent between batches of cell products, or contains specific features designed for optimization, such as codon usage. In some embodiments, the polynucleotide encoding the chimeric receptor is modified compared to a reference polynucleotide, such as by removing cryptic splice sites or hidden splice sites, to reduce RNA heterogeneity. In some embodiments, the polynucleotide encoding the chimeric receptor is codon-optimized for expression in mammals, e.g., human cells, e.g., human T cells. In some embodiments, the modified polynucleotide, when expressed in cells, results in improved, e.g., increased, or more uniform or more consistent levels of expression, e.g., surface expression.

[0195] B. Manipulated cells Also offered are methods, nucleic acids, compositions, and kits for generating genetically modified immune cells (e.g., NK cells). In some embodiments, the genetic modification involves the introduction of nucleic acids encoding the genetically modified component or other components for introduction into cells, such as components encoding gene disruption proteins or nucleic acids. Among the additional nucleic acids, e.g., genes for introduction, are genes for improving therapeutic efficacy, such as promoting the viability and / or function of the transplanted cells; genes for providing genetic markers for cell selection and / or evaluation, such as assessing survival or localization in vivo; and genes for improving safety, such as making cells more susceptible to negative selection in vivo.

[0196] i. Vectors and methods for genetic manipulation Also provided are methods, polynucleotides, compositions, and kits (including recombinant receptors (e.g., CARs) containing the binding molecule) for expressing a binding molecule (e.g., an anti-CD19 binding molecule) and for producing genetically engineered immune cells (e.g., NK cells) that express such binding molecules. In some embodiments, one or more binding molecules containing recombinant receptors (e.g., CARs) may be genetically engineered into a cell or a group of cells. Genetic engineering generally involves introducing nucleic acids encoding the recombinant or engineered component into a cell by means of retroviral transduction, transfection, or transformation, etc.

[0197] Also provided are polynucleotides and / or parts thereof, e.g., chains, encoding antibodies and chimeric antigen receptors. Some of the polynucleotides provided encode the anti-CD19 chimeric antigen receptor (e.g., antigen-binding fragment) described herein. Also provided are polynucleotides encoding one or more antibodies and / or parts thereof, e.g., the anti-CD19 antibody (e.g., antigen-binding fragment) described herein, and / or other antibodies and / or parts thereof, e.g., one or more antibodies and / or parts thereof that bind to other target antigens. Polynucleotides may also include polynucleotides that contain naturally occurring and / or non-naturally occurring nucleotides and bases (e.g., those with skeletal modifications). The terms “nucleic acid molecule,” “nucleic acid,” and “polynucleotide” may be used interchangeably and refer to polymers of nucleotides. Such polymers of nucleotides may contain natural and / or non-natural nucleotides, and include, but are not limited to, DNA, RNA, and PNA. “Nucleic acid sequence” refers to a linear sequence of nucleotides containing a nucleic acid molecule or polynucleotide. Also provided are polynucleotides with optimized codon usage.

[0198] Also provided are vectors containing polynucleotides, for example, any of the polynucleotides described herein, and cells containing the vectors, for example, cells for producing antibodies or antigen-binding fragments thereof. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector. In some embodiments, the vector is a lentiviral vector. Also provided are methods for producing antibodies or antigen-binding fragments thereof. A nucleic acid may encode an amino acid sequence including the VL region of an antibody and / or an amino acid sequence including the VH region (e.g., the light chain and / or heavy chain of an antibody). A nucleic acid may encode one or more amino acid sequences including the VL region of an antibody and / or one or more amino acid sequences including the VH region (e.g., the light chain and / or heavy chain of an antibody). In further embodiments, one or more vectors containing such polynucleotides (e.g., expression vectors) are provided. In further embodiments, host cells containing such polynucleotides are provided. In such alternative embodiments, the host cell comprises (1) a vector comprising a nucleic acid encoding an amino acid sequence including the VL region of an antibody and an amino acid sequence including the VH region of an antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence including the VL region of an antibody and a second vector comprising a nucleic acid encoding an amino acid sequence including the VH region of an antibody (e.g., transformed thereby). In some embodiments, the host cell comprises one or more vectors comprising one or more nucleic acids encoding one or more amino acid sequences including one or more antibodies and / or a portion thereof, e.g., an antigen-binding fragment thereof (e.g., transformed thereby). In some embodiments, one or more such host cells are provided. In some embodiments, a composition comprising one or more such host cells is provided. In some embodiments, one or more host cells may express different antibodies or the same antibody. In some embodiments, each host cell may express two or more antibodies.

[0199] Also provided is a method for producing an anti-CD19 chimeric antigen receptor. For recombinant production of the chimeric receptor, the nucleic acid sequence encoding the chimeric receptor antibody, for example, as described herein, can be isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acid sequences can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody). In some embodiments, a method for producing an anti-CD19 chimeric antigen receptor is provided, which involves culturing host cells containing the nucleic acid sequence encoding the antibody provided above under conditions suitable for receptor expression. In certain examples, immune cells, such as human immune cells, are used to express the provided polypeptide encoding the chimeric antigen receptor. In some examples, the immune cells are NK cells, including primary NK cells.

[0200] In some embodiments, gene transfer is achieved by transduction of immune cells (e.g., activated immune cells) and expansion in culture to a number sufficient for clinical application. In some embodiments, cells are further manipulated to promote the expression of cytokines or other factors. Various methods for introducing genetically modified components, such as antigen receptors, such as CARs, are well known and can be used in conjunction with the provided methods and compositions. Non-limiting examples of methods include those for introducing polynucleotides encoding receptors, including via viruses, such as retroviruses or lentiviruses, transduction, transposons, and electroporation.

[0201] In some embodiments, recombinant polynucleotides are transferred into immune cells (e.g., NK cells) using recombinant infectious viral particles, such as vectors derived from Simian virus 40 (SV40), adenoviruses, or adeno-associated viruses (AAVs). In some embodiments, recombinant polynucleotides are transferred into immune cells using recombinant lentiviral vectors or retroviral vectors, such as gamma retroviral vectors. In some embodiments, the retroviral vectors have long terminal repeat sequences (LTRs), such as retroviral vectors derived from Moloney's mouse leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), splenic focal-forming virus (SFFV), or human immunodeficiency virus type 1 (HIV-1). Most retroviral vectors are derived from mouse retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. Retroviruses are typically amphipathic, meaning they can infect host cells of several species, including humans. In one embodiment, the expressed gene is replaced with the retroviral gag, pol, and / or env sequences. Several exemplary retroviral lines have been described. Methods of lentiviral transduction are known and described in the art.

[0202] Additional polynucleotides, such as genes to be introduced, may include genes to improve therapeutic efficacy, such as promoting the viability and / or function of the transplanted cells; genes to provide genetic markers for cell selection and / or evaluation, such as assessing in vivo survival or localization; and genes to improve safety, such as making cells more susceptible to in vivo negative selection.

[0203] In some cases, a polynucleotide containing a nucleic acid sequence encoding a CD19-binding receptor, such as a chimeric antigen receptor (CAR), contains a signal sequence encoding a signal peptide. In some embodiments, the signal sequence may encode a signal peptide derived from a native polypeptide. In other embodiments, the signal sequence may encode a heterologous or non-native signal peptide. In some embodiments, a non-limiting example of the signal peptide includes the CD8 alpha (CD8a) signal peptide described in SEQ ID NO: 4.

[0204] In some embodiments, the vector or construct may contain a promoter and / or enhancer or regulatory element for regulating the expression of the encoded recombinant receptor. In some examples, the promoter and / or enhancer or regulatory element may be a condition-dependent promoter, enhancer, and / or regulatory element. In some examples, these elements drive the expression of the transgene.

[0205] In some embodiments, the vector or construct may contain a single promoter that drives the expression of one or more nucleic acid molecules. In some embodiments, such nucleic acid molecules, e.g., transcripts, may be multicistronic (bisistronic or tricistronic). For example, in some embodiments, the transcription unit can be operated as a bicistronic unit containing an IRES (internal ribosome entry site), thereby enabling co-expression of a gene product (e.g., encoding a chimeric receptor and membrane-bound interleukin-15) via a message from a single promoter. Alternatively, in some cases, a single promoter can direct the expression of RNA containing two or three genes (e.g., encoding a chimeric receptor and membrane-bound interleukin-15) separated from each other by a self-cleaving peptide (e.g., a 2A cleavage sequence) or a sequence encoding a protease recognition site within a single open reading frame (ORF). Thus, the ORF encodes a single polypeptide, which is cleaved into individual proteins during translation (in the case of T2A) or post-translation. In some cases, peptides, such as T2A, can cause ribosomes to skip the synthesis of peptide bonds at the C-terminus of the 2A element (ribosome skipping), resulting in separation between the end of the 2A sequence and the next downstream peptide. Many 2A elements are known. Examples of 2A peptides that can be used in the methods and polynucleotides disclosed herein are, but are not limited to, 2A peptides from foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), Tosea asigna virus (T2A, e.g., SEQ ID NO: 20 encoded by SEQ ID NO: 19), and porcine tescovirus-1 (P2A). In some embodiments, one or more different or separate promoters drive the expression of binding molecules, e.g., nucleic acid molecules encoding recombinant receptors and nucleic acids encoding membrane-bound interleukin-15.

[0206] ii. Interleukin-15 In some embodiments, any of the immune cells provided herein are engineered to express interleukin-15 (mbIL15). In some embodiments, IL15 is membrane-bound IL15. Thus, in some embodiments, any of the immune cells provided herein are engineered to express membrane-bound interleukin-15 (mbIL15). In such embodiments, expression of mbIL15 on immune cells (e.g., NK cells) enhances the cytotoxic effect of the engineered cells by increasing cell proliferation and / or lifespan. In some embodiments, IL15 is expressed from a separate cassette on a construct containing any of the CARs disclosed herein. In some embodiments, IL15 is expressed from the same cassette as any of the CARs disclosed herein.

[0207] In some embodiments, the chimeric receptor and IL15 are separated by nucleic acid sequences encoding cleavage sites, e.g., proteolytic cleavage sites or T2A, P2A, E2A, or F2A self-cleaving peptide cleavage sites. In some embodiments, the chimeric receptor and IL15 are separated by a T2A peptide (e.g., SEQ ID NO: 20, encoded by SEQ ID NO: 19). In some embodiments, IL15 is membrane-bound IL15 (mbIL15). In some embodiments, mbIL15 includes a native IL15 sequence, e.g., a human native IL15 sequence (e.g., SEQ ID NO: 22, encoded by SEQ ID NO: 21). In some embodiments, mbIL15 includes a native IL15 sequence, e.g., a human native IL15 sequence (e.g., SEQ ID NO: 22, encoded by SEQ ID NO: 21), and at least one transmembrane domain (e.g., CD8a). In some embodiments, IL15 is encoded by the nucleic acid sequence of SEQ ID NO: 21. In some embodiments, IL15 may be cleaved or modified to encode a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 21. In some embodiments, IL15 includes the amino acid sequence of SEQ ID NO: 22. In some embodiments, IL15 is cleaved or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 22.

[0208] Therefore, in some embodiments, any of the CARs described herein are encoded by the same nucleic acid sequence as mbIL15. In some embodiments, the nucleic acid sequences encoding the CAR and mbIL15 are separated by the sequence encoding T2A (e.g., SEQ ID NO: 19). In some embodiments, any of the engineered cells described herein express CD19-targeted recombinant receptors (e.g., CARs) and mbIL15.

[0209] In some embodiments, mbIL15 is membrane-bound by the fusion of IL15 to a transmembrane domain. Therefore, in some embodiments, mbIL15 includes a transmembrane domain. In some embodiments, the transmembrane domain includes a CD8a transmembrane domain. In some embodiments, the transmembrane domain includes a hinge and / or a transmembrane region. In some embodiments, the transmembrane domain includes a hinge and a transmembrane region. In some embodiments, the hinge is a CD8a hinge sequence (e.g., SEQ ID NO: 6). In some embodiments, the transmembrane region is a CD8a transmembrane region (e.g., SEQ ID NO: 8). In some embodiments, mbIL15 includes a native IL15 sequence, e.g., a human native IL15 sequence, and at least one transmembrane domain (e.g., a CD8a transmembrane domain). In some embodiments, the CD8a transmembrane domain includes the sequence of SEQ ID NO: 10. In some embodiments, mbIL15 is cleaved or modified to contain an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 23. In some embodiments, mbIL15 contains the amino acid sequence of SEQ ID NO: 23. Membrane-bound IL15 sequences are described in PCT Publications WO2018 / 183385 and WO2020 / 056045, each of which is expressly incorporated herein in whole by reference.

[0210] iii.Cell type Some embodiments of the methods and compositions provided herein relate to cells such as immune cells. In some embodiments, immune cells are engineered to express chimeric receptors that bind to antigens (e.g., CD19).

[0211] Genetic engineering has made it possible to develop approaches that utilize specific aspects of the immune system to fight disease. In some cases, immune cells from a healthy donor can be modified to specifically eliminate disease-causing cells or cells associated with the disease (e.g., B cells associated with SLE). Various types of immune cells can be used, including T cells, natural killer (NK) cells, or combinations thereof, as detailed below.

[0212] To facilitate immunotherapy for the treatment of autoimmune diseases, polynucleotides, polypeptides, and vectors encoding chimeric antigen receptors (CARs) comprising a target-binding moiety (e.g., an antigen expressed by B cells) and a cytotoxic signaling complex are provided herein. For example, some embodiments include polynucleotides, polypeptides, or vectors encoding a chimeric antigen receptor for, for example, CD19, to facilitate the targeting of immune cells against B cells involved in the pathogenesis of autoimmune diseases. Methods for treating autoimmune diseases (e.g., SLE) and other uses of such cells for immunotherapy are also provided herein. Also provided are engineered immune cells (e.g., NK cells) expressing such chimeric receptors.

[0213] In some embodiments, immune system cells are manipulated to enhance cytotoxicity against target cells, such as tumor cells. For example, immune system cells may be manipulated to contain CD19-directed CARs as described herein. In some embodiments, white blood cells or leukocytes are used because their intrinsic function is to defend the body from the proliferation of abnormal cells and infections. There are various types of white blood cells that play specific roles in the human immune system, and therefore the manipulation of cells disclosed herein is a preferred starting point. White blood cells include granulocytes and agranulocytes (those with or without granules in the cytoplasm, respectively). Granulocytes include basophils, eosinophils, neutrophils, and mast cells. Agranulocytes include lymphocytes and monocytes. Cells described herein and in other ways may be manipulated to contain CD19-directed CARs, or nucleic acids encoding CARs. In some embodiments, immune cells are manipulated to co-express membrane-bound interleukin 15 (mbIL15) costimulatory domains. In some embodiments, immune cells engineered to express CAR are engineered to express the mbIL15 domain bicistronically.

[0214] a. Month In some embodiments, immune cells include monocytes. Monocytes are a subtype of leukocyte. Monocytes can differentiate into macrophages and myeloid dendritic cells. Monocytes are involved in the adaptive immune system and perform major functions such as phagocytosis, antigen presentation, and cytokine production. Phagocytosis is the process of taking in cellular material or entire cells, and then digesting and destroying the taken-in cellular material.

[0215] In some embodiments, monocytes are positive for cell surface expression of a marker selected from the group consisting of CCR2, CCR5, CD11c, CD14, CD16, CD62L, CD68+, CX3CR1, HLA-DR, or any combination thereof. In some embodiments, monocytes are positive for cell surface expression of CD14. In some embodiments, monocytes are positive for cell surface expression of CCR2. In some embodiments, monocytes are positive for cell surface expression of CCR5. In some embodiments, monocytes are positive for cell surface expression of CD62L.

[0216] In some embodiments, monocytes are used in conjunction with one or more additional manipulated cells disclosed herein. Some embodiments of the methods and compositions disclosed herein relate to monocytes expressing CD19-binding CARs, or nucleic acids encoding CARs.

[0217] In some embodiments, monocytes are engineered to express the membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, monocytes engineered to express CAR are also engineered to express the membrane-bound interleukin 15 (mbIL15) domain (e.g., by cistronic expression). Therefore, in some embodiments, monocytes are engineered to express both CAR and mbIL15 by cistronic expression.

[0218] In some embodiments, monocytes are allogeneic cells. In some embodiments, monocytes are obtained from a donor who does not have an autoimmune disease.

[0219] b. Lymphocytes In some embodiments, immune cells include lymphocytes. Lymphocytes, another major subtype of leukocytes, include T cells (cell-mediated, cytotoxic adaptive immunity), natural killer cells (cell-mediated, cytotoxic innate immunity), and B cells (humoral, antibody-driven adaptive immunity). While B cells are manipulated according to some embodiments disclosed herein, some embodiments also relate to manipulated T cells or manipulated NK cells (in some embodiments, a mixture of T cells and NK cells derived from either the same or different donors is used). Thus, in some embodiments, immune cells include T cells. In some embodiments, immune cells include NK cells. In some embodiments, immune cells include T cells and NK cells. In some embodiments, immune cells include B cells.

[0220] In some embodiments, lymphocytes are used in conjunction with one or more additional manipulated cells disclosed herein. Some embodiments of the methods and compositions disclosed herein relate to lymphocytes expressing CD19-binding CARs, or nucleic acids encoding CARs.

[0221] In some embodiments, lymphocytes are engineered to express the membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, lymphocytes engineered to express CAR are also engineered to express the membrane-bound interleukin 15 (mbIL15) domain (e.g., by cistronic expression). Therefore, in some embodiments, lymphocytes are engineered to express both CAR and mbIL15 by cistronic expression.

[0222] In some embodiments, lymphocytes are allogeneic cells. In some embodiments, monocytes are obtained from a donor who does not have an autoimmune disease.

[0223] cT cells In some embodiments, immune cells include T cells. T cells can be distinguished from other lymphocyte subtypes (e.g., B cells or NK cells) based on the presence of T cell receptors on their cell surface.

[0224] T cells can be divided into various subtypes, including effector T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, mucosa-associated invariant T cells, and gamma delta T cells. In some embodiments, specific subtypes of T cells are manipulated. In some embodiments, T cells are positive for cell surface expression of a marker selected from the group consisting of CD3, CD4, and / or CD8. In some embodiments, T cells are positive for cell surface expression of CD3. In some embodiments, T cells are positive for cell surface expression of CD4. In some embodiments, T cells are positive for cell surface expression of CD8.

[0225] In some embodiments, CD3+ T cells are manipulated. In some embodiments, CD4+ T cells are manipulated. In some embodiments, CD8+ T cells are manipulated. In some embodiments, regulatory T cells are manipulated. In some embodiments, gamma delta T cells are manipulated. In some embodiments, a mixed pool of T cell subtypes is manipulated. For example, in some embodiments, CD4+ and CD8+ T cells are manipulated. In some embodiments, there is no particular choice regarding the T cell type manipulated to express the cytotoxic receptor complex disclosed herein. In some embodiments, certain techniques, such as the use of cytokine stimulation, are used to facilitate the expansion / collection of T cells having a specific marker profile. For example, in some embodiments, activation of specific human T cells, e.g., CD4+ T cells, CD8+ T cells, is achieved by using CD3 and / or CD28 as stimulating molecules.

[0226] In some embodiments, methods are provided for treating or preventing autoimmune diseases, comprising administering T cells expressing the cytotoxic receptor complex described herein. In some embodiments, the engineered T cells are autologous cells, while in some embodiments, the T cells are allogeneic cells. In some embodiments, the T cells are allogeneic cells. In some embodiments, the T cells are obtained from a donor who does not have an autoimmune disease.

[0227] Some embodiments of the methods and compositions disclosed herein relate to T cells engineered to express a CD19-binding CAR. In some embodiments, T cells are engineered to express a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, T cells engineered to express a CAR are also engineered to express a membrane-bound interleukin 15 (mbIL15) domain (e.g., by bisistronally). Thus, in some embodiments, T cells are engineered to express both CAR and mbIL15 by bisistronally.

[0228] In some embodiments, the immune cells include T cells and NK cells (derived from the same donor or from different donors).

[0229] d.NK cells In some embodiments, the immune cells include natural killer (NK) cells. In some embodiments, a method is provided for treating or preventing an autoimmune disease, comprising administering NK cells expressing the CD19-targeted CAR described herein. In some embodiments, the engineered NK cells are autologous cells, while in some embodiments, the NK cells are allogeneic cells. In some embodiments, the NK cells are allogeneic cells. In some embodiments, the NK cells are derived from a donor who does not have an autoimmune disease. In some embodiments, the NK cells are derived from a donor who does not have SLE.

[0230] In some embodiments, NK cells are preferred because they possess relatively high innate cytotoxicity. In some embodiments, the manipulated cells disclosed herein can further upmodulate the cytotoxic activity of NK cells, which is unexpectedly beneficial as it results in more effective activity against target cells (e.g., tumor cells or other diseased cells).

[0231] In some embodiments, NK cells are positive for cell surface expression of markers selected from the group consisting of CCR7, CD16, CD56, CD57, CD11, CX3CR1, killer Ig-like receptor (KIR), NKp30, NKp44, NKp46, or any combination thereof. In some embodiments, NK cells are positive for cell surface expression of CD16. In some embodiments, NK cells are positive for cell surface expression of CD56. In some embodiments, NK cells are positive for cell surface expression of killer Ig-like receptor.

[0232] Some embodiments of the methods and compositions described herein relate to NK cells engineered to express a CD19-binding CAR. In some embodiments, NK cells are engineered to express a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, NK cells engineered to express a CAR are also engineered to express a membrane-bound interleukin 15 (mbIL15) domain (e.g., by bisistron expression). Thus, in some embodiments, NK cells are engineered to express both CAR and mbIL15 by bisistron expression.

[0233] In some embodiments, NK cells are derived from the cell line NK-92. NK-92 cells are derived from NK cells but retain most of the activating receptors while lacking the major inhibitory receptors exhibited by normal NK cells. Some embodiments of NK-92 cells described herein relate to NK-92 cells that have been engineered to silence certain additional inhibitory receptors, such as SMAD3, and to enable upregulation of interferon-γ (IFNγ), granzyme B, and / or perforin production. Further information regarding the NK-92 cell line is disclosed in WO1998 / 49268 and U.S. Patent Application Publication 2002-0068044, which are incorporated herein by reference in their entirety.

[0234] In some embodiments, NK cells are used in combination with T cells. Therefore, in some embodiments, the immune cells include T cells and NK cells (either from the same donor or from different donors). For example, in one embodiment, primary NK cells are used in combination with primary T cells.

[0235] In any of the provided embodiments, NK cells engineered to express a CD19 CAR are further engineered to express a CAR that binds to an antigen other than CD19. In some embodiments, the antigen is associated with an autoimmune disease. For example, in some embodiments, the genetically engineered NK cells also express a CAR that binds to an antigen selected from the group consisting of BAFF-R, BCMA, CD20, CD22, CD27, CD28, CD33, CD38, CD45, CD47, CD54, CD56, CD81, CD117, CD138, CD200, FcRH5, GPRC5D, and SLAMF7. Accordingly, in some embodiments, NK cells are engineered to express anti-CD19 CARs provided herein, as well as CARs that bind to any one of BAFF-R, BCMA, CD20, CD22, CD27, CD28, CD33, CD38, CD45, CD47, CD54, CD56, CD81, CD117, CD138, CD200, FcRH5, GPRC5D, and SLAMF7. For example, in some embodiments, NK cells are engineered to express CD19-binding CARs and BCMA-binding CARs. Anti-BCMA CARs are known in the Art and include any of those described in PCT application number PCT / US2022 / 073567. In some embodiments, NK cells are engineered to express CD19-binding CARs and BAFF-R-binding CARs. In some embodiments, NK cells are engineered to express CD19-binding CARs and CD20-binding CARs. In some embodiments, NK cells are engineered to express CARs that bind to CD19 and CARs that bind to CD22. In some embodiments, NK cells are engineered to express CARs that bind to CD19 and CARs that bind to CD27. In some embodiments, NK cells are engineered to express CARs that bind to CD19 and CARs that bind to CD28. In some embodiments, NK cells are engineered to express CARs that bind to CD19 and CARs that bind to CD33. In some embodiments, NK cells are engineered to express CARs that bind to CD19 and CARs that bind to CD38.In some embodiments, NK cells are engineered to express CARs that bind to CD19 and CARs that bind to CD45. In some embodiments, NK cells are engineered to express CARs that bind to CD19 and CARs that bind to CD47. In some embodiments, NK cells are engineered to express CARs that bind to CD19 and CARs that bind to CD54. In some embodiments, NK cells are engineered to express CARs that bind to CD19 and CARs that bind to CD56. In some embodiments, NK cells are engineered to express CARs that bind to CD19 and CARs that bind to CD81. In some embodiments, NK cells are engineered to express CARs that bind to CD19 and CARs that bind to CD117. In some embodiments, NK cells are engineered to express CARs that bind to CD19 and CARs that bind to CD138. In some embodiments, NK cells are engineered to express CARs that bind to CD19 and CARs that bind to CD200. In some embodiments, NK cells are engineered to express CARs that bind to CD19 and CARs that bind to FcRH5. In some embodiments, NK cells are engineered to express CARs that bind to CD19 and CARs that bind to GPRC5D. In some embodiments, NK cells are engineered to express CARs that bind to CD19 and CARs that bind to SLAMF7.

[0236] e. Hematopoietic stem cells In some embodiments, the immune cells include hematopoietic stem cells (HSCs). In some embodiments, the HSCs are used in the methods disclosed herein. In some embodiments, the cells are engineered to express a CD19-binding CAR.

[0237] In some embodiments, HSCs are positive for cell surface expression of a marker selected from the group consisting of CD34, CD59, and CD90. In some embodiments, HSCs are positive for cell surface expression of CD34. In some embodiments, HSCs are positive for cell surface expression of CD59. In some embodiments, HSCs are positive for cell surface expression of CD90.

[0238] In some embodiments, allogeneic HSCs are used, while in some embodiments, autologous HSCs are used. In some embodiments, HSCs are used in combination with one or more additional engineered cell types disclosed herein. Some embodiments of the methods and compositions disclosed herein relate to stem cells, e.g., HSCs, engineered to express a CD19-binding CAR, or a nucleic acid encoding a CAR.

[0239] In some embodiments, HSCs are engineered to express the membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, HSCs engineered to express CAR are also engineered to express the membrane-bound interleukin 15 (mbIL15) domain (e.g., bisistronally). Therefore, in some embodiments, HSCs are engineered to bisistronally express both CAR and mbIL15.

[0240] In some embodiments, HSCs are allogeneic cells. In some embodiments, HSCs are obtained from donors who do not have autoimmune diseases.

[0241] f. Induced pluripotent stem cells In some embodiments, immune cells are derived from (or differentiated from) pluripotent stem cells (PSCs). In some embodiments, immune cells derived from induced pluripotent stem cells (iPSCs), such as NK cells, are used in the immunotherapy methods disclosed herein. For example, in some embodiments, NK cells are derived from iPSCs. In some embodiments, induced pluripotent stem cells (iPSCs) are used in the methods disclosed herein. In some embodiments, iPSCs are used to leverage their ability to differentiate and derive into non-pluripotent cells, including but not limited to CD34 cells, hematopoietic endothelial cells, HSCs (hematopoietic stem cells and progenitor cells), hematopoietic pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, NK cells, and B cells (by differentiating iPSCs or poorly differentiated cells containing one or more gene modifications at the same selected site). In some embodiments, iPSCs are used to create iPSC-derived NK cells.

[0242] Some embodiments of the methods and compositions disclosed herein relate to induced pluripotent stem cells engineered to express a CD19-binding CAR. In some embodiments, the iPSCs engineered to express the CAR are also engineered to express membrane-bound interleukin 15 (mbIL15) (e.g., by bisistron expression).

[0243] In some embodiments, the manipulated iPSCs differentiate into NK, T, or other immune cells, such as for use in compositions or methods provided herein. In some embodiments, the manipulated iPSCs differentiate into NK cells.

[0244] C. Preparation of cells for genetic manipulation In some embodiments, the preparation of the manipulated cells includes one or more culture and / or preparation steps. Cells for introducing recombinant receptors (e.g., CARs) may be isolated from a biological sample, such as one obtained from or derived from a subject. In some embodiments, the sample is an apheresis (e.g., leukocyte apheresis) sample.

[0245] In some embodiments, the subjects from whom cells are isolated do not have an autoimmune disease, do not require cell therapy, or are not scheduled to receive cell therapy. In some embodiments, the cells are isolated from subjects different from those who require cell therapy or will receive cell therapy. Therefore, in some embodiments, the cells are allogeneic to the subjects to be administered cell therapy.

[0246] In some embodiments, the subject from which cells are isolated has an autoimmune disease, requires cell therapy, or is being administered cell therapy. In some embodiments, cells are isolated from the subject to which cell therapy is being administered. Therefore, in some embodiments, the cells are autologous to the subject to which they are administered.

[0247] Samples include tissues, bodily fluids, and other samples taken directly from the subject, as well as samples obtained from one or more processing steps such as separation, centrifugation, genetic manipulation (e.g., transduction with a viral vector), washing, and / or incubation. Biological samples may be samples obtained directly from a biological source or processed samples. Biological samples include, but are not limited to, bodily fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, as well as tissue and organ samples (including processed samples derived therefrom).

[0248] In some embodiments, the sample from which cells are derived or isolated is blood or a blood-derived sample, or an apheresis (e.g., leukocyte apheresis) product, or derived therefrom. In some embodiments, cells are isolated from an apheresis (e.g., leukocyte apheresis) sample. Non-limiting samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumors, leukemia, lymphoma, lymph nodes, intestinal-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, small intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organs, and / or cells derived therefrom. In some embodiments, cells are derived from PBMCs. Samples include autologous and allogeneic sources in the context of cell therapy, e.g., adoptive cell therapy.

[0249] In some embodiments, the cells are primary cells, such as human primary cells. In some embodiments, the cells are immune cells, such as primary NK cells.

[0250] In some embodiments, cell isolation involves one or more preparation and / or inaffinity-based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents to, for example, remove unwanted components, concentrate desired components, or lyse or remove cells sensitive to a particular reagent. In some examples, cells are separated based on one or more properties such as density, adhesion, size, sensitivity to and / or resistance to a particular component.

[0251] In some cases, the cells of interest derived from the circulating blood are obtained, for example, by apheresis (e.g., leukocyte apheresis). The sample, in some embodiments, contains lymphocytes including NK cells, T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and / or platelets, and in some embodiments, contains cells other than erythrocytes and platelets.

[0252] In some embodiments, the isolation method includes the separation of different cell types based on the intracellular expression or presence of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is based on affinity or immunoaffinity. For example, isolation in some embodiments includes the separation of cells and cell populations based on the cellular expression or expression level of one or more markers, typically cell surface markers, by incubation with an antibody or binding partner that specifically binds to such markers, and then generally includes a washing step to separate the cells bound to the antibody or binding partner from the cells not bound to the antibody or binding partner.

[0253] Such separation steps can be based on positive selection, where cells bound to the reagent are retained for further use, and / or negative selection, where cells that did not bind to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some embodiments, negative selection may be particularly useful when antibodies that specifically identify cell types in a heterogeneous population are unavailable, and separation is best performed based on markers expressed by cells other than the desired population.

[0254] The isolation process does not necessarily require 100% enrichment or removal of a particular cell population or cells expressing a specific marker. For example, positive selection or enrichment of a particular type of cell, such as cells expressing a marker, refers to increasing the number or percentage of such cells, but does not require the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell, such as cells expressing a marker, refers to decreasing the number or percentage of such cells, but does not require the complete removal of all such cells.

[0255] In some cases, the separation process is performed multiple times, with fractions positively or negatively selected in one step being subjected to further separation steps, such as subsequent positive or negative selection. In some cases, cells expressing multiple markers simultaneously can be depleted in a single separation step by incubating cells with multiple antibodies or binding partners specific to each of the markers to be negatively selected. Similarly, multiple cell types can be positively selected simultaneously by incubating cells with multiple antibodies or binding partners expressed in various cell types.

[0256] For example, in some embodiments, NK cells or specific subpopulations of NK cells, such as those positive for one or more surface markers, e.g., CD56+, CCR7+, CD16+, CD57+, CD11+, CX3CR1+, killer Ig-like receptor (KIR)+, NKp30+, NKp44+, or NKp46+, or cells expressing them at high levels, are isolated by positive selection techniques or negative selection techniques. In some embodiments, NK cells are isolated by positive selection for CD56. For example, CD56+ NK cells may be positively selected using anti-CD56 conjugated magnetic beads.

[0257] In some embodiments, cells (e.g., NK cells) are enlarged in culture before, during, and / or after genetic manipulation. In some embodiments, cells are enlarged in culture before genetic manipulation. In some embodiments, cells are enlarged in culture after genetic manipulation. In some embodiments, cells are enlarged in culture before and after genetic manipulation. Methods for enlarging cells are known in the Art and include any of those described in U.S. Patent Nos. 7,435,596 and 8,026,097; and Japanese Patent Applications Nos. PCT / SG2018 / 050138; PCT / US2020 / 044033; PCT / US2021 / 071330; and PCT / US2022 / 074164.

[0258] In some embodiments, cell expansion in culture involves co-culturing cells with feeder (e.g., stimulant) cells. Therefore, in some embodiments, cells are expanded in culture before genetic manipulation by co-culturing them with feeder cells. In some embodiments, the feeder cells express IL15 (e.g., membrane-bound IL15) and 4-1BBL. In some embodiments, the feeder cells express membrane-bound interleukin 15 (mbIL15) and 4-1BBL. In some embodiments, the feeder cells do not express the MHCII molecule. In some embodiments, the feeder cells do not express the MHCII molecule. In some embodiments, the feeder cells are immune cells. In some embodiments, the feeder cells are K562 cells. Manipulated feeder cells are disclosed, for example, in international patent application PCT / SG2018 / 050138. In some embodiments, after genetic manipulation, the cells can be further expanded in culture.

[0259] In some embodiments, cell expansion in culture involves culturing cells in the presence of IL2, IL12, and / or IL18. In some embodiments, cells are cultured in the presence of IL2. In some embodiments, cells are cultured in the presence of IL12 and IL18. In some embodiments, cells are cultured in the presence of IL2, IL12, and IL18.

[0260] In some embodiments, the preparation method includes a step of freezing, for example, the cells before or after isolation, manipulation, and / or expansion. In some embodiments, the cells are cryopreserved after manipulation. In some embodiments, such as when the cells are further expanded in culture after genetic manipulation, the cells are cryopreserved after further expansion. In some embodiments, the cells are suspended in a cryopreservation solution. In some embodiments, the compositions provided herein are cryopreserved (e.g., before injection into a subject). In some embodiments, any of a variety of known cryopreservation solutions and parameters may be used.

[0261] D. Cell gene editing Provided are methods and uses of genetically modified immune cells and / or compositions thereof, including gene-edited genetically engineered immune cells. In some embodiments, immune cells are gene-edited to increase or decrease the expression of a target protein. In some embodiments, immune cells are gene-edited to increase the expression of a target protein. In some embodiments, immune cells are gene-edited to decrease the expression of a target protein. In some embodiments, the method includes gene-editing immune cells, such as increasing or decreasing the expression of a target protein. In some embodiments, the method includes gene-editing immune cells to increase the expression of a target protein. In some embodiments, the method includes gene-editing immune cells to decrease the expression of a target protein. The expression of a target protein can be reduced by disrupting the gene encoding the target protein or a portion thereof (the target gene).

[0262] Immune cells are intended to be capable of gene editing at any point before, during, and / or after genetic manipulation. In some embodiments, immune cells are gene-edited before genetic manipulation. In some embodiments, immune cells are gene-edited concurrently with genetic manipulation. In some embodiments, immune cells are gene-edited after genetic manipulation.

[0263] As discussed below, in some embodiments, gene editing is used to reduce or eliminate the expression of a target protein, for example, by disrupting a protein-coding gene. In some embodiments, gene editing can reduce the transcription of a target gene by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amount between those listed). In some embodiments, gene editing reduces the transcription of a target gene by at least about 30%. In some embodiments, gene editing reduces the transcription of a target gene by at least about 40%. In some embodiments, gene editing reduces the transcription of a target gene by at least about 50%. In some embodiments, gene editing reduces the transcription of a target gene by at least about 60%. In some embodiments, gene editing reduces the transcription of a target gene by at least about 70%. In some embodiments, gene editing reduces the transcription of a target gene by at least about 80%. In some embodiments, gene editing reduces the transcription of the target gene by at least about 90%. In some embodiments, gene editing reduces the transcription of the target gene by at least about 95%. In some embodiments, gene editing reduces the transcription of the target gene by at least about 99%. In some embodiments, the gene is completely knocked out, and the transcription of the target gene is eliminated (undetectable).

[0264] In some embodiments, gene editing can reduce the expression of a target protein by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amount between those listed). In some embodiments, gene editing reduces the expression of the target protein by at least about 30%. In some embodiments, gene editing reduces the expression of the target protein by at least about 40%. In some embodiments, gene editing reduces the expression of the target protein by at least about 50%. In some embodiments, gene editing reduces the expression of the target protein by at least about 60%. In some embodiments, gene editing reduces the expression of the target protein by at least about 70%. In some embodiments, gene editing reduces the expression of the target protein by at least about 80%. In some embodiments, gene editing reduces the expression of the target protein by at least about 90%. In some embodiments, gene editing reduces the expression of the target protein by at least about 95%. In some embodiments, gene editing reduces the expression of the target protein by at least approximately 99%. In some embodiments, the gene is completely knocked out, and the expression of the target protein is eliminated (undetectable).

[0265] In some embodiments, gene editing is used to “knock in” a target gene or otherwise increase its transcription. In some embodiments, the transcription of the target gene increases by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amount between those enumerated). In some embodiments, the transcription of the target gene increases by at least about 30%. In some embodiments, the transcription of the target gene increases by at least about 40%. In some embodiments, the transcription of the target gene increases by at least about 50%. In some embodiments, the transcription of the target gene increases by at least about 60%. In some embodiments, the transcription of the target gene increases by at least about 70%. In some embodiments, the transcription of the target gene increases by at least about 80%. In some embodiments, the transcription of the target gene increases by at least about 90%. In some embodiments, the transcription of the target gene increases by at least about 100%.

[0266] In some embodiments, gene editing is used to “knock in” or otherwise enhance the expression of a target protein. In some embodiments, the expression of the target protein can be enhanced by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amount between those enumerated). In some embodiments, the expression of the target protein is increased by at least about 30%. In some embodiments, the expression of the target protein is increased by at least about 40%. In some embodiments, the expression of the target protein is increased by at least about 50%. In some embodiments, the expression of the target protein is increased by at least about 60%. In some embodiments, the expression of the target protein is increased by at least about 70%. In some embodiments, the expression of the target protein is increased by at least about 80%. In some embodiments, the expression of the target protein is increased by at least about 90%. In some embodiments, the expression of the target protein is increased by at least about 100%.

[0267] As will be discussed in more detail below, various approaches can be employed in a given embodiment to improve or modify one or more properties of immune cells. Gene editing can be used to reduce, eliminate (e.g., knock out), or increase the expression of a target gene. For example, the transcription of a target gene and / or the translation of the protein encoded by the target gene (e.g., target protein) can be reduced, eliminated (e.g., knocked out), or increased. The target gene may be involved in the immune function of the cell or be part of a signaling pathway in which an increase or decrease in function is desired. Various gene targets are further detailed below. Disrupting specific genes in immune cells (e.g., NK cells) can increase the activity and / or persistence of those immune cells.

[0268] i. Methods of gene editing In some embodiments, gene editing of a target gene (whether knockout or knock-in) is achieved by targeted introduction of DNA breaks and subsequent DNA repair mechanisms. In some embodiments, DNA double-strand breaks are repaired by non-homologous end joining (NHEJ), in which enzymes are used to directly join the DNA ends to repair the break. NHEJ is an error-prone process. Generally, without a repair template, the NHEJ process religates the ends of the broken DNA strand, often resulting in nucleotide deletions and insertions at the break site. However, in some embodiments, double-strand breaks are repaired by homology-directed repair (HDR), which is more accurate and has the advantage of enabling sequence-specific breaks and repairs. HDR uses a homologous sequence as a template to reconstruct the DNA sequence missing at the break point, such as a vector having the desired gene element (e.g., an insertion element to disrupt the coding sequence of a TCR subunit) within a sequence homologous to the adjacent sequence of the double-strand break. As a result, the desired change (e.g., insertion) is inserted into the DSB region. The HDR path can be induced by a canonical HDR path or an alternative HDR path. Unless otherwise specified, the terms “HDR” or “homology-directed restoration” as used herein encompass both canonical HDR and alternative HDR.

[0269] Canonical HDR, or "canonical homologous-directed repair" or "cHDR," are used interchangeably and refer to the process of repairing DNA damage using homologous nucleic acids (e.g., endogenous homologous sequences such as sister chromatids; or exogenous nucleic acids such as donor templates). Canonical HDR typically acts when a large break occurs in a double-stranded break (DSB) and at least one single-stranded portion of the DNA is formed. In normal cells, canonical HDR typically involves a series of steps including break recognition, break stabilization, excision, single-stranded DNA stabilization, DNA crossover intermediate formation, crossover intermediate resolution, and ligation. The canonical HDR process requires RAD51 and BRCA2, and the homologous nucleic acid, e.g., repair template, is typically double-stranded. In canonical HDRs, a double-stranded polynucleotide, such as a double-strand repair template, is introduced, which contains a sequence homologous to the target sequence and is either directly incorporated into the target sequence or used as a template for inserting the repair template sequence or a portion of it into the target gene. After the excision of the break, repair can proceed via various pathways, such as the double Holliday junction model (also known as the double-strand break repair pathway or DSBR pathway) or the synthesis-dependent strand annealing (SDSA) pathway.

[0270] In the double Holliday junction model, strand intrusion occurs due to two single-stranded overhangs of the target sequence into homologous sequences in a double-stranded polynucleotide, such as a double-stranded donor template, resulting in the formation of an intermediate with two Holliday junctions. As new DNA is synthesized from the end of the invading strand to fill the gap resulting from the excision, the junction shifts. The end of the newly synthesized DNA is ligated to the excised end, the junction is resolved, and as a result, it is inserted into the target sequence, or a portion of the target sequence containing a gene variant. Crossover with polynucleotides, such as repair templates, can occur during junction resolution.

[0271] In the SDSA pathway, only single-stranded overhangs enter the polynucleotide (e.g., donor template), and new DNA is synthesized from the end of the entered strand to fill the gap created by the excision. Next, the newly synthesized DNA anneals to the remaining single-stranded overhang, new DNA is synthesized to fill the gap, and the strands are ligated to produce a modified DNA double helix.

[0272] Alternative HDR, or "alternative homologous directive repair," or "alternative HDR," is used interchangeably and, in some embodiments, refers to a process that repairs DNA damage using homologous nucleic acids (e.g., endogenous homologous sequences such as sister chromatids; or exogenous nucleic acids such as repair templates). Alternative HDR differs from canonical HDR in that its process utilizes a different pathway and may be inhibited by mediators of canonical HDR, such as RAD51 and BRCA2. Furthermore, alternative HDR is also distinguished by the involvement of single-stranded or nicked homologous nucleic acid templates, such as repair templates, while canonical HDR generally involves double-stranded homologous templates. In the alternative HDR pathway, a single-stranded template polynucleotide, such as a repair template, is introduced. A nick, single-strand break, or DSB at the cleavage site is mediated by a nuclease molecule to alter a desired target site, such as a gene variant of a target gene, resulting in excision at the cleavage site and the appearance of a single-stranded overhang. The incorporation of template polynucleotides, such as repair template sequences, to alter target sites on DNA typically occurs via the SDSA pathway, as described herein. In some embodiments, HDR is performed by introducing one or more agents capable of inducing DSBs and repair templates, such as single-stranded oligonucleotides, into cells. The introduction can be carried out by any suitable delivery. The conditions that enable HDR can be any conditions suitable for performing HDR in cells.

[0273] In some embodiments, gene editing is achieved by one or more of various engineered nucleases. In some embodiments, restriction enzymes are used, particularly when double-strand breaks are desired in multiple regions. In some embodiments, bioengineered nucleases are used. Depending on the embodiment, one or more zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases and / or clustered regularly spaced short-chain palindromic repeat (CRISPR / Cas9) systems are used to specifically edit a gene encoding one or more TCR subunits.

[0274] Meganucleases are characterized by their ability to recognize and cleave large DNA sequences (14–40 base pairs). In some embodiments, meganucleases from the LAGLIDADG family are used and subjected to mutagenesis and screening to generate meganuclease mutants that recognize specific sequences, such as specific sites in target genes, or other target genes disclosed herein. In some embodiments, two or more meganucleases or their functional fragments are fused to create hybrid enzymes that recognize desired target sequences within target genes.

[0275] In contrast to meganucleases, ZFNs and TALENs function based on a nonspecific DNA cleavage catalytic domain linked to a specific DNA sequence-recognizing peptide, such as a zinc finger or transcription activator-like effector (TALE). Therefore, advantageously, ZFNs and TALENs enable sequence-independent cleavage of DNA and possess a high degree of sequence specificity in target recognition. Zinc finger motifs function naturally in transcription factors to recognize specific DNA sequences for transcription. The C-terminal portion of each finger plays a role in specifically recognizing the DNA sequence. While the sequences recognized by ZFNs are relatively short (e.g., about 3 base pairs), in some embodiments, combinations of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more zinc fingers with characterized recognition sites are used, thereby enabling targeting of specific sequences. The combined ZFNs are then fused with the catalytic domain of an endonuclease such as FokI (FokI heterodimer as appropriate) to induce target DNA cleavage.

[0276] Transcription activator-like effector nucleases (TALENs) are specific DNA-binding proteins characterized by a series of 33 or 34 amino acid repeats. Similar to ZFNs, TALENs are fusions of a nuclease's DNA-cleavage domain and TALE domain, enabling sequence-independent introduction of double-strand DNA breaks through highly accurate target site recognition. TALENs can induce double-strand breaks at target sites, which are repaired by error-prone non-homologous end junctions (NHEJs), resulting in gene disruption through the introduction of small insertions or deletions. Advantageously, TALENs are used in several embodiments, at least partially, due to their high specificity in DNA binding, reduced off-target effects, and ease of constructing the DNA-binding domain.

[0277] CRISPR (Clustered Regular Interval Short-Chain Palindromic Repeats) is a genetic element used by bacteria to protect themselves from viruses. Repeats are short sequences derived from the viral genome and incorporated into the bacterial genome. Cas (CRISPR-associated protein) processes these sequences and cleaves matching viral DNA sequences. By introducing a plasmid containing the Cas gene and specifically constructed CRISPR into eukaryotic cells, the eukaryotic genome can be cleaved at any desired location. Additional information regarding CRISPR can be found in U.S. Patent Publication 2014 / 0068797, which is incorporated herein by reference.

[0278] In some embodiments, CRISPR is used to disrupt a target gene. Depending on the embodiment and which target gene is edited, a class 1 or class 2 Cas is used. In some embodiments, a class 1 Cas is used, and the Cas type is selected from the following types: I, IA, IB, IC, ID, IE, IF, IU, III, IIIA, IIIB, IIIC, IIID, IV, IVA, IVB, and combinations thereof. In some embodiments, the Cas is selected from the group consisting of Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1, and combinations thereof. In some embodiments, the Cas is Cas3. In some embodiments, a class 2 Cas is used, and the Cas type is selected from the following types: II, IIA, IIB, IIC, V, VI, and combinations thereof. In some embodiments, the Cas is selected from the group consisting of Cas9, Csn2, Cas4, Cas12a (formerly known as Cpf1), C2c1, C2c3, Cas13a (formerly known as C2c2), Cas13b, Cas13c, CasX, CasY, and combinations thereof. In some embodiments, the Cas is Cas9. In some embodiments, a class 2 CasX is used, and CasX can form a complex with a guide nucleic acid, and the complex can bind to target DNA, and the target DNA includes a non-target strand and a target strand. In some embodiments, a class 2 CasY is used, and CasY can bind to and modify the target nucleic acid and / or polypeptides associated with the target nucleic acid.

[0279] ii. Target genes In some embodiments, immune cells are gene-edited in target genes. In some embodiments, editing of target genes advantageously confers enhanced growth, cytotoxicity, and / or persistence to the edited cells. For example, in some embodiments, immune cells are gene-edited to increase IL-15 levels and / or signaling. While we do not wish to be bound by theory, it is conceivable that gene-editing cells to increase IL-15 levels and / or signaling could eliminate the need to provide the subject with fludarabine-containing lymphocyte apheresis before administering the genetically modified cells. Specifically, in immune cells gene-edited to increase IL-15 levels and / or signaling, the increased bioavailability of IL-15 mediated by fludarabine is not intended. Therefore, in some embodiments, immune cells (e.g., NK cells) are gene-edited in target genes to increase IL-15 levels, for example, by reducing or eliminating the expression of cytokine-induced SH2-containing protein (Cis) (e.g., by disrupting the CISH gene encoding Cis). In some embodiments, immune cells (e.g., NK cells) are genetically edited in target genes to increase IL15 signaling, for example, by reducing or eliminating the expression of cytokine-induced SH2-containing protein (Cis) (e.g., by disrupting the CISH gene encoding Cis).

[0280] As a non-limiting example, IL-15 is a positive regulator of NK cells and can enhance one or more of the following: NK cell homing, NK cell migration, NK cell expansion / proliferation, NK cell cytotoxicity, and / or NK cell persistence, as disclosed herein. In CD8+ T cells, CISH has been shown to actively silence TCR signaling to maintain tumor tolerance, and CISH is a downstream negative regulator of IL-15 receptor signaling (Palmer et al., J. Exp. Med. (2015) 212(12):2095-2113). In NK cells and T cells, CISH plays a role in checkpoint maturation and proliferation (Delconte et al., Nature Immunol (2016) 17:816-24). Thus, according to some embodiments, gene editing of CISH can increase the persistence, proliferation, and / or cytotoxicity of immune cells (e.g., NK cells), or otherwise enhance their efficacy, as disclosed herein.

[0281] In some embodiments, CISH gene editing activates or inhibits a wide variety of pathways. CIS proteins act as negative regulators of IL15 signaling, for example, by inhibiting the JAK-STAT signaling pathway. These pathways typically lead to the transcription of IL15-responsive genes (including CISH). In some embodiments, disruption of CISH deinhibits JAK-STAT (e.g., JAK1-STAT5) signaling and enhances the transcription of IL15-responsive genes. In some embodiments, disruption of CISH results in enhanced signaling via mammalian target of rapamycin (mTOR), and consequently, increased gene expression related to cellular metabolism and respiration. In some embodiments, disruption of CISH results in an IL15-induced increase in the expression of IL-2Rα (CD25) rather than IL-15Rα or IL-2 / 15Rβ, enhanced NK cell membrane binding of IL15 and / or IL2, increased phosphorylation of STAT-3 and / or STAT-5, and increased expression of anti-apoptotic proteins such as Bcl-2. In some embodiments, CISH disruption results in IL15-induced upregulation of selected genes related to mitochondrial function (e.g., electron transport chain and cellular respiration) and the cell cycle. Thus, in some embodiments, gene-edited CISH disruption enhances the cytotoxicity and / or persistence of NK cells, at least partially through metabolic reprogramming. In some embodiments, negative regulators of cellular metabolism, such as TXNIP, are downregulated in response to CISH disruption. In some embodiments, promoters for cell survival and proliferation, including BIRC5 (Survivin), TOP2A, CKS2, and RACGAP1, are upregulated after CISH disruption, while antiproliferative or pro-apoptotic proteins, such as TGFB1, ATM, and PTCH1, are downregulated.In some embodiments, CISH disruption alters the state of signaling by or through one or more of the following: CXCL-10, IL2, TNF, IFNg, IL13, IL4, Jnk, PRF1, STAT5, PRKCQ, IL2 receptor beta, SOCS2, MYD88, STAT3, STAT1, TBX21, LCK, JAK3, IL& receptor, ABL1, IL9, STAT5A, STAT5B, Tcf7, PRDM1, and / or EOMES (e.g., activation or inactivation).

[0282] In some embodiments, CISH editing confers an enhanced ability to homing NK cells to target sites. In some embodiments, CISH editing confers an enhanced ability to NK cells to move within tissues in response to chemoattractants, for example, or to detach from repellents. In some embodiments, CISH editing confers an enhanced activation ability to NK cells, thereby enabling them to exert antitumor effects, for example. In some embodiments, CISH editing confers an enhanced proliferative capacity to NK cells, thereby enabling, in some embodiments, to generate a robust number of NK cells from donor blood samples. Furthermore, in such embodiments, NK cells edited for CISH and manipulated to express CARs are more easily, robustly, and consistently expanded in culture. In some embodiments, CISH gene editing confers enhanced cytotoxicity to NK cells. In some embodiments, editing of CISH synergistically enhances the cytotoxic effect of immune cells expressing CARs.

[0283] In some embodiments, CISH expression is knocked down or knocked out by gene editing of the CISH gene, for example, using CRISPR-Cas editing. Thus, in some embodiments, immune cells (e.g., NK cells) are gene-edited at the CISH gene. In other embodiments, small interfering RNAs, antisense RNAs, TALENs, or zinc fingers are used. Information regarding CISH editing is described, for example, in International Patent Applications PCT / US2023 / 060850 and PCT / US2020 / 035752, which are incorporated herein by reference in their entirety.

[0284] In some embodiments, gene editing reduces CISH transcription by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amount between those enumerated). In some embodiments, gene editing reduces CISH transcription by at least about 30%. In some embodiments, gene editing reduces CISH transcription by at least about 40%. In some embodiments, gene editing reduces CISH transcription by at least about 50%. In some embodiments, gene editing reduces CISH transcription by at least about 60%. In some embodiments, gene editing reduces CISH transcription by at least about 70%. In some embodiments, gene editing reduces CISH transcription by at least about 80%. In some embodiments, gene editing reduces CISH transcription by at least about 90%. In some embodiments, gene editing reduces CISH transcription by at least about 95%. In some embodiments, gene editing reduces CISH transcription by at least about 99%.

[0285] In some embodiments, gene editing can reduce Cis expression by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amount between those listed). In some embodiments, gene editing reduces Cis expression by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amount between those listed). In some embodiments, gene editing reduces Cis expression by at least about 30%. In some embodiments, gene editing reduces Cis expression by at least about 40%. In some embodiments, gene editing reduces Cis expression by at least about 50%. In some embodiments, gene editing reduces Cis expression by at least about 60%. In some embodiments, gene editing reduces Cis expression by at least about 70%. In some embodiments, gene editing reduces Cis expression by at least about 80%. In some embodiments, gene editing reduces Cis expression by at least about 90%. In some embodiments, gene editing reduces Cis expression by at least about 95%. In some embodiments, gene editing reduces Cis expression by at least about 99%.

[0286] In some embodiments, immune cells (e.g., NK cells) are gene-edited to reduce or eliminate the expression of alternative or additional target genes, such as transforming growth factor-beta receptor 2 protein (TGFbR2) and / or Casitas B lineage lymphoma-b protein (Cbl-b). In some embodiments, immune cells are gene-edited to reduce the expression of TGFbR2. In some embodiments, immune cells are gene-edited to reduce the expression of Cbl-b. The expression of any other target protein, or any combination of target proteins, can be reduced or eliminated, for example, by disrupting the gene encoding the target protein.

[0287] II. Compositions and Formulations Also provided are compositions (including pharmaceutical compositions and formulations) comprising genetically engineered immune cells (e.g., NK cells) that express CD-19-targeting CARs. Also provided are compositions (including pharmaceutical compositions and formulations) comprising genetically engineered NK cells that express any of the CD19-targeting CARs described herein.

[0288] Provided are pharmaceutical formulations comprising genetically engineered NK cells expressing CD19-directed CARs, multiple genetically engineered NK cells expressing CD19-directed CARs, and / or additional agents for concomitant treatment or therapy. Pharmaceutical compositions and formulations generally comprise one or more pharmaceutically acceptable carriers or excipients. In some embodiments, the composition comprises at least one additional therapeutic agent.

[0289] The term "pharmaceutical preparation" refers to a preparation that is in a form that enables the biological activity of the active ingredient contained therein, and that does not contain any additional ingredients that are unacceptably toxic to the person to whom the preparation is administered.

[0290] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical preparation other than the active ingredient that is non-toxic to the target substance. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0291] In some embodiments, the choice of carrier is partially determined by the specific cells, binding molecules, and / or antibodies, and / or the method of administration. Thus, a variety of suitable formulations exist. For example, the pharmaceutical composition may contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The preservative or mixture thereof is typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Pharmacochemically acceptable carriers are generally non-toxic to the recipient at the dosage and concentration used and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); and low molecular weight (approximately 10 residues). Polypeptides (less than 100%); proteins, e.g., serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, e.g., sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, e.g., polyethylene glycol (PEG).

[0292] In some embodiments, a buffer is included in the composition. Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffers is used. The buffer or mixture is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administerable pharmaceutical compositions are known.

[0293] The antibody formulations described herein may include lyophilized formulations and aqueous solutions. The formulation or composition may also contain two or more active ingredients useful for a specific indication, disease, or condition treated with the binding molecule or cell, preferably active ingredients having complementary activity to the binding molecule or cell, such that their activities do not adversely affect each other. Such active ingredients are preferably present in combination in amounts effective for the intended purpose. Accordingly, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., methotrexate or rituximab. In some embodiments, cells or antibodies are administered in the form of salts, e.g., pharmaceutically acceptable salts. Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, sulfuric acid, and organic acids such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, and aryl sulfonic acids, e.g., p-toluenesulfonic acid.

[0294] The pharmaceutical composition contains, in some embodiments, manipulated cells in an amount effective to treat or prevent a disease or condition, such as a therapeutically or prophylactically effective amount. Therapeutic or prophylactic effectiveness is monitored, in some embodiments, by periodic evaluation of the treated subject. Depending on the condition, repeated administration over several days or more is necessary until the desired suppression of disease symptoms is achieved. However, other drug regimens may also be useful and can be determined. The desired dosage may be delivered by a single bolus, multiple bolus, or sequential infusion of the composition.

[0295] Formulations include those for oral administration, intravenous administration, intraperitoneal administration, subcutaneous administration, pulmonary administration, transdermal administration, intramuscular administration, intranasal administration, buccal administration, sublingual administration, or suppository administration. In some embodiments, the drug or cell population is administered to the target by intravenous, intraperitoneal, or subcutaneous injection using peripheral systemic delivery.

[0296] In some embodiments, the composition is provided as a sterile liquid formulation (e.g., an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition), which in some embodiments may be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions are somewhat more convenient to administer, particularly by injection. The liquid composition may contain a carrier, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0297] Sterile injectable solutions can be prepared by incorporating a drug or cells into a mixture with a solvent, such as a suitable carrier, diluent, or excipient (e.g., sterile water, saline, glucose, dextrose, etc.). Formulations for in vivo administration are typically sterile. Sterility can be easily achieved, for example, by filtration through a sterile filtration membrane. In some embodiments, the dose of manipulated cells to be administered is contained in a cryopreserved composition. In some embodiments, the composition is administered after thawing the cryopreserved composition.

[0298] III. Methods and Use Provided herein are methods and uses of genetically modified NK cells, as well as their pharmaceutical compositions and formulations, for purposes such as the treatment of autoimmune diseases (e.g., SLE) involving any of the CD19-related conditions. Also provided herein are methods and uses of genetically modified NK cells, as well as their pharmaceutical compositions and formulations, for purposes such as reducing CD19-expressing cells (e.g., B cells) in subjects having a disease or condition involving any of the CD19-related conditions. Also provided herein are methods and uses of genetically modified NK cells, as well as their pharmaceutical compositions and formulations, for purposes such as reducing the level of autoantibodies in subjects having a B cell-mediated condition (e.g., autoimmune disease).

[0299] In some embodiments, what is also provided is a method for reconfiguring a B cell compartment (e.g., a peripheral B cell compartment) in an object having a B cell-mediated state. In some embodiments, the reconfiguration of the B cell compartment is achieved when the majority of regrowing B cells in an object treated by the method provided herein are non-class-switched (e.g., IgM and / or IgD isotype) B cells. In some embodiments, more than 70%, more than 80%, more than 90%, or more than 95% of regrowing B cells in an object treated by the method provided herein are non-class-switched (e.g., IgM and / or IgD isotype). In some embodiments, more than 70% of regrowing B cells in an object treated by the method provided herein are non-class-switched (e.g., IgM and / or IgD isotype). In some embodiments, more than 80% of regrowing B cells in an object treated by the method provided herein are non-class-switched (e.g., IgM and / or IgD isotype). In some embodiments, more than 90% of the regrowing B cells in subjects treated by the methods provided herein are non-class-switched (e.g., IgM and / or IgD isotypes). In some embodiments, more than 95% of the regrowing B cells in subjects treated by the methods provided herein are non-class-switched (e.g., IgM and / or IgD isotypes). In some embodiments, the isotype of the regrowing B cells is evaluated about 30 days, about 45 days, about 60 days, about 75 days, or about 90 days after administration of the last dose of the composition containing immune cells expressing anti-CD19 CAR. In some embodiments, the isotype of the regrowing B cells is evaluated about 30 days after administration of the last dose of the composition containing immune cells expressing anti-CD19 CAR. In some embodiments, the isotype of the regrowing B cells is evaluated about 45 days after administration of the last dose of the composition containing immune cells expressing anti-CD19 CAR. In some embodiments, the isotype of regrowing B cells is evaluated approximately 60 days after administration of the last dose of the composition containing immune cells expressing anti-CD19 CAR.In some embodiments, the isotype of regrowing B cells is evaluated approximately 75 days after administration of the last dose of the composition containing immune cells expressing anti-CD19 CAR. In some embodiments, the isotype of regrowing B cells is evaluated approximately 90 days after administration of the last dose of the composition containing immune cells expressing anti-CD19 CAR. In some embodiments, the regrowing B cells are regrowing peripheral B cells.

[0300] Such methods, for example, treatment methods and uses, include administering genetically modified NK cells, such as multiple genetically modified NK cells expressing a provided anti-CD19 recombinant receptor (e.g., CAR), to a subject. Such methods and uses include, for example, therapeutic methods and uses, the administration of genetically modified NK cells or compositions containing them to a subject having a B-cell mediated disease, such as an autoimmune disease. Thus, such methods and uses also include therapeutic methods and uses, for example, the administration of genetically modified NK cells or compositions containing them to a subject having an autoimmune disease (e.g., SLE). In some embodiments, the cells and / or compositions are administered in an amount effective for treating the disease. Such methods and uses also include therapeutic methods and uses, for example, the administration of genetically modified NK cells or compositions containing them to a subject determined to be at risk of autoimmune disease or at risk of recurrence thereof. In some embodiments, the subject is determined to be at risk of autoimmune disease. Thus, in some embodiments, the composition is administered in an amount effective for preventing the disease. In some embodiments, the subject is determined to be at risk of recurrence of autoimmune disease. Therefore, in some embodiments, the composition is administered in an amount effective in preventing disease relapse (e.g., recurrence).

[0301] Also provided herein are such methods and procedures, and the use of genetically modified NK cells in the preparation of pharmaceuticals for carrying out such therapeutic methods. In some embodiments, the method is carried out by administering cells or compositions containing them to a subject who has, had, or is suspected of having a B cell-mediated disease (e.g., an autoimmune disease). Thus, in some embodiments, the method is carried out by administering cells or compositions containing them to a subject who has, had, or is suspected of having an autoimmune disease. In some embodiments, the method or use thereby treats the autoimmune disease in the subject. Also provided herein are the use of any of the compositions, such as the pharmaceutical compositions provided herein, for the treatment of autoimmune diseases, such as their use in treatment regimens.

[0302] As used herein, “treatment” (and its grammatical variations such as “treat” or “treating”) means the complete or partial improvement or reduction of a disease, condition, or disorder, or any associated symptoms, adverse effects, outcomes, or phenotype. Desired effects of treatment include, but are not limited to, symptom relief, attenuation of any direct or indirect pathological consequences of the disease, a decrease in the rate of disease progression, improvement or mitigation of the disease state, and remission or improved prognosis. In some embodiments, desired effects of treatment include partial renal remission (PRR), complete renal remission (CRR), a reduction in the number of relapses, and / or an extension of the interval between relapses. These terms do not imply a complete cure of the disease, or the complete elimination of any symptoms, or an effect on all symptoms or outcomes.

[0303] As used herein, “delaying the onset of disease” means delaying, preventing, slowing, stabilizing, suppressing, and / or postponing the onset of a disease (e.g., SLE). This delay can be of varying lengths depending on the disease and / or the medical history of the subject being treated. In some embodiments, the molecules and compositions provided are used to delay the onset of disease or to slow the progression of disease. Sufficient or substantial delay may effectively encompass prevention, in the sense that the subject does not develop the disease.

[0304] As used herein, "preventing" includes providing prevention with respect to the onset or recurrence of a disease in subjects who may be predisposed to the disease but have not yet been diagnosed with the disease.

[0305] As used herein, “suppress” a function or activity means reducing the function or activity compared to other identical conditions except for the condition or parameter of interest, or compared to a different condition.

[0306] The “effective amount” of a drug, such as a pharmaceutical preparation, binding molecule, antibody, cell, or composition, in the context of administration, refers to the amount / quantity and duration of medication that is effective in achieving the desired outcome, such as a therapeutic or prophylactic result.

[0307] The “therapeutic effective amount” of a drug, such as a pharmaceutical formulation, binding molecule, antibody, cell, or composition, refers to the amount effective in the dosage and duration required to achieve a desired therapeutic outcome, such as the treatment of a disease, condition, or disorder, and / or the pharmacokinetic or pharmacodynamic effects of the treatment. The therapeutic effective amount may vary depending on factors such as the disease condition of the subject, age, sex, and weight, and the cell population to be administered. In some embodiments, the methods provided include administering an effective amount, such as a therapeutic effective amount of cells and / or composition.

[0308] The “preventively effective dose” refers to the amount of medication that is effective in the dosage and duration required to achieve the desired preventive outcome. Typically, though not always, the preventive dose is less than the therapeutically effective dose because the preventive dose is used in subjects in the pre- or early stages of the disease. Alternatively, the preventive dose may be used in subjects to prevent recurrence of the disease (e.g., relapse), in which case the preventively effective dose may be the same as or equal to the therapeutically effective dose.

[0309] As used herein, “Subject” or “Individual” means a mammal. In some embodiments, “mammal” includes humans, non-human primates, livestock and farm animals, as well as zoo animals, game animals, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, monkeys, etc. In some embodiments, the subject is a human. In some embodiments, the subject is a human at least 18 years of age. In some embodiments, the subject is a human at least 12 years of age.

[0310] Methods for administering cells for cell therapy are known and may be used in connection with the methods and compositions provided.

[0311] The diseases or conditions treated may be any in which the expression of an antigen is associated with and / or involved in the pathogenesis of a disease state or disorder, for example, any in which the antigen causes, exacerbates, or otherwise contributes to such a disease, condition, or disorder. Non-limiting examples of diseases and conditions include B cell-mediated diseases and / or autoimmune diseases. Non-limiting examples of antigens include antigens associated with various diseases and conditions that may be treated, such as CD19. Among the diseases treated are any autoimmune diseases in which CD19 is associated with and / or involved in the pathogenesis of the disease.

[0312] In some embodiments, autoimmune diseases include, but are not limited to, systemic lupus erythematosus (SLE), lupus nephritis (LN), and lupus including CNS lupus, inflammatory bowel disease (IBD, e.g., Crohn's disease or ulcerative colitis), rheumatoid arthritis (RA; e.g., juvenile rheumatoid arthritis), ANCA-associated vasculitis, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, Chagas disease, Grave's disease, Wegener's granulomatosis, polyarteritis nodosa, Sjögren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis (MS), psoriasis, IgA nephropathy, IgM polyneuritis, vasculitis, diabetes mellitus, Raynaud's syndrome, antiphospholipid syndrome, Goodpasture disease, Kawasaki disease, autoimmune hemolytic anemia, myasthenia gravis (MG), or progressive glomerulonephritis. In some embodiments, the subject may exhibit symptoms of two or more autoimmune diseases. In some embodiments, the autoimmune disease is systemic lupus erythematosus (SLE). In some embodiments, the autoimmune disease is SLE with renal involvement (e.g., lupus nephritis (LN)). In some embodiments, the autoimmune disease is lupus nephritis (LN). In some embodiments, the autoimmune disease is CNS lupus. In some embodiments, the autoimmune disease is IBD. In some embodiments, the autoimmune disease is Crohn's disease. In some embodiments, the autoimmune disease is ulcerative colitis. In some embodiments, the autoimmune disease is vasculitis. In some embodiments, the autoimmune disease is ANCA vasculitis (AAV). In some embodiments, the autoimmune disease is autoimmune encephalitis (AE). In some embodiments, the autoimmune disease is ITP. In some embodiments, the autoimmune disease is TTP. In some embodiments, the autoimmune disease is autoimmune thrombocytopenia. In some embodiments, the autoimmune disease is Chagas disease. In some embodiments, the autoimmune disease is Grave's disease. In some embodiments, the autoimmune disease is Wegener's granulomatosis. In some embodiments, the autoimmune disease is polyarteritis nodosa. In some embodiments, the autoimmune disease is Sjögren's syndrome. In some embodiments, the autoimmune disease is pemphigus vulgaris. In some embodiments, the autoimmune disease is psoriasis. In some embodiments, the autoimmune disease is IgA nephropathy.In some embodiments, the autoimmune disease is membranous nephropathy (MN). In some embodiments, the autoimmune disease is IgM polyneuropathy. In some embodiments, the autoimmune disease is vasculitis. In some embodiments, the autoimmune disease is diabetes mellitus. In some embodiments, the autoimmune disease is Raynaud's syndrome. In some embodiments, the autoimmune disease is antiphospholipid syndrome. In some embodiments, the autoimmune disease is Goodpasture's disease. In some embodiments, the autoimmune disease is Kawasaki disease. In some embodiments, the autoimmune disease is autoimmune hemolytic anemia. In some embodiments, the autoimmune disease is myasthenia gravis (MG). In some embodiments, the autoimmune disease is progressive glomerulonephritis. In some embodiments, the autoimmune disease is acquired immunodeficiency syndrome (AIDS). In some embodiments, the autoimmune disease is Addison's disease. In some embodiments, the autoimmune disease is alopecia areata. In some embodiments, the autoimmune disease is celiac disease. In some embodiments, the autoimmune disease is chronic inflammatory demyelinating polyneuropathy (CIDP). In some embodiments, the autoimmune disease is Guillain-Barré syndrome. In some embodiments, the autoimmune disease is Hashimoto's thyroiditis. In some embodiments, the autoimmune disease is pernicious anemia. In some embodiments, the autoimmune disease is psoriasis. In some embodiments, the autoimmune disease is psoriatic arthritis. In some embodiments, the autoimmune disease is reactive arthritis. In some embodiments, the autoimmune disease is rheumatoid arthritis (RA). In some embodiments, the autoimmune disease is refractory RA. In some embodiments, RA is refractory to TNF inhibitors. In some embodiments, the autoimmune disease is multiple sclerosis (MS). In some embodiments, MS is primary progressive MS (PPMS). In some embodiments, MS is secondary progressive MS (SPMS). In some embodiments, MS is relapsing-remitting MS (RRMS). In some embodiments, the subject does not have antiphospholipid syndrome.

[0313] In some embodiments, the autoimmune disease includes scleroderma. In some embodiments, the autoimmune disease is scleroderma. In some embodiments, the autoimmune disease includes systemic sclerosis (also known as systemic scleroderma). In some embodiments, the autoimmune disease is systemic sclerosis (also known as systemic scleroderma). In some embodiments, the autoimmune disease includes focal scleroderma. In some embodiments, the autoimmune disease is focal scleroderma.

[0314] In some embodiments, the autoimmune disease includes myositis (also known as IIM). In some embodiments, the autoimmune disease is myositis (also known as IIM). In some embodiments, the autoimmune disease is selected from the group consisting of anti-synthetase syndrome (ASSD), overlap myopathy (OM), dermatomyositis (DM), clinical myopathic dermatomyositis, juvenile myositis (JM), necrotizing myositis (NM; e.g., necrotizing autoimmune myositis (or immune-mediated necrotizing myositis)), polymyositis (PM), and sporadic inclusion body myositis (sIBM). In some embodiments, the autoimmune disease is ASSD. In some embodiments, the autoimmune disease is OM. In some embodiments, the autoimmune disease is DM. In some embodiments, the autoimmune disease is JM. In some embodiments, the autoimmune disease is NM. In some embodiments, the autoimmune disease is PM. In some embodiments, the autoimmune disease is sIBM.

[0315] In some embodiments, the autoimmune disease includes vasculitis. In some embodiments, the autoimmune disease is vasculitis. In some embodiments, the vasculitis is large vasculitis. In some embodiments, the vasculitis is medium vasculitis. In some embodiments, the vasculitis is small vasculitis. In some embodiments, the vasculitis is anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis. In some embodiments, ANCA vasculitis is granulomatosis with polyangiitis (GPA). In some embodiments, ANCA vasculitis is microscopic polyangiitis (MPA). In some embodiments, ANCA vasculitis is eosinophilic granulomatosis with polyangiitis (EGPA).

[0316] In some embodiments, the autoimmune disease includes myasthenia gravis (MG). In some embodiments, the autoimmune disease is MG. In some embodiments, MG is ocular MG. In some embodiments, MG is early-onset systemic MG. In some embodiments, MG is late-onset MG.

[0317] In some embodiments, the autoimmune disease is autoimmune encephalitis (AE). In some embodiments, the AE includes an antibody against an intracellular antigen (e.g., anti-Hu or anti-GAD65). In some embodiments, the AE includes an autoantibody against an extracellular epitope of an ion channel, receptor, and / or other related protein (e.g., anti-NMDA receptor).

[0318] In some embodiments, the subject has a persistent or relapsing disease after treatment with, for example, a B-cell targeting agent (e.g., anti-BAFF antibody, anti-CD19 antibody, or anti-CD20 antibody). In some embodiments, the subject is effectively treated by administration even though the subject has become resistant to the previous B-cell targeting agent. In some embodiments, the subject has not relapsed but is determined to be at risk of relapse, such as having a high risk of relapse, and therefore the composition is administered prophylactically, for example, to reduce the likelihood of relapse or to prevent relapse.

[0319] In some embodiments, the subject has received one or more prior treatments before initiating administration of genetically modified NK cells. In some embodiments, the subject has received at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty or more prior treatments. In some embodiments, the subject has received at least three, four, five, six, seven, eight, nine, or ten or more prior treatments. In some embodiments, the subject has received at least one prior treatment. In some embodiments, the subject has received at least two prior treatments. In some embodiments, the subject has received at least three prior treatments. In some embodiments, the subject has received at least four prior treatments.

[0320] In some embodiments, the subjects were relapsed or refractory to one or more prior treatments. In some embodiments, prior treatments included B-cell targeting agents (e.g., anti-BAFF antibodies, anti-CD19 antibodies, anti-CD20 antibodies, or anti-CD22 antibodies), immunosuppressants, steroids (e.g., corticosteroids), nonsteroidal anti-inflammatory drugs (NSAIDs), antimalarial agents, or HSCT. In some embodiments, prior treatments included B-cell targeting agents. In some embodiments, one or more prior treatments included immunosuppressants. In some embodiments, one or more prior treatments included steroids (e.g., corticosteroids). In some embodiments, one or more prior treatments included nonsteroidal anti-inflammatory drugs (NSAIDs). In some embodiments, one or more prior treatments included antimalarial agents. In some embodiments, one or more prior treatments included HSCT.

[0321] In some embodiments, cell therapy is performed by allogeneic transplantation, in which case the cells are isolated from and / or otherwise prepared from a subject other than the subject receiving or ultimately receiving cell therapy, e.g., a first subject. In such embodiments, the cells are then administered to a different subject of the same species, e.g., a second subject. Thus, in some embodiments, the cells are allogeneic with respect to the subject being treated.

[0322] In some embodiments, the subjects to whom the cells, cell populations, or compositions are administered are primates such as humans. In some embodiments, the subjects to whom the cells, cell populations, or compositions are administered are non-human primates. In some embodiments, non-human primates are monkeys (e.g., cynomolgus macaques) or apes. In some embodiments, the subjects are non-primate mammals, such as rodents (e.g., mice, rats). The subjects may be male or female and may be any appropriate age, including infants, young children, adolescents, adults, and elderly subjects. In some embodiments, the subjects are child subjects (e.g., boys or adolescents). For example, in some embodiments, the subjects are under 18 years of age. In some embodiments, the subjects are about 12 to about 18 years of age. In some embodiments, the subjects are at least 12 years of age. In some embodiments, the subjects are about 14 to about 18 years of age. In some embodiments, the subjects are at least 14 years of age. In some embodiments, the subjects are about 16 to about 18 years of age. In some embodiments, the subjects are at least 16 years of age. It is expected that pediatric subjects may have worse outcomes, more severe disease, and / or lower compliance with treatment regimens compared to adult subjects (e.g., subjects at least 18 years old).

[0323] In some embodiments, the subjects are adults. In some embodiments, the subjects are between 18 and 65 years old. In some embodiments, the subjects are humans who are at least 18 years old.

[0324] In some embodiments, subjects are selected for treatment based on a disease activity index (e.g., the subject's score on the disease activity index). In some embodiments, the method includes selecting subjects for treatment based on a disease activity index (e.g., the subject's score on the disease activity index). Any disease activity index described in this Section III may be used.

[0325] In some embodiments, subjects do not have CNS lesions such as neuropsychiatric systemic lupus erythematosus (NPSLE) (also known as CNS lupus). In some embodiments, subjects do not have active CNS lupus at the time of administration of the genetically modified NK cell composition. In some embodiments, subjects did not have active CNS lupus within one year prior to administration of the genetically modified NK cell composition. In some embodiments, active CNS lupus includes aseptic meningitis, ataxia, CNS vasculitis, cranial neuropathy, demyelinating syndrome, optic neuritis, psychosis, seizures, transverse myelitis, or any combination thereof. In some embodiments, subjects have CNS lesions such as NPSLE.

[0326] In some embodiments, the subjects do not have lupus nephritis (LN). In some embodiments, the subjects have SLE with renal lesions (e.g., LN). In some embodiments, the subjects have class III or class IV LN as defined, for example, by the History of International Society of Nephrology / Rental Pathology Society (ISN / RPS). Bajema et al., Kidney Int. 2018;93(4):789-96. In some embodiments, the subjects have class III or class IV LN within about 6 months or about 7 months prior to administration of the genetically engineered NK cell composition. In some embodiments, the subjects have class III or class IV LN (with or without class V LN). In some embodiments, the subjects have class III LN. In some embodiments, the subjects have class IV LN. In some embodiments, the subjects do not have class V LN. In some embodiments, the subjects have class V LN. In some embodiments, LN includes a urinary protein-creatinine ratio of 2000 mg / g (or equivalent) or higher at the time of administration of the genetically modified NK cell composition to the subject. In some embodiments, active LN is defined as proteinuria of 1500 mg / 24 hours or higher when assessed by 24-hour urine collection, and is defined, for example, by a urinary protein-creatinine ratio (UPCR) of 1.5 mg / mg or higher. In some embodiments, active LN is defined as a urinary protein:creatinine ratio (UPCR) of 7.0 g / g or higher, or proteinuria of 1.5 g / day or higher. In some embodiments, the subject did not require induction therapy for an autoimmune disease (e.g., SLE) within approximately one year prior to administration of the genetically modified NK cell composition. In some embodiments, the subject did not have histological evidence of diffuse proliferative glomerulonephritis within approximately 12 weeks prior to administration of the genetically modified NK cell composition. In some embodiments, the subject has lupus nephritis (LN).

[0327] In some embodiments, at the time of administration of the manipulated NK cell composition, the subjects have a score of 10 or higher on the European League Against Rheumatism (EULAR) / American College of Rheumatology (ACR) 2019 classification criteria for SLE. Aringer et al., Arthritis Rheumatol (2019) 71(9):1400-12. In some embodiments, at the time of administration of the manipulated NK cell composition, the subjects have a score of 10 or higher on the 2012 SLICC criteria for SL. Petri et al., Arthritis Rheum (2012) 64:2677-86. In some embodiments, at the time of administration of the manipulated NK cell composition, the subjects have a Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) score of 4 or higher. In some embodiments, at the time of administration of the engineered NK cell composition, the subjects have a Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) score of 6 or higher. Gladman et al., J Rheumatol (2002) 29(2):288-91. In some embodiments, at the time of administration of the engineered NK cell composition, the subjects are positive for one or more of the following: antinuclear antibody (ANA), anti-double-stranded DNA (anti-dsDNA) antibody, and anti-Smith (anti-Sm) antibody. In some embodiments, at the time of administration of the engineered NK cell composition, the subjects are positive for ANA. In some embodiments, ANA positivity is defined by an ANA titer of 1:80 or higher in a sample from the subject (e.g., serum sample). In some embodiments, at the time of administration of the engineered NK cell composition, the subjects are positive for anti-dsDNA antibody. In some embodiments, at the time of administration of the engineered NK cell composition, the subjects are positive for anti-Sm antibody. Methods for determining whether a subject is positive for ANA, anti-dsDNA antibodies, and anti-Sm antibodies are known and described in the art.

[0328] In some embodiments, the subject has multiple sclerosis (MS), which is defined, for example, by the 2017 McDonald diagnostic criteria (Thompson et al., Lancet Neurol. (2018) 17:162-73). In some embodiments, MS is RRMS. In some embodiments, MS is SPMS. In some embodiments, MS is PPMS. In some embodiments, the subject has a one-year history of disability progression and any two of the following: (1) one or more T2 lesions characteristic of MS in one or more typical brain regions (periventricular, cortical, or subcortical, or occult), (2) two or more T2 lesions in the spinal cord, and (3) the presence of CSF-specific oligoclonal bands. In some embodiments, prior treatment includes an anti-CD20 antibody (e.g., ocrelizumab or rituximab). In some embodiments, prior treatment includes a selective sphingosine-1-phosphate receptor 1 and 5 modulator (e.g., siponimod). In some embodiments, the pretreatment includes a DNA intercalator (e.g., mitoxantrone). In some embodiments, the pretreatment includes a purine analog (e.g., cladribine).

[0329] In some embodiments, the subject has myasthenia gravis (MG). In some embodiments, MG is ocular MG. In some embodiments, MG is generalized MG. In some embodiments, the subject has an MG-ADL score of 4 or higher. In some embodiments, the subject has an MG-ADL score of 6 or higher. In some embodiments, the subject has an MG-ADL score of 8 or higher. In some embodiments, the subject has an MG-ADL score of 10 or higher. In some embodiments, the subject has MGFA class II, III, or IV. In some embodiments, the subject has MGFA class II. In some embodiments, the subject has MGFA class III. In some embodiments, the subject has MGFA class IV. In some embodiments, the subject has an anti-AChR antibody. In some embodiments, the subject has an anti-MuSK antibody. In some embodiments, the subject has an anti-LRP4 antibody. In some embodiments, prior treatment includes thymectomy. In some embodiments, prior treatment includes an anti-CD20 antibody (e.g., rituximab). In some embodiments, prior treatment includes an antimetabolite (e.g., methotrexate). In some embodiments, the prior treatment includes an anti-complement antibody (e.g., an anti-C5 antibody). In some embodiments, the prior treatment includes an immune checkpoint inhibitor (e.g., an anti-PD1 antibody, an anti-PDL1 antibody, or a CTLA antibody). In some embodiments, the prior treatment includes an AchE inhibitor (e.g., edrophonium chloride).

[0330] In some embodiments, the subject has IIM (also known as myositis). In some embodiments, the subject has a suspected or confirmed (≥55%) diagnosis of idiopathic inflammatory myopathy according to the 2017 ACR / EULAR Classification Criteria (Lundberg, Tjarnlund et al. 2017). In some embodiments, the subject has an MMT score of ≤80. In some embodiments, the myositis is anti-synthetase syndrome. In some embodiments, the myositis is dermatomyositis. In some embodiments, the subject has a skin rash. In some embodiments, the subject has proximal upper and lower limb weakness. In some embodiments, the subject has anti-SAE antibodies. In some embodiments, the subject has anti-Mi2 antibodies. In some embodiments, the subject has anti-MDA5 antibodies. In some embodiments, the subject has anti-NXP2 antibodies. In some embodiments, the subject has anti-TIF1 antibodies. In some embodiments, the myositis is juvenile myositis. In some embodiments, the myositis is necrotizing myositis. In some embodiments, the subject has anti-SRP antibodies. In some embodiments, the subject has an anti-HMGCR antibody. In some embodiments, the myositis is polymyositis. In some embodiments, the subject has an anti-PM-Scl antibody. In some embodiments, the subject has an anti-Scl-70 antibody. In some embodiments, the subject has an anti-Ku antibody. In some embodiments, the subject has an anti-RNP antibody. In some embodiments, the subject has an anti-Ro / SSA antibody or an anti-La / SSB antibody. In some embodiments, the myositis is sporadic inclusion body myositis (sIBM). In some embodiments, the subject has an anti-CN1A antibody. In some embodiments, prior treatment includes intravenous immunoglobulin (IVIg). In some embodiments, prior treatment includes a co-stimulatory modifier (e.g., abatacept).

[0331] In some embodiments, the subject has scleroderma. In some embodiments, the scleroderma is systemic scleroderma (also known as systemic sclerosis). In some embodiments, the subject is classified as having systemic sclerosis with a total score of 9 or higher according to the 2013 ACR / EULAR classification criteria. In some embodiments, the subject has active disease. In some embodiments, active disease is defined as an MRSS score of 15 or higher. In some embodiments, the subject has an anti-centromere antibody. In some embodiments, the subject has an anti-topoisomerase 1 antibody. In some embodiments, the subject has an anti-RNA polymerase III antibody. In some embodiments, the scleroderma is focal scleroderma. In some embodiments, prior treatment includes immunosuppressive therapy (e.g., methotrexate, cyclophosphamide, mycophenolate mofetil, cyclosporine, azathioprine). In some embodiments, prior treatment includes a calcium channel blocker. In some embodiments, prior treatment includes an endothelin receptor antagonist. In some embodiments, prior treatment includes a PDE5 inhibitor. In some embodiments, prior treatment includes one or more of the following: prokinetic agents, proton pump inhibitors, ACE inhibitors, anticoagulants, prostacyclins, phototherapy, and steroids (e.g., corticosteroids).

[0332] In some embodiments, the subject has vasculitis. In some embodiments, the subject has anti-neutrophil cytoplasmic antibody (ANCA). In some embodiments, the subject has ANCA-associated vasculitis (AAV). In some embodiments, AAV is GPA. In some embodiments, AAV is MPA. In some embodiments, the subject has renal focal vasculitis. In some embodiments, AAV is EGPA. In some embodiments, the subject has anti-proteinase-3 (PR3-ANCA). In some embodiments, the subject has anti-myeloperoxidase (MPO-ANCA). In some embodiments, prior treatment includes steroids (e.g., glucocorticoids). In some embodiments, prior treatment includes immunosuppressants (e.g., cyclophosphamide, methotrexate, mycophenolate mofetil). In some embodiments, prior treatment includes anti-CD20 antibodies (e.g., rituximab). In some embodiments, prior treatment includes IVIg.

[0333] In some embodiments, the presence or level of antibodies is determined by analysis of a biopsy, such as a muscle biopsy. In some embodiments, the presence or level of antibodies is determined in a blood sample. In some embodiments, if antibodies are present in a blood sample from a subject, the subject is said to be serologically positive for the antibodies.

[0334] In some embodiments, the subject was diagnosed with an autoimmune disease (e.g., SLE or LN) at least about 18 weeks before or at least about 30 weeks before administering the composition. In some embodiments, the subject was diagnosed with an autoimmune disease at least about 18 weeks, at least about 20 weeks, at least about 22 weeks, at least about 24 weeks, at least about 26 weeks, at least about 28 weeks, or at least about 30 weeks before administering the composition. In some embodiments, the subject was diagnosed with an autoimmune disease at least about 20 weeks before administering the composition. In some embodiments, the subject was diagnosed with an autoimmune disease at least about 21 weeks before administering the composition. In some embodiments, the subject was diagnosed with an autoimmune disease at least about 22 weeks before administering the composition. In some embodiments, the subject was diagnosed with an autoimmune disease at least about 23 weeks before administering the composition. In some embodiments, the subject was diagnosed with an autoimmune disease at least about 24 weeks before administering the composition. In some embodiments, the subjects were diagnosed with an autoimmune disease at least about 25 weeks prior to administration of the composition. In some embodiments, the subjects were diagnosed with an autoimmune disease at least about 26 weeks prior to administration of the composition. In some embodiments, the subjects were diagnosed with an autoimmune disease at least about 27 weeks prior to administration of the composition. In some embodiments, the subjects were diagnosed with an autoimmune disease at least about 28 weeks prior to administration of the composition. In some embodiments, the subjects were diagnosed with an autoimmune disease at least about 29 weeks prior to administration of the composition. In some embodiments, the subjects were diagnosed with an autoimmune disease at least about 30 weeks prior to administration of the composition. In some embodiments, the subjects had SLE at least about 6 months prior to administration of the manipulated NK cell composition.

[0335] In some embodiments, the subject has not been previously treated with CAR T cells (e.g., CD19 CAR T cells). Therefore, in some embodiments, the subject is CAR T cell (e.g., CD19 CAR T cell) naive. In some embodiments, the subject has been previously treated with CAR T cells (e.g., CD19 CAR T cells). Therefore, in some embodiments, the subject is exposed to CAR T cells (e.g., CD19 CAR T cells). In some embodiments, the subject is relapsed / refractory to CAR T cells (e.g., CD19 CAR T cells). In some embodiments, the CAR T cells are CD19 CAR T cells.

[0336] In some embodiments, the subject has not been previously treated with CAR NK cells (e.g., CD19 CAR NK cells). Therefore, in some embodiments, the subject is CAR NK cell (e.g., CD19 CAR NK cell) naive. In some embodiments, the subject has been previously treated with CAR NK cells (e.g., CD19 CAR NK cells). Therefore, in some embodiments, the subject is exposed to CAR NK cells (e.g., CD19 CAR NK cells). In some embodiments, the subject is relapsed / refractory to CAR NK cells (e.g., CD19 CAR NK cells). In some embodiments, the CAR NK cells are CD19 CAR NK cells.

[0337] In some embodiments, the subjects have not been previously treated with CD19-targeted therapy. In some embodiments, the subjects have not been previously treated with mesenchymal stem cell therapy. In some embodiments, the subjects have not undergone solid organ transplantation. In some embodiments, the subjects have not undergone hematopoietic stem cell transplantation.

[0338] Genetically engineered cells can be administered by any suitable means, e.g., bolus injection, injection, e.g., intravenous or subcutaneous injection, intraocular injection, periorbital injection, subretinal injection, intravitreous injection, transseptal injection, subscleral injection, choroidal injection, intracavitary injection, subpyramidal injection, subtenon injection, posterior bulbar muscle injection, peribulbar muscle injection, or posterior cruciate delivery. In some embodiments, cells are administered parenterally, intrapulmonaryly, intranasally, and intrafocally, if desired for local treatment. Parenteral administrations include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. In some embodiments, a given amount is administered by a single bolus of cells.

[0339] In some embodiments, the administration of the genetically modified cell composition or any additional therapy, such as lymphocyte apheresis and / or combination therapy, is carried out by outpatient delivery. In some embodiments, the administration of the genetically modified cell composition is carried out by outpatient delivery. In some embodiments, the administration of the first dosing cycle is carried out by outpatient delivery. In some embodiments, the administration of subsequent dosing cycles is carried out by outpatient delivery. In some embodiments, the administration of each dosing cycle is carried out by outpatient delivery. For the prevention or treatment of a disease, the appropriate dosage of cells or compositions containing them may depend on the type of disease being treated, the type of binding molecule or recombinant receptor, the severity and course of the disease, whether the binding molecule or recombinant receptor is administered for preventive or therapeutic purposes, prior treatment, the patient's clinical history and response to the recombinant receptor or cells, and the discretion of the attending physician.

[0340] In some embodiments, the dose and / or frequency of administration are determined based on efficacy and / or response. In some embodiments, efficacy and / or response are determined by any measure known in the art, including those described in Arora et al., Arthritis Care Res (2020) 72(S10):27-46. In some examples, the dose and / or frequency of administration are determined by the expansion and persistence of recombinant receptors or cells in the blood and / or bone marrow. In some embodiments, the dose and / or frequency of administration are determined based on the presence of autoantibodies in a sample from the subject.

[0341] In certain embodiments, treatment of a subject with the genetically modified cells described herein achieves, for example, one, two, three, or four or more of the following effects: (i) reduction or improvement of the severity of the disease or associated symptoms; (ii) reduction of the duration of the disease-related symptoms; (iii) protection against the progression of the disease or associated symptoms; (iv) regression of the disease or associated symptoms; (v) protection against the onset or occurrence of the disease-related symptoms; (vi) protection against the recurrence of the disease-related symptoms; (vii) reduction of the length of hospitalization of the subject; (viii) reduction of the length of hospitalization; (ix) increase in the survival time of the subject with the disease; (x) reduction in the number of disease-related symptoms; and (xi) enhancement, improvement, supplement, complement, or strengthening of the preventive or therapeutic effect of another therapy.

[0342] Non-limiting examples of the ability of anti-CD19 CAR-expressing NK cells provided herein to exert cytotoxicity against CD19-expressing target cells are described in Morisot et al., J ImmunoTher Canc (2020)8(suppl. 3):Abstract 127 and PCT applications PCT / US2020 / 020824 and PCT / US2023 / 069403, respectively, which are incorporated herein by reference in their entirety.

[0343] In some embodiments, the method includes administering a dose of engineered cells or a composition containing engineered cells. In some embodiments, the method includes administering one, two, or three doses of engineered cells or a composition containing engineered cells. In some embodiments, engineered cells or a composition containing engineered cells can be used in a treatment regimen, and the treatment regimen (e.g., administration cycle) includes administering a dose of engineered cells or a composition containing engineered cells. In some embodiments, engineered cells or a composition containing engineered cells can be used in a treatment regimen, and the treatment regimen includes administering one, two, or three doses of engineered cells or a composition containing engineered cells. In some embodiments, the dose may contain, for example, a specific number or range of recombinant receptor-expressing immune cells (e.g., NK cells), for example, any number of such cells as described herein. In some embodiments, a composition containing a dose of cells can be administered. In some embodiments, the number, quantity, or proportion of CAR-expressing cells in a cell population or cell composition can be evaluated, for example, by detecting surrogate markers by flow cytometry or other means, or by detecting the binding of labeled molecules, such as labeled antigens that can specifically bind to the binding molecules or receptors provided herein.

[0344] The dose of immune cells such as NK cells can be determined for a given subject based on their body weight, type and condition of the disease, and the desired aggressiveness of the treatment, but depending on the embodiment, it may be about 10 per kg. 5 Approximately 10 cells per kg 12 This range is up to a single cell (for example, 10 5 ~10 7 pieces, 10 7 ~10 10 pieces, 10 10 ~10 12 (Individuals and their overlapping range). In one embodiment, a dose-escalation regimen is used. In some embodiments, for example, about 1 × 10 6 Cells per kg ~ approximately 1 × 10⁻⁶ 8NK cells are administered in the range of individual cells / kg.

[0345] In some embodiments, each dose in the administration cycle is approximately 1 × 10⁻⁶ 6 CAR-expressing NK cells per kilogram (kg) ~ approximately 1 × 10⁻⁶ 8 Contains CAR-expressing NK cells / kg....

Claims

1. A method for treating an autoimmune disease, comprising administering to a subject having an autoimmune disease a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), wherein the genetically engineered NK cells are allogeneic to the subject.

2. A method for reducing B cells in a subject, comprising administering to a subject having an autoimmune disease a composition containing natural killer (NK) cells genetically modified to express a chimeric antigen receptor (CAR) that binds to CD19, wherein the genetically modified NK cells are allogeneic to the subject.

3. (i) administering a composition containing NK cells genetically engineered to express CAR to a target of lymphocyte apheresis therapy before administering the composition; or (ii) The method according to claim 1, wherein the subject is administered lymphocyte apheresis before administration of a composition comprising NK cells genetically engineered to express CAR.

4. The method according to claim 3, wherein the lymphocyte apheresis therapy comprises the administration of cyclophosphamide but does not comprise the administration of fludarabine.

5. The method according to claim 1, wherein the autoimmune disease is systemic lupus erythematosus (SLE).

6. The method according to claim 1, wherein the autoimmune disease is lupus nephritis (LN) or includes lupus nephritis.

7. The method according to claim 1, wherein the autoimmune disease includes scleroderma, myositis, vasculitis, multiple sclerosis (MS), myasthenia gravis (MG), rheumatoid arthritis (RA), thyroid disease, type 1 diabetes, or any combination thereof.

8. The method according to claim 1, wherein the autoimmune disease includes scleroderma, and the autoimmune disease may also include systemic sclerosis (SSc).

9. The method according to claim 1, wherein the autoimmune disease may include myositis, and the autoimmune disease may include anti-synthetase syndrome.

10. The method according to claim 1, wherein the autoimmune disease includes vasculitis, and the autoimmune disease may include ANCA-associated vasculitis (AAV).

11. CAR, (a) Extracellular antigen-binding domain; (b) transmembrane domain; and (c) Intracellular signal transduction domain The method according to claim 1, including the method described in claim 1.

12. The method according to claim 11, wherein the extracellular antigen-binding domain comprises a heavy chain variable region (VH) comprising CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences described in SEQ ID NOs: 24, 25, and 26, respectively; and a light chain variable region (VL) comprising CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences described in SEQ ID NOs: 27, 28, and 29, respectively.

13. The method according to claim 12, wherein VH comprises the amino acid sequence described in SEQ ID NO: 35 and VL comprises the amino acid sequence described in SEQ ID NO:

36.

14. The method according to claim 11, wherein the extracellular antigen-binding domain is a single-chain variable fragment (scFv) containing the amino acid sequence described in SEQ ID NO:

37.

15. The method according to claim 11, wherein the transmembrane domain includes a CD8 alpha transmembrane region.

16. The method according to claim 11, wherein the transmembrane domain comprises the amino acid sequence described in SEQ ID NO:

8.

17. The method according to claim 11, wherein the intracellular signaling domain comprises the intracellular signaling region and the CD3 zeta domain of OX40.

18. The method according to claim 17, wherein the intracellular signaling region of OX40 comprises the amino acid sequence described in SEQ ID NO:

14.

19. The method according to claim 17, wherein the CD3 zeta domain comprises the amino acid sequence described in SEQ ID NO:

16.

20. The method according to claim 1, wherein CAR comprises the amino acid sequence described in SEQ ID NO:

38.

21. The method according to claim 1, wherein NK cells genetically engineered to express CAR also express membrane-bound interleukin-15 (mbIL15).

22. The method according to claim 21, wherein mbIL15 comprises the amino acid sequence described in SEQ ID NO:

40.

23. The method according to claim 21, wherein CAR and mbIL15 are bisistronally encoded by the same nucleic acid molecule, and the nucleic acid sequences encoding CAR and mbIL15 may be separated by the nucleic acid sequence encoding the T2A peptide.

24. The method according to claim 1, wherein a composition comprising NK cells genetically engineered to express CAR is administered to a subject in a dosing regimen comprising a dosing cycle.

25. The method according to claim 24, wherein the administration cycle comprises a first dose, a second dose, and a third dose of a composition comprising NK cells genetically engineered to express CAR.

26. The method according to claim 25, wherein the second dose is administered to the subject approximately two to four days after the first dose has been administered to the subject.

27. The method according to claim 26, wherein the third dose is administered to the subject approximately two to four days after the second dose has been administered to the subject.

28. The method according to claim 25, wherein a first dose is administered on approximately day 0 of the administration cycle, a second dose is administered on approximately day 3 of the administration cycle, and a third dose is administered on approximately day 7 of the administration cycle.

29. Each dose in the administration cycle is approximately 1 x 10 ８ Individual CAR-expressing NK cells ~ approximately 1 × 10⁶ １０ A single CAR-expressing NK cell, or approximately 3 × 10⁶ cells. ８ Individual CAR-expressing NK cells ~ approximately 3 × 10⁶ ９ The method according to claim 25, comprising CAR-expressing NK cells (each including both ends).

30. Each dose in the administration cycle is approximately 3 x 10 ８ A single CAR-expressing NK cell, approximately 1 × 10⁶ ９ A single CAR-expressing NK cell, or approximately 1.5 × 10⁶ cells. ９ The method according to claim 25, comprising 1 CAR-expressing NK cell.

31. Each dose in the administration cycle is about 2×10 ９ CAR-expressing NK cells or about 2.5×10 ９ The method according to claim 25, comprising CAR-expressing NK cells.

32. (i) Each of the first, second, and third doses of the administration cycle is approximately 1 × 10 ９ A single CAR-expressing NK cell, approximately 1.5 × 10⁶ ９ A single CAR-expressing NK cell, approximately 2 × 10⁶ ９ A single CAR-expressing NK cell, or approximately 2.5 × 10⁶ cells. ９ Contains individual CAR-expressing NK cells; (iii) The method according to claim 25, wherein a second dose is administered to the subject approximately three days after the first dose has been administered to the subject, and a third dose is administered to the subject approximately four days after the second dose has been administered to the subject.

33. A method for treating an autoimmune disease, comprising administering to a subject having an autoimmune disease a composition comprising natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds to CD19, (i) A composition comprising NK cells genetically engineered to express CAR is administered to a subject in a dosing regimen comprising a dosing cycle, wherein the dosing cycle comprises a first dose, a second dose, and a third dose of the composition; (ii) Each of the first, second, and third doses of the administration cycle is approximately 1 × 10 ９ A single CAR-expressing NK cell, approximately 1.5 × 10⁶ ９ A single CAR-expressing NK cell, approximately 2 × 10⁶ ９ A single CAR-expressing NK cell, or approximately 2.5 × 10⁶ cells. ９ Contains individual CAR-expressing NK cells; (iii) A method in which a second dose is administered to the subject approximately three days after the first dose has been administered, and a third dose is administered to the subject approximately four days after the second dose has been administered.

34. A method for treating or preventing an autoimmune disease, comprising administering a composition containing natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR) to a subject who has, is suspected of having, or is determined to be at risk of having an autoimmune disease, (i) CAR (a) Extracellular antigen-binding domain; (b) transmembrane domain; and (c) Intracellular signal transduction domain Includes, (ii) A composition comprising NK cells genetically engineered to express CAR is administered to a subject in a dosing regimen including a dosing cycle; (iii) Before administering a composition containing NK cells genetically engineered to express CAR to a subject, the subject has received lymphocyte apheresis; (iv) A method of lymphocyte apheresis that includes the administration of cyclophosphamide but does not include the administration of fludarabine.

35. A method for treating systemic lupus erythematosus (SLE), comprising administering to a subject having SLE a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), (i) The subject has received lymphocyte apheresis before administration of a composition containing NK cells genetically engineered to express CAR; (ii) A method of lymphocyte apheresis that includes the administration of cyclophosphamide but does not include the administration of fludarabine.

36. A method for treating lupus nephritis (LN), comprising administering to a subject having LN a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR), (i) The subject has received lymphocyte apheresis before administration of a composition containing NK cells genetically engineered to express CAR; (ii) A method of lymphocyte apheresis that includes the administration of cyclophosphamide but does not include the administration of fludarabine.

37. A method for treating an autoimmune disease, comprising administering to a subject having an autoimmune disease a composition comprising natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds to CD19, (i) The subject has received lymphocyte apheresis before administration of a composition containing NK cells genetically engineered to express CAR; (ii) Lymphocyte apheresis therapy includes administration of cyclophosphamide but does not include administration of fludarabine; (iii) A method for selecting an autoimmune disease from the group consisting of scleroderma, myositis, and vasculitis.

38. Lymphocyte apheresis therapy is administered at approximately 500 mg / m². ２ ~Approx. 1500mg / m ２ Lymphocyte apheresis therapy, including administration of cyclophosphamide, is administered at a single dose of approximately 1000 mg / m². ２ The method according to any one of claims 34 to 37, which may include the administration of cyclophosphamide.

39. Lymphocyte apheresis therapy involves administering approximately 1000 mg / m² of the composition about three days before administration. ２ The method according to any one of claims 3, 4 and 34-38, comprising the administration of cyclophosphamide.

40. A method for treating an autoimmune disease, comprising administering to a subject having an autoimmune disease a composition comprising natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds to CD19, (i) A composition comprising NK cells genetically engineered to express CAR is administered to a subject in a dosing regimen comprising a dosing cycle, wherein the dosing cycle comprises a first dose, a second dose, and a third dose of the composition; (ii) Each of the first, second, and third doses of the administration cycle is approximately 1 × 10 ９ Individual CAR-expressing NK cells ~ approximately 2.5 × 10⁻⁶ ９ Contains individual CAR-expressing NK cells; (iii) The second dose is administered to the subject approximately 2 to 4 days after the first dose has been administered, and the third dose is administered to the subject approximately 2 to 4 days after the second dose has been administered; Approximately three days before administration of a composition containing NK cells genetically engineered to express (iv)CAR, the subject received a single dose of approximately 1000 mg / m². ２ A method involving the administration of lymphocyte apheresis therapy consisting of cyclophosphamide.

41. The method according to any one of claims 1 to 24 and 37 to 40, wherein the subject has relapsed after treatment with a prior line of treatment for an autoimmune disease and / or is refractory to a prior line of treatment for an autoimmune disease.

42. The method according to claim 41, wherein the pretreatment line comprises two, three, or four pretreatment lines.

43. The method according to claim 41, wherein the prior treatment line comprises a corticosteroid, an immunosuppressant, an antimalarial agent, a B-cell targeting agent, hematopoietic stem cell transplantation (HSCT), or any combination thereof.

44. The method according to any one of claims 3, 4, and 34-43, wherein the subject is administered a corticosteroid before, during, and / or after (i) lymphocyte apheresis and / or (ii) administration of the composition, and the corticosteroid may contain a glucocorticoid.

45. The method according to any one of claims 3, 4 and 34-44, wherein the subject is administered an immunosuppressant before, during, and / or after lymphocyte apheresis and / or administration of the composition, the immunosuppressant may include anti-thymocyte globulin (ATG), mammalian targeted rapamycin inhibitor (mTOR), calcineurin inhibitor, or any combination thereof.

46. The method according to any one of claims 1 to 45, wherein the subject does not have CNS lupus.

47. The method according to claim 24, wherein the administration regimen comprises or consists of two, three, four, or five administration cycles.

48. A method for preparing a subject with an autoimmune disease for treatment with a composition comprising natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds to CD19, wherein the subject is administered lymphocyte apheresis before the composition is administered, the lymphocyte apheresis comprising cyclophosphamide.

49. Lymphocyte apheresis therapy is administered at approximately 500 mg / m². ２ ~Approx. 1500mg / m ２ It contains cyclophosphamide, and lymphocyte apheresis therapy is administered in a single dose of approximately 1000 mg / m². ２ The method according to claim 48, which may also contain cyclophosphamide.

50. Lymphocyte apheresis therapy involves administering a single dose of approximately 1000 mg / m² of the composition about three days prior to administration. ２ The method according to claim 48 or claim 49, comprising cyclophosphamide.

51. The method according to any one of claims 48 to 50, wherein NK cells genetically engineered to express a CD19-binding CAR also express membrane-bound interleukin-15 (mbIL15).

52. The method according to any one of claims 1 to 51, wherein, among a plurality of subjects treated according to the method, the number of peripheral B cells in the subjects is reduced on average by at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% compared to subjects that were not treated according to the method, as appropriate.

53. A method for reducing B cells in a subject with a B cell-mediated disease, comprising administering to the subject a composition containing natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds to CD19, (i) A composition comprising NK cells genetically engineered to express CAR is administered to a subject in a dosing regimen including a dosing cycle; (ii) A method that reduces peripheral B cells in the subject by at least about 90%; the peripheral B cells are significantly reduced in the subject during the duration of the administration cycle; and / or at least about 75% of the regrowing peripheral B cells are non-class-switched B cells.

54. The method according to any one of claims 1 to 53, wherein, among a plurality of subjects treated according to the method, the number of peripheral B cells in the subjects is significantly reduced within about 10 days, about 15 days, or about 30 days after administration of a first dose of a composition containing NK cells genetically engineered to express CAR to the subjects, compared with subjects that were not treated according to the method as appropriate.

55. The method according to any one of claims 1 to 54, wherein, among a plurality of subjects treated according to the method, the number of peripheral B cells in the subjects is significantly reduced for at least about 15 days, at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, or at least about 9 months after the final dose of a composition containing NK cells genetically engineered to express CAR, compared to subjects that were not treated according to the method, as appropriate.

56. The method according to any one of claims 1 to 55, wherein about three months, about six months, about nine months, and / or about twelve months after administration of a final dose of a composition comprising NK cells genetically engineered to express CAR to the subject, at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the peripheral B cells in the subject are naive B cells.

57. The method according to claim 56, wherein the naive B cells are non-class-switched and the naive B cells may be IgM or IgD isotypes.

58. Use of a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR) for treating subjects with autoimmune diseases, wherein the NK cells are allogeneic to the subject.

59. Use of a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR) for reducing B cells in a subject with an autoimmune disease, wherein the NK cells are allogeneic to the subject.

60. The use of a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR) for reducing peripheral B cells in subjects with B cell-mediated diseases, (i) A composition comprising NK cells genetically engineered to express CAR, for administration to a subject in a dosing regimen including a dosing cycle; (ii) Peripheral B cells are reduced by at least about 90% in the subject; peripheral B cells are significantly reduced in the subject during the administration cycle; or at least about 75% of regrowing peripheral B cells are non-class-switched B cells.

61. The use of lymphocyte apheresis for preparation of subjects with autoimmune diseases for treatment with a composition comprising natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds to CD19, wherein (i) the lymphocyte apheresis is administered to the subject before the composition is administered to the subject; and (ii) the lymphocyte apheresis comprises cyclophosphamide but does not contain fludarabine.

62. CAR, (a) Extracellular antigen-binding domain; (b) transmembrane domain; and (c) Intracellular signal transduction domain The use according to any one of claims 58 to 61, including the use described in any one of claims 58 to 61.

63. The use of a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR) for the treatment or prevention of autoimmune disease in subjects who have, are suspected of having, or have been determined to be at risk of autoimmune disease, (i) CAR (a) Extracellular antigen-binding domain; (b) transmembrane domain; and (c) Intracellular signal transduction domain Includes, (ii) Prior to administering a composition containing NK cells genetically engineered to express CAR, the subject has received lymphocyte apheresis; (iii) Use of lymphocyte apheresis, which includes the administration of cyclophosphamide but does not include the administration of fludarabine.

64. The use of a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR) for reducing B cells in subjects who have, are suspected of having, or have been determined to be at risk of autoimmune disease, (i) CAR (a) Extracellular antigen-binding domain; (b) transmembrane domain; and (c) Intracellular signal transduction domain Includes, (ii) Prior to administering a composition containing NK cells genetically engineered to express CAR, the subject has received lymphocyte apheresis; (iii) Use of lymphocyte apheresis, which includes the administration of cyclophosphamide but does not include the administration of fludarabine.

65. The use according to any one of claims 62 to 64, wherein the extracellular antigen-binding domain comprises a heavy chain variable region (VH) comprising CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences described in SEQ ID NOs: 24, 25, and 26, respectively; and a light chain variable region (VL) comprising CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences described in SEQ ID NOs: 27, 28, and 29, respectively.

66. The use according to any one of claims 62 to 65, wherein the transmembrane domain includes a CD8 alpha transmembrane region.

67. The use according to any one of claims 62 to 66, wherein the intracellular signaling domain comprises the intracellular signaling region and the CD3 zeta domain of OX40.

68. The use of a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR) for treating subjects with autoimmune diseases, wherein the CAR is (a) Heavy chain variable regions (VH) comprising CDR-1, CDR-2, and CDR-3, respectively, containing the amino acid sequences described in SEQ ID NOs: 24, 25, and 26; and extracellular antigen-binding domains comprising light chain variable regions (VL) comprising CDR-1, CDR-2, and CDR-3, respectively, containing the amino acid sequences described in SEQ ID NOs: 27, 28, and 29; (b) CD8 alpha transmembrane domain; and (c) Intracellular signaling domain of OX40 and intracellular signaling domain including CD3 zeta domain Includes, use.

69. The use of a composition comprising natural killer (NK) cells genetically engineered to express a CD19-binding chimeric antigen receptor (CAR) for treating subjects with autoimmune diseases, (i) CAR (a) an extracellular antigen-binding domain comprising a heavy chain variable region (VH) containing the amino acid sequence described in SEQ ID NO: 35, and a light chain variable region (VL) containing the amino acid sequence described in SEQ ID NO: 36; (b) Transmembrane domains including the CD8 alpha transmembrane region; and (c) Intracellular signaling domain of OX40 and intracellular signaling domain including CD3 zeta domain Including; (ii) A composition comprising NK cells genetically engineered to express CAR is formulated for administration in a dosing regimen comprising a dosing cycle, wherein the dosing cycle comprises a first dose, a second dose, and a third dose of the composition; (iii) Each of the first, second, and third doses of the administration cycle is approximately 1 × 10 ９ Individual CAR-expressing NK cells ~ approximately 2.5 × 10⁻⁶ ９ Contains individual CAR-expressing NK cells; (iv) Use in which the second dose is administered to the subject approximately three days after the first dose has been administered, and the third dose is administered to the subject approximately four days after the second dose has been administered.

70. The use according to any one of claims 58 to 69, wherein the autoimmune disease includes systemic lupus erythematosus (SLE).

71. The use according to any one of claims 58 to 70, wherein the autoimmune disease includes lupus nephritis (LN).

72. The use according to any one of claims 58 to 69, wherein the autoimmune disease includes scleroderma, myositis, vasculitis, multiple sclerosis (MS), myasthenia gravis (MG), rheumatoid arthritis (RA), thyroid disease, type 1 diabetes, or any combination thereof.

73. The use according to any one of claims 60 to 72, wherein the genetically modified NK cells are allogeneic to the target.

74. The use according to any one of claims 58 to 73, wherein, among a plurality of subjects treated with a composition comprising NK cells genetically engineered to express CAR, the number of peripheral B cells in the subjects is reduced on average by at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%.

75. The use according to any one of claims 58 to 74, wherein, among a plurality of subjects treated with a composition comprising NK cells genetically engineered to express CAR, the number of peripheral B cells in the subjects is significantly reduced for at least about 15 days, at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, or at least about 9 months after the final dose of the composition comprising NK cells genetically engineered to express CAR.

76. Lymphocyte apheresis therapy is administered at approximately 500 mg / m². ２ ~Approx. 1500mg / m ２ It contains cyclophosphamide, and lymphocyte apheresis therapy is administered in a single dose of approximately 1000 mg / m². ２ The use according to any one of claims 61, 63-67 and 70-75, which may include cyclophosphamide.

77. Lymphocyte apheresis therapy involves administering approximately 1000 mg / m² of the composition about three days before administration. ２ The use according to claim 76, comprising cyclophosphamide.

78. The use according to any one of claims 58 to 77, wherein NK cells genetically engineered to express a CD19-binding CAR also express membrane-bound interleukin-15 (mbIL15).

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